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https://openalex.org/W3111672162	https://www.medgen-journal.ru/jour/article/download/1669/1305	Russian	null	On the issue of timely application of high-throughput sequencing in ophthalmogenetic practice	Medicinskaâ genetika	2,020	cc-by	1,576	К вопросу о своевременном применении методов высокопроизводительного секвенирования в офтальмогенетической практике Марахонов А.В., Васильева Т.А., Мишина И.А., Репина С.А., Гарифуллина С.А., Щагина О.А., Вассерман Н.Н., Куцев С.И., Кадышев В.В., Зинченко Р.А. ФГБНУ «Медико-генетический научный центр имени академика Н. П. Бочкова» 115522, г. Москва, ул. Москворечье, д. 1 Наследственная офтальмологическая патология представляет собой генетически неоднородную группу заболеваний, кото- рые проявляются либо как изолированное глазное расстройство, либо как симптом наследственных синдромов (хромосом- ных или моногенных). Диагностический поиск в некоторых случаях наследственной офтальмологической патологии может быть трудоемким и дорогостоящим. Наиболее сложная ситуация возникает в случае продолжающейся беременности и необходимости дородовой диагностики. В настоящем сообщении рассмотрена необходимость своевременного примене- ния методов высокопроизводительного секвенирования (ВПС) в офтальмогенетической практике. Схожесть клинических проявлений наследственной офтальмопатологии затрудняет верификацию конкретных нозологических форм. В сложных случаях, как представляется, ВПС является методом выбора, и это может существенно ускорить постановку точного диа- гноза и оценку генетического риска в семье. Ключевые слова: NGS, HTS, клинический полиморфизм Ключевые слова: NGS, HTS, клинический полиморфизм Для цитирования: Марахонов А.В., Васильева Т.А., Мишина И.А., Репина С.А., Гарифуллина С.А., Щагина О.А., Вассерман Н.Н., Куцев С.И., Кады- шев В.В., Зинченко Р.А. К вопросу о своевременном применении методов высокопроизводительного секвенирования в офтальмогенетической практике. Медицинская генетика 2020; 19(8): 31-32. DOI: 10.25557/2073-7998.2020.08.31-32 Автор для корреспонденции: Марахонов А.В.; e-mail: marakhonov@generesearch.ru Финансирование. Исследование поддержано грантом РФФИ № 19-015-00122 и выполнено в рамках Государственного задания Мини- стерства науки и высшего образования. Автор для корреспонденции: Марахонов А.В.; e mail: marakhonov@generesearch.ru Финансирование. Исследование поддержано грантом РФФИ № 19-015-00122 и выполнено в рамках Государственного задания Мини- стерства науки и высшего образования. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Поступила: 20.05.2020 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Поступила: 20.05.2020 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Поступила: 20.05.2020 On the issue of timely application of high-throughput sequencing in ophthalmogenetic practice Marakhonov A.V., Vasilyeva T.A., Mishina I.A., Repina S.A., Garifullina S.A., Shagina O.A., Vasserman N.N., Kutsev S.I., Kadyshev V.V., Zinchenko R.A. Research Centre for Medical Genetics Moskvorechie Str., 1, Moscow, 115522, Russia Marakhonov A.V., Vasilyeva T.A., Mishina I.A., Repina S.A., Garifullina S.A., Shagina O.A., Vasserman N.N., Kutsev S.I., Kadyshev V.V., Zinchenko R.A. Marakhonov A.V., Vasilyeva T.A., Mishina I.A., Repina S.A., Garifullina S.A., Shagina O.A., Vasserman N.N., Kutsev S.I., Kadyshev V.V., Zinchenko R.A. Research Centre for Medical Genetics Moskvorechie Str., 1, Moscow, 115522, Russia Inherited ophthalmological pathology is a heterogeneous group of diseases that manifests itself either as an isolated eye disorder or as a symptom of hereditary syndromes (chromosomal or monogenic). Thus, diagnostic search in some cases of ophthalmologic pathology can be laborious and expensive. The most difficult situation may arise when antenatal diagnosis is required during the ongoing pregnancy. In the present report the necessity of timely application of high-throughput sequencing methods in ophthal- mogenetic practice is considered. Polymorphism of inherited ophthalmologic pathology may seriously hinder making an accurate diagnosis and make it time consuming and expensive. In complex cases, HTS appears to be a method of choice, and it can signifi- cantly speed up diagnosis confirmation of and genetic risk assessment. Keywords: NGS, HTS, clinical polymorphism For citation: Marakhonov A.V., Vasilyeva T.A., Mishina I.A., Repina S.A., Garifullina S.A., Shagina O.A., Vasserman N.N., Kutsev S.I., Kadyshev V.V., Zinchenko R.A. On the issue of timely application of high-throughput sequencing in ophthalmogenetic practice. Medical genetics. 2020; 19(8): 31-32 (In Rus). DOI: 10.25557/2073-7998.2020.08.31-32 Corresponding author: Marakhonov A.V.; e-mail: marakhonov@generesearch.ru Funding. Supported by RFBR grant 19-015-00122 and the State Assignment of the Ministry of Science and Higher Education of the Russian Federation. Conflict of Interest. Authors declare no conflict of interest. Accepted: 20.05.2020 Conflict of Interest. Authors declare no conflict of interest. Accepted: 20.05.2020 МЕДИЦИНСКАЯ ГЕНЕТИКА. 2020. №8 31 краткие сообщения ло, что плод имеет мужской пол и является гемизигот- ным по данному варианту. При проведении прерыва- ния беременности в сроке 21-22 недель развились ос- ложнения, что привело к гистерэктомии. П риход высокопроизводительного секвенирова- ния (ВПС) в клиническую практику привел к из- менению парадигмы в диагностической оценке пациентов с подозрением на редкие генетические забо- левания [1]. Тем не менее, его применение в клиниче- ской практике все еще остается не слишком широким, зачастую в силу причин, не связанных с клинической не- обходимостью, таких как все еще относительно высокая стоимость исследования, а также временные затраты. П Можно предположить, что при более ранней диагно- стике семья могла бы иметь гораздо больше возможно- стей избежать угрожающих жизни осложнений у женщи- ны, а также предотвратить психологическую трагедию в семье. Клинический полиморфизм и вариабельная экс- прессивность наследственной офтальмологической па- тологии сильно затрудняют установление точного диа- гноза и отнимают много времени и средств на постанов- ку диагноза. Литература 1. Hamilton A., Tétreault M., Dyment D.A., et al. Concordance be- tween whole‐exome sequencing and clinical Sanger sequencing: im- plications for patient care. Mol Genet Genomic Med 2016; 4: 504-512. 1. Hamilton A., Tétreault M., Dyment D.A., et al. Concordance be- tween whole‐exome sequencing and clinical Sanger sequencing: im- plications for patient care. Mol Genet Genomic Med 2016; 4: 504-512. 2. Bolz H.J. Despite Challenges and Pitfalls: How Ophthalmology Ben- efits From the Use of Next-Generation Sequencing. Klin Monbl Au- h lkd 2018 23 2 8 263 1. Hamilton A., Tétreault M., Dyment D.A., et al. Concordance be- tween whole‐exome sequencing and clinical Sanger sequencing: im- plications for patient care. Mol Genet Genomic Med 2016; 4: 504-512. 2. Bolz H.J. Despite Challenges and Pitfalls: How Ophthalmology Ben- efits From the Use of Next-Generation Sequencing. Klin Monbl Au- genheilkd 2018; 235: 258−263. 1. Hamilton A., Tétreault M., Dyment D.A., et al. Concordance be- tween whole‐exome sequencing and clinical Sanger sequencing: im- plications for patient care. Mol Genet Genomic Med 2016; 4: 504-512. 2. Bolz H.J. Despite Challenges and Pitfalls: How Ophthalmology Ben- efits From the Use of Next-Generation Sequencing. Klin Monbl Au- genheilkd 2018; 235: 258−263. 2. p p ; 2. Bolz H.J. Despite Challenges and Pitfalls: How Ophthalmology Ben- efits From the Use of Next-Generation Sequencing. Klin Monbl Au- genheilkd 2018; 235: 258−263. Цель: провести медико-генетическое консульти- рование и ДНК-диагностику направленной в ФГБНУ «МГНЦ» семьи для прогнозирования риска рождения больного ребенка. 3. g 3. Васильева Т.А., Воскресенская А.А., Хлебникова О.В., Позде- ева Н.А., Марахонов А.В., Зинченко Р.А. Дифференциальная диагностика наследственных форм врожденной аниридии с по- зиций современной генетики. Вестник Российской академии ме- дицинских наук 2017; 72(4): 233-241 References 1. Hamilton A., Tétreault M., Dyment D.A., et al. Concordance between whole‐exome sequencing and clinical Sanger sequencing: implications for patient care. Mol Genet Genomic Med 2016; 4: 504-512. p ; 2. Bolz H.J. Despite Challenges and Pitfalls: How Ophthalmology Ben- efits From the Use of Next-Generation Sequencing. Klin Monbl Au- genheilkd 2018; 235: 258−263. Проведено секвенирование полного экзома, сек- венирование по Сэнгеру. 3. g ; 3. Vasilyeva T.A., Voskresenskaya A.A., Khlebnikova O.V., Pozdeyeva N.A., Marakhonov A.V., Zinchenko R.A. Differentsial’naya diagnostika nasledstvennykh form vrozhdennoy aniridii s pozitsiy sovremennoy genetiki [Genetic Approaches to Differential Diagnosis of Hereditary Forms of Congenital Aniridia]. Vestnik Rossiyskoy akademii meditsinskikh nauk [Annals of the Russian Academy of Medical Sciences]. 2017;72 (4):233–241. doi: 10.15690/vramn834 (In Russ.) Пациенты и методы у ( ) 4. Марахонов А.В., Васильева Т.А., Воскресенская А.А., Кады- шев В.В., Поздеева Н.А., Шилова Н.В., Браславская С.И., Хлеб- никова О.В., Зинченко Р.А., Куцев С.И. Опыт применения ме- дицинской технологии диагностики врожденной аниридии в ФГБНУ МГНЦ М д 2017; 16(11): 23 26 Проведено обследование семьи, в которой у ребен- ка, мальчика 6 лет, были диагностированы врожден- ная аниридия, глаукома, отслоение сетчатки, тяжелая задержка психомоторного развития и отсутствие ре- чи, а также было проведено несколько офтальмологи- ческих хирургических операций. Ранее у пораженно- го ребенка был проведен поиск мутаций в гене PAX6 и изучены вариации числа копий локуса 11p13, кото- рые не выявили никаких изменений [4]. Мать ребен- ка беременна, срок гестации 7-8 недель. ФГБНУ «МГНЦ». Медицинская генетика 2017; 16(11): 23−26. 5. Dickinson J.L., Sale M.M., Passmore A., et al. Mutations in the NDP gene: contribution to Norrie disease, familial exudative vitreoretinopathy and retinopathy of prematurity. Clin Exp Ophthalmol 2006; 34: 682−688. Marakhonov A.V., Vasilyeva T.A., Mishina I.A., Repina S.A., Garifullina S.A., Shagina O.A., Vasserman N.N., Kutsev S.I., Kadyshev V.V., Zinchenko R.A. Представляется, что в сложных случаях ВПС является методом выбора, который может существенно ускорить установление диагноза и оценку генетическо- го риска. Это важно в случае крайне редких заболеваний, которые не известны врачам и офтальмологам. По данным ВОЗ доля наследственных заболева- ний глаз в структуре офтальмоогической патологии составляет не менее 35%, они приводят в 65−70% слу- чаев к инвалидности. Исследования наследственной офтальмопатологии затруднены ввиду широкой ге- нетической гетерогенности (аллельной, локусной) и выраженного клинического полиморфизма [2,3]. При всей очевидности основного направительного диагноза заболевания, обусловленного вариантами нуклеотидной последовательности в каком-то одном гене, необходимо всегда рассматривать возможность направления пациента на полноэкзомное секвениро- вание в качестве первого шага диагностики. Результаты Учитывая отсутствие точного диагноза у поражен- ного мальчика, для расчета генетического риска было выполнено полноэкзомное секвенирование, которое позволило выявить ранее не описанную нонсенс-му- тацию p.G129X в гене NDP, ассоциированном с болез- нью Норри (OMIM#310600) [5]. Последующая валида- ция варианта подтвердила идентифицированную заме- ну, унаследованную по X-сцепленному рецессивному типу. Исследование амниотической жидкости показа- / 4. Marakhonov A.V., Vasilyeva T.A., Voskresenskaya A.A., Kadyshev V.V., Pozdeyeva N.A., Shilova N.V., Braslavskaya S.I., Khlebnikova O.V., Zinchenko R.A., Kutsev S.I. Opyt primeneniya meditsinskoy tekhnologii diagnostiki vrozhdennoy aniridii v FGBNU «MGNC» [Application of medical technology for the diagnosis of congenital aniridia at the Research Centre for Medical Genetics]. Meditsinskaya genetika [Medical Genetics] 2017; 16(11): 23-26. (In Russ.) 5. Dickinson J.L., Sale M.M., Passmore A., et al. Mutations in the NDP gene: contribution to Norrie disease, familial exudative vitreoretinopathy and retinopathy of prematurity. Clin Exp Ophthalmol 2006; 34: 682−688. МЕДИЦИНСКАЯ ГЕНЕТИКА. 2020. №8 МЕДИЦИНСКАЯ ГЕНЕТИКА. 2020. №8 32
https://openalex.org/W4324377416	https://www.qeios.com/read/H3CWR6/pdf	English	null	Review of: "Relevance of Medical Ethics in Public Health: Case Study of Polio Eradication"	null	2,023	cc-by	866	Qeios, CC-BY 4.0 · Review, March 15, 2023 Review of: "Relevance of Medical Ethics in Public Health: Case Study of Polio Eradication" Vandana Prasad Potential competing interests: No potential competing interests to declare. This is very important article written by domain experts that have been involved in this issue for decades. It contains extremely significant information and analytical insights and raises critical questions that have remained unanswered to this day even though suffering has been caused to so many as a result of a failure of public health diligence. One main critique I have is with respect to readability for the relatively lay reader; the entire article is peppered with short staccato categorical and judgemental statements without reference that appear in the middle of sections that are otherwise dealing with facts and evidence that would lead the reader to make up their own minds about the ethical failures of the public health intervention being analysed. This not only disturbs the flow; it also creates unnecessarily a sense of bias on the part of the authors. Some examples; and there are many, are the following sentences ”Harm through public health is stark", the sentence “In summary, the vaccine-choice did not conform to principles of medical ethics” - which comes in the section on “The beginnings and progress of Global Polio Eradication Initiative” , “ Compassion was conspicuously absent”. The point is not to do away with the views of the authors, but to include these views in the conclusion after the case has been presented. There are other issues with respect to article flow and segmentation with a high level of repetition, disjointedness and also overlap. To give an instance: the introduction starts with a definition of terms, breaks into arguments against the OPV briefly, and ends with a sentence hanging in the air about ““Equity refers to the fair distribution of both benefits and risks if any”' that has no links to anything related to any maldistribution before or after it, till another section is read close to the end of the article. The suggestion would be to keep the introduction to explaining concepts, provide the factual detail, explain the historical context and conclude with the opinions and questions that are peppered throughout. By the time the conclusion is reached, the readers are well inclined to agree since the facts would have been placed on board. Qeios ID: H3CWR6 · https://doi.org/10.32388/H3CWR6 Review of: "Relevance of Medical Ethics in Public Health: Case Study of Polio Eradication" In terms of a related but more substantive issue, one of the main premises of the article seems to be to say that public health has hitherto been kept independent of medical ethics and that should not be the case. This premise is displayed by statements like “As a humanitarian mission, polio eradication is ‘public good’ -- we tend to value it above medical ethics. Basis of such confidence is the perception that public health can do no harm to individuals” . As a public health practitioner, the reviewer does not find this statement quite correct or fair. At a minimum it would require a reference for it not to seem highly biased. It is customary to do a cost-benefit analysis for public health interventions. Whether it is done well or not is a different issue. The domain of ethics in public health is a known entity. To suggest that ethics is given Qeios ID: H3CWR6 · https://doi.org/10.32388/H3CWR6 1/2 Qeios, CC-BY 4.0 · Review, March 15, 2023 atment in medicine than in public health is not borne out by practice and current theory. better treatment in medicine than in public health is not borne out by practice and current theory. Instead, the question that is asked elsewhere in the article - ”Was polio eradication public health or clinical trial?” is exactly the challenge that public health deserves to face. This should get much more leverage in this article rather than the usage of a fairly misplaced distinction between medicine and public health, human rights and public good. However, the title too displays something of a contradiction since clinical trials are well within the domain of public health itself… The question might have been absolutely spot-on as whether this was a public health intervention or a stealthy national population-level field trial that escaped the ethical rigours supposedly placed upon clinical trials. Public health as a discipline is not conceptually at fault here - its practice is as amenable to politics, negligence, vested interests as any other, including the field of medicine, that in many other contexts, has even more hegemony than public health. The article proves more than sufficiently that the case for using OPV for polio eradication is simply poor public health; by all existing public health standards. This choice has been unethical by dint of its lack of transparency and concern for the suffering of persons affected by VAPD/VDVP. Qeios ID: H3CWR6 · https://doi.org/10.32388/H3CWR6 Review of: "Relevance of Medical Ethics in Public Health: Case Study of Polio Eradication" The conclusions and recommendations for due public health processes; including the basic evaluation of existing evidence, as well as processes that examine ethics are perfectly valid. Qeios ID: H3CWR6 · https://doi.org/10.32388/H3CWR6 2/2
https://openalex.org/W2036839434	https://europepmc.org/articles/pmc1976925?pdf=render	English	null	Cancer surveys - Advances & prospects in clinical epidemiological and laboratory oncology	British journal of cancer	1,984	cc-by	777	BOOK REVIEWS Cancer surveys - Advances & prospects in clinical epidemiological and laboratory oncology-Vol. 2, No. 3 Precancer. J.J. DECROSSE, U.K., Oxford University Press, 171 pp. 1983. Current topics in microbiology and immunology - Retroviruses 1. M. COOPER, P.H. HOFSCHNEIDER, H. KOPROWSKI, F. MELCHERS, R. RoTT, H.G. SCHWEIGER, P.K. VOGT & R. ZINKERNAGEL, USA, Springer Press, 146 pp, 1983, $41.40. ISBN 3 540 12167 6. Current topics in microbiology and immunology - Retroviruses 1. M. COOPER, P.H. HOFSCHNEIDER, H. KOPROWSKI, F. MELCHERS, R. RoTT, H.G. SCHWEIGER, P.K. VOGT & R. ZINKERNAGEL, USA, Springer Press, 146 pp, 1983, $41.40. ISBN 3 540 12167 6. "Precancer" is a difficult subject to discuss and an issue of Cancer Surveys, devoted to this topic, is welcome. The approach is selective, with an editorial overview followed by contributions dealing with the cervix uteri, breast, oral cavity, lungs, stomach, large bowel and bladder. A final section discusses some experimental approaches. Emphasis is placed on the diversity of methods which can be used to describe precancerous lesions in' terms other than those of conventional morphology. They include cytogenetics, quantitation of cellular DNA using flow cytometry, immunoperoxidase and other methods for displaying viral and tissue antigens, and (more speculatively) some of the techniques of modern molecular biology such as in situ nucleic acid hybridization. A lot of interesting material is presented in the individual sections such as the growing evidence of an association between human papillomavirus (HPV) infection and condyloma acuminatum in the uterine cervix, current views on the aetiology of gastric cancer and on the adenoma -. carcinoma sequence in the large intestine, and changes in local mucosal expression of ABH and T antigens in bladder cancer. Useful resumes are given of familiar problems such as the nature of oral leukoplakia, the histogenesis of lung cancer and the biological significance of the diverse elements that constitute mammary "fibrocystic disease". This volume contains chapters which deal with avian endogenous viruses sarcoma viruses and acute leukaemia viruses; there is a section on retrovirus replication and integration, whilst the murine system is represented by discussions of leukaemia viruses and Abelson virus. The first impression is one of surprise that there is no chapter on Rous sarcoma virus; however this "prototype model" is referred to so often as a comparison with the other viruses that the reader soon becomes familiar with several of the properties of RSV. Br. J. Cancer (1984), 50, 129-131 Br. J. Cancer (1984), 50, 129-131 BOOK REVIEWS In general, the chapters are well written and not so long as to become tedious. There are several discussions of cellular oncogenes which are informative and of great current interest. The biggest disappointment is the chapter on murine leukaemia viruses. This contains an enormous amount of information but fails to satisfactorily draw the facts together into unifying or summary statements. One problem which occurs throughout the book is the appearance of undefined jargon which could cause problems for the uninitiated reader. p In spite of this reservation, I enjoyed reading this book. It should prove extremely useful to many readers who are interested in a relatively recent overview of retroviruses and their oncogenes. Certain criticisms may be made. Descriptions of morphological changes could in several instances have been replaced by photomicrographs without distorting the deliberately broad balance of presentation. Specific topics which are not discussed in detail include precancer in the oesophagus, endometrium, skin and liver - the last of which (in particular) provides opportunities to consider some reasonably plausible analogies between precancer in man and some reasonably plausible analogies between precancer in man and experimental animals. The genetics of precancerous conditions are inadequately considered. A discussion of the problems posed by precancer to the epidemiologist would have been useful. J. Arrand Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester 20. J. Arrand Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester 20. Molecular aspects of anti-cancer drug action - Topics in molecular and structural biology 3. S. NEIDLE & M.J. WARING, UK, Macmillan Press, x+404 pp, 1983, £45.00. ISBN 0 333 31556 1. The very large volume of literature associated with cancer related topics is largely concerned with the clinical, biological or biochemical aspects of the disease. To the synthetic chemist who originates the drug and begins the chain of discovery events leading to the successful application of a drug, there is little published information to guide his creative or innovative ideas to synthesise new classes of potentially useful drugs. Cancer surveys are already established as a valuable series of reviews; this new number on precancer generally maintains the high quality which we have come to expect. R.L. Carter Institute for Cancer Research, Sutton, Surrey.
https://openalex.org/W4210977786	https://www.mdpi.com/1099-4300/24/2/261/pdf?version=1644975936	English	null	Plasma-like Description for Elementary and Composite Quantum Particles	Entropy	2,022	cc-by	8,443	entropy entropy Citation: Akhmeteli, A. Plasma-like Description for Elementary and Composite Quantum Particles. Entropy 2022, 24, 261. https:// doi.org/10.3390/e24020261 Academic Editors: Andrei Khrennikov, Karl Svozil and Rosario Lo Franco Article Andrey Akhmeteli LTASolid Inc., Houston, TX 77042, USA; akhmeteli@ltasolid.com Abstract: Schrödinger noticed in 1952 that a scalar complex wave function can be made real by a gauge transformation. The author showed recently that one real function is also enough to describe matter in the Dirac equation in an arbitrary electromagnetic or Yang–Mills ﬁeld. This suggests some “symmetry” between positive and negative frequencies and, therefore, particles and antiparticles, so the author previously considered a description of one-particle wave functions as plasma-like collections of a large number of particles and antiparticles. The description has some similarities with Bohmian mechanics. This work offers a criterion for approximation of continuous charge density distributions by discrete ones with quantized charge based on the equality of partial Fourier sums, and an example of such approximation is computed using the homotopy continuation method. An example mathematical model of the description is proposed. The description is also extended to composite particles, such as nucleons or large molecules, regarded as collections including a composite particle and a large number of pairs of elementary particles and antiparticles. While it is not clear if this is a correct description of the reality, it can become a basis of an interesting model or useful picture of quantum mechanics. Keywords: quantum; plasma; antiparticle; homotopy continuation Citation: Akhmeteli, A. Plasma-like Description for Elementary and Composite Quantum Particles. Entropy 2022, 24, 261. https:// doi.org/10.3390/e24020261 Academic Editors: Andrei Khrennikov, Karl Svozil and Rosario Lo Franco entropy entropy 1. Introduction Recent progress in quantum information processing puts a new emphasis on foun- dations of quantum theory. However, it is probably safe to say that there is currently no consensus on the interpretation of quantum theory [1–4]. This suggests that no existing interpretation is completely satisfactory, so the formal description discussed in this work may be of some interest, if not as a “how actually” model, then at least as a “how possibly” model ([5], §3.3). The description is intuitive; uses some notions of quantum ﬁeld theory, such as vacuum polarization; and does not seem to have problems with wave function spreading, although it implies that the wave function has something to do with charge distribution. There are some similarities with de Broglie–Bohm interpretation (Bohmian mechanics), especially for composite particles. Bohmian mechanics is sometimes consid- ered not just as an interpretation, but also as another picture of quantum mechanics and a basis for computational methods [6]. This can be also a way to assess the description of this work. Received: 29 November 2021 Accepted: 7 February 2022 Published: 10 February 2022 Received: 29 November 2021 Accepted: 7 February 2022 Published: 10 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. There is a well-known analogy between quantum particles and plasma: the dispersion relation for the Klein–Gordon equation (c = ¯h = 1) ω2 = m2 + k2 (1) (1) Copyright: © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). is similar to the dispersion relation for waves in a simple plasma model ω2 = ω2 p + k2. (2) (2) However, to expand this analogy, we need a description of both negatively and positively charged particles.The description is based on the little-known possibility of using a real, https://www.mdpi.com/journal/entropy Entropy 2022, 24, 261. https://doi.org/10.3390/e24020261 Entropy 2022, 24, 261 2 of 13 rather than complex, wave function to describe charged particles. Schrödinger [7] noticed that a charged scalar ﬁeld can be made real by a gauge transformation, in spite of “the widespread belief about ‘charged’ ﬁelds requiring complex representation.” As a general- ization of this result, it was shown recently (see in [8–10]; see also in [11], pp. 1. Introduction 24–25, [12]) that, in a general case, one can use just one real function instead of the four complex components of the Dirac spinor in the Dirac equation in an arbitrary electromagnetic ﬁeld at the expense of getting a fourth-order partial differential equation. A similar result can be derived for the Dirac equation in a non-Abelian gauge ﬁeld [13]. Using one real wave function instead of complex functions introduces some “sym- metry” between positive and negative frequencies and, thus, particles and antiparticles. Therefore, a tentative description of such (one-particle electron) wave function was given in [14,15]: the wave function can describe N + 1 electrons and N positrons, where N is very large. This collection of particles and antiparticles has the same total charge (and mass) as an electron, and the value of the wave function at some point (or some combination of the wave function and its derivatives at the point) is a measure of both “vacuum polarization” and the density of probability of ﬁnding an electron at this point (ﬁnding a positron at that point is also possible, but probably requires much more energy). An electron found during a measurement can be any of the N + 1 electrons. The results of the measurement on the speciﬁc collection can depend, say, on the precise coordinates of the particles in the collec- tion. One can consider such a collection as a “dressed” electron with a well-deﬁned total charge. The description assumes trajectories of the “bare” electrons and positrons, but the uncertainty principle is valid for the “dressed” electron. The charge density distribution of the “dressed” electron is deﬁned by all charges of the “bare” electrons and positrons and can be very close to the charge density distribution built from the traditional wave function (see Section 2.1). If an electron is removed from the collection (for example, a pre- cise position measurement means high momentum uncertainty, as a result, some particle acquires high speed, quickly leaves the collection, and the area around the place vacated by the particle will tend to be ﬁlled with the surrounding particles) and the energy of the remaining collection is not high enough for the collection to manifest as pairs, it is difﬁcult to tell the collection from vacuum. This may be the source of discreteness emphasized in [16]. 1. Introduction The col- lections are difﬁcult to visually tell from each other, but the total charges of the collections are one and zero electron charge, respectively. Figure 1 is similar to M. Strassler’s Figure 3 at [22], but the ﬁgure here describes an electron, rather than a nucleon (which is a composite particle), and the size of the collection is deﬁned by the size of the volume where the wave function does not vanish. The description has obvious similarities with the Bohmian interpretation. Let us discuss them for a single-particle system (as it is understood both traditionally and in the Bohmian interpretation [23], because there are always numerous particles in the plasma-like description). If the system is described by a wave function, the latter deﬁnes current lines, which can be regarded as possible particle trajectories in both the Bohmian interpreta- tion and the plasma-like description. There is only one particle and one trajectory in the Bohmian interpretation (other trajectories are realized in other instances of the statistical ensemble [23]), and there are numerous particles and trajectories in the plasma-like de- scription. However, there is more similarity in the case of a composite particle, where the trajectory of the composite particle itself seems singled-out/different from the trajectories of the surrounding elementary fermions/antifermions in the plasma-like description (see Section 3). ) While single-particle systems are very important (for example, they are sufﬁcient to describe the double-slit experiment), it is necessary to discuss many-particle systems (cf. V. Vedral’s comments [24] on the approach in [25]). The author does not have a complete description of second quantization for such systems, but for fermions the Pauli principle can emerge in the following way: for identical wave functions, the relevant collections of discrete charges have identical or very similar coordinates of the charges and thus a combination of such collections can have a very high energy, for example, due to Coulomb interaction. Note that matter and radiation (and, eventually, Fermi and Bose ﬁelds) are treated differently in the plasma-like description. 1. Introduction It is also important to note that spreading of wave packets (which complicates, e.g., the de Broglie’s double solution approach [17]) is not problematic for this description. This description suggests an analogy between plasma and elementary particles. Such analogy was discussed long ago. For example, Vigier [18] considered a vacuum containing fermionic and antifermionic ﬁelds, and compared it to a plasma (see also references in Section 3). It is difﬁcult to say if the vast body of work on quark–gluon plasma is directly relevant to interpretation of quantum theory as quark-gluon plasma is a high-temperature or high- density state of matter [19]. The description of this article only covers the unitary evolution of quantum theory, but not the wave function collapse. On the one hand, the author would like to essentially limit the discussion to matters of mathematical physics, on the other hand, there are now some indications that unitary evolution may be sufﬁcient to describe experiments. For ex- ample, Schlosshauer reviewed experimental data and concluded [20]: “(i) the universal validity of unitary dynamics and the superposition principle has been conﬁrmed far into the mesoscopic and macroscopic realm in all experiments conducted thus far; (ii) all observed “restrictions” can be correctly and completely accounted for by taking into account envi- ronmental decoherence effects; (iii) no positive experimental evidence exists for physical state-vector collapse”. Furthermore, Allahverdyan e.a. theoretically studied dynamics of a spin interacting with a quantum model of a measuring apparatus and concluded [21] that “uniqueness of the outcome of each run and reduction can be derived from the Hamiltonian dynamics of the macroscopic pointer alone”, and “recurrences might still occur, but at inaccessibly large times”. Entropy 2022, 24, 261 3 of 13 One can object that the mass of such a collection of a large number of particles and antiparticles would be too large, as each pair should have a mass of at least two electron masses, but it is not necessarily so, as the energy of an electron and a positron that are very close to each other can be signiﬁcantly less than their energy when the distance between them is large. The above description is illustrated by Figure 1, where electrons and positrons are represented by minus and plus signs, respectively. Collections (a) and (b) there are identical except for an extra electron (represented by a blue minus sign) in collection (a). 1. Introduction Second quantization for bosons can be performed using the same approach as in [15], Section 4 (the approach using a generalized Carleman linearization [26,27] was proposed by nightlight): for a rather general system of nonlinear differential equations of evolution in a 3-dimensional space, the set of solutions can be embedded into the set of solutions of a system of linear differential equations of evolution in the Fock space with a Hamiltonian built using boson operators. In this article, we offer a tentative resolution of some important issues arising in this description. In particular, we try to answer the following questions: • How can a continuous charge density distribution be approximated by a discrete one with a quantized charge? • How can a continuous charge density distribution be approximated by a discrete one with a quantized charge? q g • What mathematical model (equations of motion) can underlie such a description? • How can the description be extended to composite particles? • What are some implications of the obvious analogy with plasma? Entropy 2022, 24, 261 4 of 13 (a) (b) Figure 1. Collections (a,b) have 201 and 200 electrons, respectively, and 200 positrons each. (a) (b) (a) (b) Figure 1. Collections (a,b) have 201 and 200 electrons, respectively, and 200 positrons each. 2. Methods and Results 2.1. Approximation of a Continuous Charge Density Distribution by a Discrete One with a Quantized Charge 2.1. Approximation of a Continuous Charge Density Distribution by a Discrete One with a Quantized Charge How accurately can a continuous charge density distribution for a speciﬁc wave function with a total charge equal to one electron charge be approximated by a collection of discrete charges with values of ±1 electron charge? It is obvious that a Fourier expansion of a point-like charge density distribution contains arbitrarily high spatial frequencies, whereas high-spatial-frequency Fourier components of smooth charge density distributions tend to zero fast; therefore, it is probably impossible to approximate a continuous charge density distribution by a ﬁnite number of discrete quantized charges with a good accuracy. However, quantum ﬁeld theories are typically considered to be just effective theories: “. . . we are now used to the idea that there are important interactions at many different energy scales, some of them probably so large that we cannot see them, directly. Certainly not now. Perhaps not ever. Nevertheless, we can use an effective ﬁeld theory to describe physics at a given energy scale, E, to a given accuracy, ϵ, in terms of a quantum ﬁeld theory with a ﬁnite set of parameters. We can formulate the effective ﬁeld theory without any reference to what goes on at arbitrarily small distances [28].” Therefore, we can look for collections of discrete charges with values of ±1 electron charge that have the same Fourier components with spatial frequencies below some limit value as the initial smooth charge density distribution. Let us illustrate this approach in the one-dimensional case. We assume that the smooth charge density distribution is periodic, e.g., with a period of 2π, so we consider it on a segment [−π, π]. Let us choose the following function for our example: f (x) = 15 2π6 (x2 −π2)2(x + π 8 ) (3) (3) Z π −π f (x)dx = 1 (4) (4) (the total charge in the charge density distribution is +1 (electron charge)). The charge density distribution was chosen not to be non-negative everywhere, as we have in mind applications not just to the Schrödinger equation and the Dirac equation, but also to the Klein–Gordon equation, where the charge density does not have to be of the same sign everywhere. 2. Methods and Results Adding equations u2 n + v2 n = 1, Entropy 2022, 24, 261 6 of 13 we obtain a system of kc + ks + 2j + 1 polynomial equations for 2(2j + 1) unknowns un and vn: we obtain a system of kc + ks + 2j + 1 polynomial equations for 2(2j + 1) unknowns un and vn: 1 π 2j+1 ∑ n=1 (−1)n+1 1 2((un + ivn)k + (un −ivn)k) = ak, 1 ≤k ≤kc, 1 π 2j+1 ∑ n=1 (−1)n+1 1 2i((un + ivn)k −(un −ivn)k) = bk, 1 ≤k ≤ks, u2 n + v2 n = 1, 1 ≤n ≤2j + 1. (9) (9) It seems likely (but the author has not proven) that, given some values of kc and ks, this system will always have some real solutions for sufﬁciently large j, or, in other words, it is always possible to ﬁnd a discrete charge density distribution with a sufﬁciently large number of point-like particles that has the same kc + ks Fourier components as the initial smooth charge density distribution. However, the polynomial system is very complicated for large values of kc, ks, and j, and it is difﬁcult to ﬁnd such discrete distribution. It is possible though to do that numerically, using the powerful homotopy continuation method for polynomial systems (see [29] and references there). The idea of homotopic continuation is as follows [30]. If there is a square system of polynomial equations F = ( f1(x), . . . , fm(x)), where x = (x1, . . . , xm), f1(x), . . . , fm(x) are the left-hand sides of the equations, and the right-hand sides equal zero, one uses the homotopy Ht = (1 −t)G + tF, t ∈[0, 1], (10) (10) connecting a start system G = H0 with the target system F = H1. If we differentiate Ht(x(t)) = 0 with respect to t, we obtain ∂Ht ∂x x′(t) + ∂Ht ∂t x=x(t) = 0. (11) (11) This yields a system of ordinary differential equations for x(t), which can be solved numer- ically, if one knows a solution of the start system. 2. Methods and Results Entropy 2022, 24, 261 5 of 13 L id h F i i Let us consider the Fourier expansion: Let us consider the Fourier expansion: p f (x) = 1 2a0 + ∞ ∑ k=1 (ak cos(kx) + bk sin(kx)), (5) where ak = 1 π Z π −π f (y) cos(ky)dy, bk = 1 π Z π −π f (y) sin(ky)dy. (6) Figure 2. The smooth charge density distribution to be approximated by a collection of discrete quantized charges. f (x) = 1 2a0 + ∞ ∑ k=1 (ak cos(kx) + bk sin(kx)), (5) (5) ak = 1 π Z π −π f (y) cos(ky)dy, bk = 1 π Z π −π f (y) sin(ky)dy. (6) (6) Figure 2. The smooth charge density distribution to be approximated by a collection of discrete quantized charges. We will try to ﬁnd a discrete charge density distribution approximating f (x). Let us assume that this distribution describes 2j + 1 particles with coordinates xn, including j + 1 electrons and j positrons, so the discrete charge density distribution is g(x) = 2j+1 ∑ n=1 (−1)n+1δ(x −xn). (7) (7) Let us demand that the k-th cosine Fourier components of distribution g(x) coincide with ak for 1 ≤k ≤kc (the zeroth cosine Fourier component of g(x) automatically coincides with a0 due to Equation (4)) and that the k-th sine Fourier components of distribution g(x) coincide with bk for 1 ≤k ≤ks, where kc and ks are some natural numbers. Let us introduce the following notation: un = cos(xn), vn = sin(xn). As cos(kxn) = 1 2((cos(xn) + i sin(xn))k + (cos(xn) −i sin(xn))k) = 1 2((un + ivn)k + (un −ivn)k), sin(kxn) = 1 2i((cos(xn) + i sin(xn))k −(cos(xn) −i sin(xn))k) = 1 2i((un + ivn)k −(un −ivn)k), (8) cos(kxn) and sin(kxn) can be expressed as polynomials of un and vn, and the equality of kc + ks Fourier components of g(x) to Fourier components ak and bk of f (x) can be expressed as kc + ks polynomial equations for un and vn. 2. Methods and Results While the typical goal of the homotopy continuation method is ﬁnding all the solutions of the target polynomial system, our goal is quite limited: we would like to ﬁnd just one solution of the target system to get an idea of how a discrete charge density distribution with a quantized charge can approximate a smooth distribution. For this reason, we build the start system G and its solution as follows. We generate 2j + 1 random values ˜xn, such that −π ≤˜xn ≤π, and obtain a sequence ¯xn by sorting ˜xn in ascending order. Then, we construct sequences ¯un = cos( ¯xn) and ¯vn = sin( ¯xn) and obtain the start system by replacing ak and bk in the right-hand sides of the ﬁrst kc + ks equations of system (9) by the values of their left-hand sides after substitution un = ¯un, vn = ¯vn. Obviously, ¯un, ¯vn is a solution of the start system, so we can build a solution of system (9) using homotopy continuation. y y ( ) g py We obtained such a solution for kc = 25, ks = 24, j = 24 (the target and start systems are square for these values, with 98 unknowns and 98 equations, each) using the solver from [29] (the latest version and documentation can be found at [31]). The results are presented in Figure 3. The calculated coordinates of the discrete charges are given in the Appendix A. Note that the partial Fourier sums coincide for the smooth and discrete charge density distributions. Let us also note that the difference between the smooth distribution and its partial Fourier sum is less than 0.1% of the maximum value of the distribution. Empirically, the charges are mostly arranged in pairs; clusters of three charges are denoted by the ellipses (this choice of the clusters is rather arbitrary, but may be useful as an illustration). It is interesting that the solution of the start system was generated by choosing random coordinates between −π and π and did not display many pairs/clusters. The pairs/clusters in the solution of the target system probably appeared because the charge density distribution to be approximated was smooth. 7 of 13 7 of 13 Entropy 2022, 24, 261 The approach of this section can be useful for interpretation of quantum phase-space distribution functions [32], such as the Wigner distribution function, which are not nec- essarily non-negative. 2. Methods and Results It is also interesting to compare the approach with the initial Schrödinger’s interpretation of the wave function (see, e.g., in [33,34] and references therein). Schrödinger’s interpretation of e\|ψ\|2 as charge density meets some objections. For example, A. Khrennikov noted ([35], p. 23): “Unfortunately, I was not able to ﬁnd in Schrödinger’s papers any explanation of the impossibility to divide this cloud into a few smaller clouds, i.e., no attempt to explain the fundamental discreteness of the electric charge.” The plasma-like description suggests that e\|ψ\|2 (and its analogs for the Klein– Gordon and Dirac equations) is just a smoothed charge density, and the description is immune to the above objection. Figure 3. The smooth charge density distribution (green) and the discrete charges of the approximat- ing discrete charge density distribution. The charges are mostly arranged in pairs; the clusters of three charges are encircled by ellipses. Figure 3. The smooth charge density distribution (green) and the discrete charges of the approximat- ing discrete charge density distribution. The charges are mostly arranged in pairs; the clusters of three charges are encircled by ellipses. 2.2. An Example Mathematical Model (23) (23) Let us now deﬁne a smoothed initial charge density at x0 = x0 in: Let us now deﬁne a smoothed initial charge density at x0 = x0 in: g0 sm(x0 in, x) = (2π)−3 2 Z \|k\|<λ G0(x0 in, k) exp(ikx)dk, (24) (24) where λ > 0 is a large cutoff constant. Let us construct the smoothed initial current density at x0 = x0 in as follows (the smoothing process is chosen rather arbitrarily, but based on the requirement that the directions of the 4-velocities of the particles and the 4-potentials of the electromagnetic ﬁeld in the same point coincide; up to a factor, the smoothed charge density is a sum of smoothed delta-functions describing particles): gi sm(x0 in, x) = Bi(x0 in, x) B0(x0 in, x) g0 sm(x0 in, x). (25) (25) Now, let the initial 4-current density be equal to the smoothed 4-current density: jµ(x0 in, x) = gµ sm(x0 in, x). (26) (26) Let us assume that Bµ(x0, x) and the temporal derivatives ˙Bµ(x0, x) are deﬁned everywhere in the 3D space for x0 = x0 in in such a way that □B0(x0 in, x) −Bν ,ν0(x0 in, x) = B,i 0,i(x0 in, x) −˙Bi ,i(x0 in, x) = j0(x0 in, x). (27) (27) As was shown in [15], Section 2, if Bµ and ˙Bµ are deﬁned in the entire 3D space at some point in time x0, ¨Bµ(x0, x) can be calculated from Equations (18)–(20), so the Cauchy problem can As was shown in [15], Section 2, if Bµ and ˙Bµ are deﬁned in the entire 3D space at some point in time x0, ¨Bµ(x0, x) can be calculated from Equations (18)–(20), so the Cauchy problem can be posed and Bµ can be calculated in the entire spacetime (we use the following notation: ˙Y = Y,0 and ¨Y = Y,00 are the ﬁrst and second temporal derivatives of Y, correspondingly). The trajectories of the particles/antiparticles can be calculated using the condition ˙xn(x0) = B(x0,xn) B0(x0,xn). B (x ,xn) On the one hand, the above equations of motions coincide with those of scalar electro- dynamics for some choice of initial conditions, on the other hand, one can expect that for a large cut-off constant the current will be close to that of a collection of point particles. 2.2. An Example Mathematical Model Let us deﬁne an example mathematical model (equations of motion) of collections of charged particles and antiparticles interacting with electromagnetic ﬁeld. As we want the experimental predictions of such a model to be as close as possible to those of quantum theory, we will use scalar electrodynamics (the Klein–Gordon–Maxwell electrodynamics) as our starting point. Its equations of motion are as follows [7]: (∂µ + ieAµ)(∂µ + ieAµ)ψ + m2ψ = 0, (12) □Aµ −Aν ,νµ = jµ, (13) jµ = ie(ψ∗ψ,µ −ψ∗ ,µψ) −2e2Aµψ∗ψ. (14) (12) (13) (14) The metric signature is (+, −, −, −), and □= ∂µ∂µ is the d’Alembertian. The complex charged matter ﬁeld ψ in scalar electrodynamics (Equations (12)–(14)) can be made real by a gauge transformation (at least locally), and the equations of motion in the relevant gauge (unitary gauge) for the transformed four-potential of electromagnetic ﬁeld Bµ and real matter ﬁeld ϕ are as follows [7]: □ϕ −(e2BµBµ −m2)ϕ = 0, (15) □Bµ −Bν ,νµ = jµ, (16) jµ = −2e2Bµϕ2. (17) (15) (16) (17) (16) (16) (17) Using a substitution Φ = ϕ2, one can obtain [15] Using a substitution Φ = ϕ2, one can obtain [15] □Φ −1 2 Φ,µΦ,µ Φ −2(e2BµBµ −m2)Φ = 0, (18) (18) Entropy 2022, 24, 261 8 of 13 □Bµ −Bν ,νµ = jµ, (19) □Bµ −Bν ,νµ = jµ, (19) jµ = −2e2BµΦ. (20) (19) □Bµ B,νµ jµ, (19) jµ = −2e2BµΦ. (20) (20) Let us start building the model emulating scalar electrodynamics. We assume that we have N + 1 particles with charge e and N antiparticles with charge −e. The trajectory of the n-th particle/antiparticle is xn(x0). Thus, the charge density distribution is g0(x0, x) = e 2N+1 ∑ n=1 (−1)n+1δ(x −xn(x0)). (21) (21) Here, xn(x0) = (x1 n(x0), x2 n(x0), x3 n(x0)) is the 3D coordinate of the n-th particle/antiparticle at the time point x0. Greek indices run from 0 to 3 and Latin indices run from 1 to 3, unless they denote the particle number. Particles have odd numbers, and antiparticles have even numbers. Let us present g0(x) as a 3D Fourier integral: g0(x0, x) = (2π)−3 2 Z G0(x0, k) exp(ikx)dk, (22) (22) where G0(x0, k) = (2π)−3 2 Z g0(x0, x) exp(−ikx)dx. 2.2. An Example Mathematical Model g p p The current in this example model is not always time-like, but this problem is inher- ited from scalar electrodynamics and can probably be overcome if we start with spinor Entropy 2022, 24, 261 9 of 13 electrodynamics (Dirac–Maxwell electrodynamics). This issue was discussed for the de Broglie–Bohm interpretation of the Klein–Gordon ﬁeld [36–39]. In the present article, how- ever, the Klein–Gordon equation is regarded just as a reasonably decent approximation for electrons. As the model of this section emulates arbitrarily well (for a sufﬁciently large cut-off constant) a quantum theory—the Klein–Gordon electrodynamics (scalar electrodynamics), one can reasonably expect that the model should successfully describe a wide spectrum of quantum phenomena. Modeling of speciﬁc experiments, such as the double-slit experiment, is left for future work. It is not clear if this model can describe pair creation or annihilation. One probably needs to study continuation of the solutions at the points x where B0(x) = 0. Let us emphasize again that one needs to choose the cutoff constant to fully deﬁne the mathematical model. While this constant can be arbitrarily high, the model has problems at very high temporal/spatial frequencies once this choice is made. However, these problems seem similar to those of standard quantum ﬁeld theories [28]. In the example mathematical model, the electromagnetic ﬁeld guides numerous parti- cles/antiparticles, whereas the particles/antiparticles act as a source of the electromagnetic ﬁeld. In comparison, previously the author showed that the Klein–Gordon–Maxwell elec- trodynamics in the unitary gauge allows natural elimination of the particle wave function and describes independent evolution of the electromagnetic ﬁeld. Therefore, the electro- magnetic ﬁeld can be regarded as the guiding ﬁeld in the Bohmian interpretation [14,15,25]. 2.3. Extension to Composite Particles Such description is closer to the de Broglie–Bohm interpretation and the Couder experiment [45] for composite particles than, e.g., for electrons, as a composite particle Entropy 2022, 24, 261 10 of 13 is different from other particles in the collection, and it seems natural to single out its trajectory from all trajectories of the particles in the collection. is different from other particles in the collection, and it seems natural to single out its trajectory from all trajectories of the particles in the collection. 2.3. Extension to Composite Particles - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + Figure 4. 2.4. The Plasma Analogy There is an obvious analogy between this description and plasma. As the dispersion relation for the Klein–Gordon equation ω2 = m2 + k2 (in a natural system of units) is similar to a dispersion relation of a simple plasma model, such analogy was used previously (see, e.g., in [18,46,47]). This analogy illustrates the effective long-range interaction within a collection. Let us try to use this analogy to get an idea of the density of particles in a collection modeling what is perceived as one particle in traditional quantum experiments. If ne is the electron density in the collection, the plasma frequency ωp in the electron–positron plasma is √ 2 times greater than the traditional plasma frequency [48], i.e., ωp = s 8πnee2 me (28) (28) (we do not consider any renormalization of mass and charge in this preliminary treatment). It is natural to suggest that this plasma frequency is equal (maybe on the order of magnitude) to the angular frequency of Zitterbewegung 2mec2 ¯h [49,50], so we obtain ne = m3 ec4 2π¯h2e2 = mec ¯h 3 c¯h 2πe2 = 1 2πα ¯h mec −3 , (29) (29) where α = e2 ¯hc is the ﬁne structure constant. Thus, ne ≈3.8 · 1032cm−3 or 21.8 per cube with an edge length equal to the reduced Compton wavelength ¯h mec ≈3.86 · 10−11cm. The high electron density suggests that there is low energy per particle of a collection. Let us also note that in this context the Zitterbewegung frequency plays a role of a “natural frequency”, rather than a frequency of some “internal clock” [51]. The plasma analogy suggests that more complex equations of motion can be useful for quantum theory, e.g., some analogs of the Vlasov equation. 2.3. Extension to Composite Particles A composite particle (the blue disk) and a collection of pairs. Figure 4. A composite particle (the blue disk) and a collection of pairs. 2.3. Extension to Composite Particles Let us try to resolve the following problem of the description. Composite particles, such as nucleons or large molecules, also demonstrate quantum properties [40,41]. It is however difﬁcult to imagine that molecule–antimolecule pairs play a signiﬁcant role in diffraction of large molecules (creation of such pairs is possible, but much less proba- ble than creation of electron–positron pairs). However, composite particles take part in some interactions (for example, electromagnetic or strong interactions), so the description can be modiﬁed as follows in that case: composite particles are accompanied by a large collection of fermion–antifermion pairs (for example, electron-positron pairs for electro- magnetic interactions and quark–antiquark pairs for strong interactions; in some situations, it can be difﬁcult to tell such pairs from force carriers, such as photons or gluons). Such fermions/pairs/force carriers are present at all locations where the wave function tradition- ally describing the composite particle does not vanish, so the dimensions of the collection are not limited by the range of the interaction (for example, the short range of strong interaction). Thus, the composite particle can be detected at all locations where the wave function does not vanish, although at most locations it is fermions/pairs/force carriers of the collection that interact directly with the instrument, not the composite particle itself. Such a composite particle with a collection of pairs is illustrated by Figure 4. Such a composite particle with a collection of pairs is illustrated by Figure 4. This does not mean that composite particles require an approach that is fundamentally different from that for elementary particles. Fundamentally, composite particles consist of elementary particles, which can be described as collections of particles and antiparticles, but some part of them forms a bound state, so the “bare” composite particle (the blue disk in Figure 4) retains its individuality. Thus, Figure 4 is just a higher-level (less detailed) picture. Let us note that in the processes of particle diffraction and interference, the mo- mentum transfer is deﬁned by Fourier components of matter distribution in the crystal lattice/diffraction grating/screen [42–44], so the mass of the incident particle plays a “pas- sive” role: for the same momentum transfer, the effective de Broglie wavelength is shorter for a particle of a larger mass. 3. Conclusions We considered a possible description of one-particle wave functions as plasma-like collections of particles and antiparticles, and proposed an approach to approximating smooth charge density distributions by discrete ones with quantized charge based on the requirement that partial Fourier sums are equal for the initial and the approximating Entropy 2022, 24, 261 11 of 13 11 of 13 distributions. An example of an approximating discrete distribution was computed using the homotopy continuation method for polynomial systems. distributions. An example of an approximating discrete distribution was computed using the homotopy continuation method for polynomial systems. py p y y An example mathematical model based on the Klein–Gordon–Maxwell electrodynam- ics (scalar electrodynamics) is proposed. The description was extended to composite particles, and some implications of the plasma analogy were derived. One cannot be sure that this description correctly describes reality, but even if it does not, it provides an interesting model or useful picture of quantum mechanics and an approach to understanding quantum randomness. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The author is grateful to A. V. Gavrilin, A. Yu. Kamenshchik, T. G. Khunjua, A. D. Shatkus, and A. Tarasevitch for their interest in this work and valuable remarks. The author is also grateful to anonymous reviewers for valuable comments. Conﬂicts of Interest: The author declares no conﬂicts of interest. Conﬂicts of Interest: The author declares no conﬂicts of interest. Conﬂicts of Interest: The author declares no conﬂicts of interest. Appendix A pp In the following table, the coordinates of the discrete charges in Figure 3 are given for reference. In the following table, the coordinates of the discrete charges in Figure 3 are given for reference. −3.1126741447007592777837555796944 −2.8775683647214638678316386267493 −2.6434375832598523581831400447478 −1.9336327385447378036758712466442 −1.6936070421273554090248068336325 −2.1719682464297679903185420586677 −1.4519956803651471460404417607211 −1.2088029171600329445514113518049 −0.96395311532928105718506480243987 −0.71762228955760307445232301075533 −0.47125651467198634491476595790667 −0.22858211104030609796912348102868 0.86186952364267439824952086701953 0.00616783249506095738298247885632 0.23137130395092737580909635004611 0.44789490782808160879397312969880 0.65746934747098210872577876522952 1.2615668189213491342215825540197 1.4597980397241581466451663147530 2.0643955075009215796102922944881 1.6587989484672624969753714473643 1.8599046587703446165936780433590 2.2734402898434140647729061709433 2.7088031793185310430667064345281 2.9363544347316877716974068736781 2.9530344365605025711769473845442 −3.0951381769952591512118001250174 −2.8614098696120820985572576736353 −2.1882892356297157247876068745650 −2.6326516523345556425794778026752 −1.9710273543645197236521756279888 −1.7555235230550466106754786539860 −1.5403236844404032536985003739319 −1.1039117628153018022432733933447 −1.3237515410575950384914085327471 −0.87873421856160715534322966231588 −0.64632138885302414208246702251485 0.33294945631962504080286722164668 −0.40611981918357315216289810648891 −0.16042959567225122871033380542859 0.08676186721721802846687332761214 0.57756516301030844804631542526482 0.82076073717919350910333838443007 1.3035691589932125510920278727478 2.0187559332910846063814218989366 1.7817077278249609050042692119912 1.5432590795101890022517254564541 2.2542171585208840682836715659146 2.7204794077160776393522523646147 5. 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https://openalex.org/W4200393735	https://informatio.fic.edu.uy/index.php/informatio/article/download/360/374	Spanish; Castilian	null	Al encuentro de la palabra: consideraciones para las prácticas de promoción de lectura en la escuela como escenario para la memoria y la ética	Informatio	2,021	cc-by	10,325	1Biblioteca Nacional de Colombia, Colombia. Correo electrónico: paolaisabelroa@gmail.com 1Biblioteca Nacional de Colombia, Colombia. Correo electrónico: paolaisabelroa@gmail.com 2Asociación Colombiana de Literatura Infantil y Juvenil (ACLIJ), Colombia. Correo electrónico: silvia.castrillon@gmail.com Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 ISSN: 2301-1378 DOI: 10.35643/Info.26.2.5 DOI: 10.35643/Info.26.2.5 Al encuentro de la palabra: consideraciones para las prácticas de promoción de lectura en la escuela como escenario para la memoria y la ética Meeting with the word: Considerations for reading promotion practices in school as a setting for memory and ethic Encontrando a Palavra: Considerações sobre as Práticas de Promoção da Leitura na Escola como Cenário de Memória e Ética Paola Isabel Roa1 ORCID: 0000-0002-4714-6889 Silvia Castrillón2 ORCID: 0000-0003-0141-614X Paola Isabel Roa1 ORCID: 0000-0002-4714-6889 Silvia Castrillón2 ORCID: 0000-0003-0141-614X Resumo Este artigo propõe uma reflexão sobre as práticas de promoção da leitura e da escrita como importantes mediações pedagógicas e culturais nos processos de recuperação emocional e de elaboração de experiências em crianças e jovens vítimas de situações de violência, como deslocamento, abandono, recrutamento forçado , extrema pobreza, violência doméstica, entre outros. Com o sentido ético e a memória como focos conceituais, abordamos as experiências de leitura de crianças e jovens para dar conta das possibilidades que a palavra abre para o encontro e o reconhecimento de si, dos outros e do mundo. Palavras chave: PROMOÇÃO DA LEITURA E DA ESCRITA; ÉTICA; MEMÓRIA; MEDIAÇÕES PEDAGÓGICAS E CULTURAIS. Fecha de recibido: 31/05/2021 Fecha de aceptado: 07/09/2021 Resumen Este artículo propone una reflexión sobre las prácticas de promoción de lectura y escritura como mediaciones pedagógicas y culturales importantes dentro de los procesos de recuperación emocional y elaboración de experiencias en niños, niñas y jóvenes víctimas de situaciones de violencia, como el desplazamiento, el abandono, el reclutamiento forzado, la extrema pobreza, el maltrato intrafamiliar, entre otras. Con el sentido ético y la memoria a modo de focos conceptuales, nos aproximamos a las experiencias de lectura de niños, niñas y jóvenes con el fin de dar cuenta de las posibilidades que abre la palabra para el encuentro y el reconocimiento de sí mismos, de los otros y del mundo. Palabras clave: PROMOCIÓN DE LECTURA Y ESCRITURA; ÉTICA; MEMORIA; MEDIACIONES PEDAGÓGICAS Y CULTURALES. 87 87 Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 Abstract This article proposes a reflection on the practices of reading and writing as important pedagogical and cultural mediations within the processes of emotional recovery and elaboration of experiences in children and young people who are victims of violence, such as displacement, abandonment, forced recruitment, extreme poverty, domestic abuse, etc. With the ethical sense and memory as conceptual focuses, we approach the reading experiences of children and young people to give an account of the possibilities that the word opens for the encounter and recognition of themselves, others and the world. Keywords: PROMOTION OF READING AND WRITING; ETHIC; MEMORY; PEDAGOGICAL AND CULTURAL MEDIATIONS. 1. Introducción Durante los años 2008 y 2009, Asolectura (Asociación Colombiana de Lectura y Escritura) desarrolló la propuesta «Grupos de lectura con niños, niñas y jóvenes víctimas de la violencia en Colombia», con el apoyo del Fondo de IBBY para Niños en Crisis. Esta propuesta pretendía ―a partir de la creación de grupos de lectura en voz alta, su acompañamiento y observación permanente― desarrollar un componente de formación para maestros sobre la manera en que las prácticas de lectura y escritura y las prácticas para su promoción constituyen herramientas importantes dentro de los procesos de recuperación emocional y elaboración de experiencias en niños, niñas y jóvenes víctimas de situaciones de violencia, como 88 88 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 el desplazamiento, el abandono, el reclutamiento forzado, la extrema pobreza, el maltrato intrafamiliar, entre otras. El sentido ético y la memoria fueron los focos conceptuales desde los que se abordó la reflexión sobre la necesidad del acceso a prácticas de lectura y escritura; a la vez, estas categorías orientaron el trabajo en los diferentes grupos de lectura en voz alta y en los seminarios con maestros realizados a lo largo de un año y medio. El interés por involucrar en forma directa estos aspectos en la construcción de conocimiento sobre la lectura y la escritura y su promoción parte de la necesidad que identificamos de transformar las representaciones que sobre las prácticas de promoción de lectura y escritura tiene la sociedad. Estas prácticas se asocian de manera permanente con fines recreativos o lúdicos, o se limitan al desarrollo de habilidades para la decodificación y reproducción de textos escritos. En el diseño y el desarrollo del proyecto nos preocupó siempre la forma en que era posible consolidar una práctica de promoción de lectura dentro de diferentes poblaciones, no como alternativa para la diversión y el uso del tiempo libre, sino desde la necesidad. Es decir, nuestro propósito radicaba en articular las prácticas de lectura y escritura con aquellas necesidades de los órdenes simbólico, político, educativo y emocional que configuran la identidad de las personas y que se revierten en sus interacciones con otros. 1. Introducción Además, el contexto de violencia colombiano demanda, a nuestra manera de ver, que las organizaciones que se ocupan de los niños y las niñas en proceso de formación ―tanto del sector público como del privado, regulares o no- propongan caminos plurales e igualitarios e incorporen a su trabajo fines asociados a una verdadera formación ética, ciudadana y política. Los grupos de lectura se desarrollaron con poblaciones de niños, niñas y jóvenes de las siguientes instituciones: Fundación San Antonio ―Programa Redes: Escuelas para el Tiempo Libre―; Alta Consejería para la Presidencia de la República; Secretaría de Integración Social ―Plan de Atención Integral para Población Habitante de Calle―; Idipron (Instituto Distrital para la Protección de la Niñez y la Juventud); Casa del Menor Trabajador; Secretaría de Gobierno ―Unidades de Atención para Población Desplazada―; Fundación Funicaf (Fundación Infantil Caminos de Amor y Fe). 89 89 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 2. Preliminar. Marco teórico Nos reconocemos en nuestras historias. Es gracias a las palabras y a la capacidad de narrar lo que nos sucede que podemos hacernos a una idea de quiénes somos, y es en el acto de la escucha atenta de historias diferentes a la propia como se posibilita el descubrimiento de los otros y de lo otro. Narrando y dejando que nos narren ―pero sobre todo deteniéndonos para trascender e interpretar los fragmentos de esas narraciones de las que somos espectadores― nos vamos haciendo a palabras, a imágenes, a recuerdos, a sensaciones con los que podemos construir un lugar desde el cual enunciar, comprender y transformar el mundo; un lugar propio y distinto a cualquier otro, pero compartido con otros. Un lugar en el que resuenan las voces que narran; un lugar habitado por lo que somos, que se ensancha o se agota de acuerdo con las palabras, las imágenes, los recuerdos, las sensaciones, los juicios y la belleza que pongamos, o no, en movimiento. Un lugar desde el cual observar el mundo, para decir de él, para tejer la memoria, para acoger nuestras experiencias y para acoger a los otros; en resumen, ese lugar de lo ético en nuestra existencia. Es acerca de estas cuestiones que queremos hablar en este documento: sobre la manera como la lectura, la escritura y su promoción enriquecen -al estar fundamentadas en la palabra- las condiciones que hacen posible la construcción de ese espacio desde el que los seres humanos construyen su memoria y su sentido ético; y sobre la necesidad de que para estos fines la escuela asuma de forma más decidida la responsabilidad de poner a las niñas, los niños y los jóvenes en contacto con materiales simbólicos que alimenten sus imaginarios, su lenguaje, su capacidad de expresión y su sentido crítico. Se trata, entonces, de proponer una reflexión sobre el acceso a la cultura escrita, la literatura y el goce estético como mecanismos sólidos y plurales que alimentan la forma en que los niños y las niñas hacen frente a la realidad y construyen sus entornos vitales. Con esto, las prácticas de promoción de lectura sobre las que nos proponemos hablar distan mucho de las concepciones que las sitúan en procesos instruccionales o recreativos, sin posicionamientos reflexivos ni críticos frente a lo que se lee y se escribe. La siguiente es una apuesta por: 90 90 Informatio 26(2), 2021, pp. 2. Preliminar. Marco teórico 87-111 Informatio ISSN: 2301-1378 Nadine Gordimer expresa: Así, con esta gigantesca brecha entre la palabra y el ser del hombre, la posibilidad de tránsito entre diferentes realidades y formas de explicar el mundo entre los seres humanos desaparece y, con ella, la idea de una educación para la vida, para la construcción de un sentido vital que permita hablar del aprendizaje desde lógicas diferentes a las de la técnica y las competencias para su uso. Señalamos en muchas ocasiones la relación entre palabra y educación, o entre lenguaje y educación, asumiendo al lenguaje no solo como el sistema de signos que permite la transmisión de informaciones y la comunicación entre las personas, sino también como la base sobre la cual los seres humanos construyen e interpretan la realidad. Es más, nos acogemos aquí a las nociones que conciben el lenguaje como la forma de la realidad misma. «El contenido de la vida está en el lenguaje de la vida» (Mate, 2003, p. 57), de tal manera que la educación ―en cuanto ejercicio para conocer y explorar los lenguajes con los cuales los seres humanos han explicado el mundo y sus fenómenos― no se encuentra solo al servicio del acceso al conocimiento científico y positivo que ha construido la humanidad, sino que también tiene que ver ―en primera instancia desde una perspectiva ética― con el lenguaje como expresión del devenir de la vida, como la manifestación de la experiencia que significa ser receptor de la realidad, de la concreta y convencional y, a la vez, de la realidad de eso que no está disponible siempre para ser dicho y nombrado ni para ser objeto del conocimiento, sino que se presenta como una realidad sujeta a la experiencia de cada ser humano. Toda praxis pedagógica tiene la función de dotar al recién llegado de un aparato simbólico con el que pueda hacer frente a lo «indisponible» de la existencia humana, a todas aquellas cuestiones imposibles de eludir que 91 91 91 Informatio 26(2), 2021, pp. 87-111 ISSN: 2301-137 nunca pueden responderse técnicamente: el dolor, la muerte, el mal, el sentido de la vida… es decir, la (Mèlich, 2002). Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 nunca pueden responderse técnicamente: el dolor, la muerte, el mal, el sentido de la vida… es decir, la (Mèlich, 2002). Nadine Gordimer expresa: En esta lógica del sentido ético es necesario plantear también que esta realidad y este sentido de la vida construidos en el lenguaje no son en ninguna medida procesos que los individuos alimentan en soledad para después vincularlos o sumarlos a los de otros individuos ―teniendo como resultado la construcción de sociedad―, sino que es gracias a la interacción con otros como se erige el sentido primero de ser humano; es en el encuentro y en la receptividad de lo desconocido, de lo otro, que el yo adquiere sentido. Gracias a la mirada y a la escucha que los otros nos otorgan nos constituimos en sujetos humanos, se dotan de sentido nuestra acción y nuestra capacidad de nombrar el mundo para decir de él, para escuchar e interpretar la experiencia de los otros y así establecer redes de sentido social que enmarquen y nutran nuestros intercambios. Si lo «ético» es fundante, es porque nos constituimos a nosotros mismos a partir del otro. Antes de la presencia interpelante del otro somos, sí, pero pura existencia, sueño prolongado. El otro es el que nos saca de nuestro autismo y nos convierte en seres vivos. No somos seres vivos antes de que el otro interrumpa el sueño de nuestra existencia vacía (Mate, 2003, p. 53). En lo referente a la memoria, la presente propuesta y la práctica de la que partió buscan constituirse en una contribución para fundamentar el trabajo sobre la reconstrucción de memoria desde el escenario pedagógico. Para ello, la reflexión y el trabajo estuvieron ubicados también en los terrenos del lenguaje, de la imaginación y de lo simbólico, con el aporte definitivo de la literatura, en la medida en que no se asumió la memoria como un mecanismo para la acumulación de información o como medio para acceder al pasado a través del recuerdo, sino como un proceso de intercambio entre los seres humanos de sus narraciones del pasado, de sus interpretaciones y de sus reflexiones sobre el presente y el futuro en permanente retroalimentación. En lo referente a la memoria, la presente propuesta y la práctica de la que partió buscan constituirse en una contribución para fundamentar el trabajo sobre la reconstrucción de memoria desde el escenario pedagógico. Nadine Gordimer expresa: Para ello, la reflexión y el trabajo estuvieron ubicados también en los terrenos del lenguaje, de la imaginación y de lo simbólico, con el aporte definitivo de la literatura, en la medida en que no se asumió la memoria como un mecanismo para la acumulación de información o como medio para acceder al pasado a través del recuerdo, sino como un proceso de intercambio entre los seres humanos de sus narraciones del pasado, de sus interpretaciones y de sus reflexiones sobre el presente y el futuro en permanente retroalimentación. En el diseño de las acciones del proyecto y de los componentes de formación de maestros nos referimos a la memoria, a hacer memoria, como un diálogo siempre abierto al que todos los seres humanos -los vivos, los muertos, los silenciosos, los atemorizados, las víctimas y los victimarios- deben tener acceso para construir el sentido de lo que fue, de lo que es y de lo que será, con un alto grado de responsabilidad y con poder dilucidador frente a los discursos homogéneos y 92 92 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 autoritarios ―tanto privados como públicos―, que fijan versiones de los hechos ocurridos y a su vez establecen derroteros para pensar el presente. Nos interesa pensar la relación entre educación, memoria y ética a través de la movilización del lenguaje simbólico por medio de la lectura y la escritura. Por un lado, porque identificamos ―durante el proceso de exploración y acercamiento a las instituciones encargadas de prestar atención a las poblaciones en situación de vulnerabilidad y en los espacios de conversación y formación con maestros― que son muy pocos los escenarios en los que sentir, argumentar, escuchar y leer tienen cabida, pues, por el contrario, se privilegian las relaciones unidireccionales para la transmisión de información, el aprendizaje técnico y el asistencialismo terapéutico intrascendente. Por otro lado, porque consideramos que es en este replegarse silencioso de la palabra y del mundo simbólico que la escuela, las instituciones culturales y buena parte de la sociedad se convencen de que solo con los conocimientos de tipo instrumental y técnico los niños, las niñas y los jóvenes podrán hacer frente a las demandas de la vida actual y al futuro. Nadine Gordimer expresa: Habría que tener en cuenta que en un país como Colombia el futuro depende, en gran medida, de un orden de cosas que tiene que ver con la vida, con su conservación, con la imperiosa necesidad de que ningún otro niño, niña o joven devenga en victimario, y con el establecimiento de procesos de memoria que hagan posible ―a partir del diálogo y el intercambio de lo que significa para cada uno estar vivo― reconocernos entre quienes compartimos el presente para forjar una responsabilidad con aquellos que han sido víctimas y que se relegan a un pasado que se entiende como lugar para el olvido. 3. La autosuficiencia de la autonomía La necesidad de abrir una discusión alrededor de la lectura y la escritura como trabajos para la memoria y la ética en la escuela surge de una preocupación sobre la forma en la que el concepto de autonomía y subjetividad se está apoderando del discurso escolar contemporáneo vinculando dichas categorías, por un lado, a la demanda de seres competitivos que sepan desempeñarse dentro de un mundo globalizado ―para quienes, dentro de la lógica de las desigualdades sociales, las posibilidades de garantizar una calidad de vida dependen de las destrezas laborales e intelectuales con las que puedan competir con otros en la búsqueda del 93 93 93 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 éxito―, y, por otro lado, a la movilización del culto al yo, a la individualidad y al interés personal como verdad y punto de partida para la comprensión de todo lo que acontece. Se trata de una comprensión que se reduce a entender lo que está delante de sí a partir de significados ya dados e inmóviles que refuerzan la comodidad consigo mismo; o, en el caso de la sociedad de masas, de un culto al yo determinado por una autocontemplación que, a la par que reduce todo a lo conocido y explicado por la colectividad, uniforma a los individuos. Incluso en aquellos casos en los que los jóvenes pretenden distinguirse mediante acciones contestatarias sus alternativas también son determinadas y capitalizadas por la sociedad dominante mediante las industrias culturales, que los conduce otra vez a tendencias gregarias que reducen las posibilidades de miradas críticas y plurales sobre la realidad. La desaparición del individuo consiste en que los seres humanos están dejando de ser únicos e irremplazables, válidos por sí mismos, para convertirse en seres cada vez más parecidos entre ellos y más anónimos, cuyas actividades, comportamiento y pensamiento están moldeados socialmente para servir a los intereses del todo social. Durante las horas de trabajo son trabajadores, y en sus ratos de ocio consumidores de las distracciones que la sociedad les impone. Si alguien busca todavía experiencias más libres, las agencias de turismo le gestionan salidas a la naturaleza; si uno pretende huir a la contracultura o la vida bohemia, el departamento de cultura pone a su disposición cursos introductorios. Todas las esferas de la vida caen bajo la administración social y no hay donde escapar (Tafalla, 2003). En verdad, este famoso «otro» es presentable únicamente si es un buen otro; es decir, ¿qué otra cosa sino el mismo que nosotros mismos? ¡Respeto a las diferencias, claro que sí! Pero a reserva de que el diferente sea demócrata parlamentario, partidario de la economía de mercado, sostén de la libertad de opinión, feminista, ecologista… Lo que también puede decirse así: yo 3. La autosuficiencia de la autonomía La autonomía y la subjetividad que se pretenden en la actualidad (cuyo discurso está siendo reproducido en gran medida por la escuela y los planes de educación del Estado) resultan ser criterios que se alimentan para legitimar la interacción entre las personas solo a través de su individualidad y competitividad y mediante los intercambios productivos para fines útiles inmediatos. Se fomentan tipos de relaciones en las que se desconocen elementos esenciales de la vida en comunidad y de la capacidad de experiencia común para transformarse mutuamente; relaciones en las que la ética se subordina a tolerar al otro, siempre y cuando se mantenga a distancia. Oportuno resulta citar lo que señala Alain Badiou (2004): 94 94 94 Informatio 26(2), 2021, pp. 87-111 ISSN: 2301-1378 respeto las diferencias, en la medida en que quien difiere de mí respete exactamente como yo dichas diferencias. Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 respeto las diferencias, en la medida en que quien difiere de mí respete exactamente como yo dichas diferencias. Se subestiman los escenarios en los que la expresión de la subjetividad no es el éxito y el avance a codazos con respecto a los demás, sino la forma como se pone en diálogo la diferencia constitutiva de cada ser con una pluralidad[1] en la que discurren múltiples miradas del mundo, diferentes mecanismos para comprender lo que ocurre en lo cotidiano. En el caso de esta experiencia, estos escenarios están dados por la lectura en voz alta, la lectura compartida, el diálogo y los diversos encuentros con la palabra y la literatura. 4. La experiencia Buscamos promover en el ámbito escolar y en el de la atención a población vulnerable una reflexión sobre la lectura y la escritura como mediaciones pedagógicas potentes en la educación simbólica y social; prácticas de lectura que al involucrar el mundo simbólico, la imaginación, la narración y la interpelación crítica contribuyan a enriquecer la formación de niños, niñas y jóvenes en lo concerniente a su sentido ético[2]. A partir del trabajo de promoción de lectura con 15 grupos -de los 20 que estaban programados al inicio- de niños, niñas, jóvenes y jóvenes adultos en situación de desplazamiento, extrema pobreza o desmovilización de los grupos armados, se adelantó una observación cualitativa de la que son producto el presente documento, la propuesta de criterios para la selección de repertorios de lectura Pluralidad, literatura y mediación, y un informe detallado del proceso de trabajo con estas poblaciones. Así mismo, fue fundamental el trabajo realizado con cerca de doscientos maestros y maestras del sector público de Bogotá y de otras regiones del país mediante seminarios de formación en los que se pusieron en discusión los criterios abordados en la presente propuesta. Estos seminarios se constituyeron también en escenarios en los que se dio inicio a la divulgación y reflexión de las cuestiones aquí planteadas. También fueron insumos para este trabajo las observaciones y reflexiones originadas en los Clubes de Lectores ―promovidos por Asolectura junto con la Secretaría de Cultura, Recreación y Deporte―, el programa Escritores a la Escuela ―realizado por Asolectura para la Secretaría de Educación en el año 95 95 95 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 2007― y Argos Construye Lectores, programa apoyado por la Fundación Argos que tuvo lugar durante 2009. retorno El hecho de que las prácticas de lectura y escritura en la escuela y los espacios culturales tengan implicaciones en la formación del sentido ético de niños, niñas y jóvenes requiere, por un lado, de la reflexión para la transformación y el enriquecimiento de la concepción predominante que tenemos de la ética ―un supuesto convencional y rígido desde el que se pauta el deber ser de conductas, valores y emociones― y, por otro, de la adopción, como consecuencia de la transformación de dicha concepción, de una postura frente a la ética que la sitúe en lo cotidiano -en el cotidiano encuentro de los humanos con los humanos-, que lo trascienda y asuma esa postura como una noción viva y móvil en la que nuestro actuar se configura en estrecho vínculo con los otros y con el proyecto común en el que nos jugamos una vida digna. Es decir, un horizonte de significación en el que, a diferencia de los decálogos con los que se acostumbra juzgar y justificar los comportamientos, se enmarquen las condiciones para el intercambio de las diferencias, para el encuentro de sí mismo con la particularidad del otro. En las diferentes fases de trabajo con maestros y mediadores de lectura, en especial en los seminarios y las observaciones de prácticas de promoción de lectura, identificamos que la referencia directa que se establece entre la lectura y la ética es la que tiene relación con la moraleja, la prédica y la fijación del sentido de lo que es leído por el adulto. En esta dirección, las obras literarias y los materiales de lectura son evaluados por los maestros desde categorías del orden de los valores y con expectativas que privilegian las moralejas y las enseñanzas explícitas. Se da poca cabida a argumentos complejos y a historias que problematicen la realidad y los órdenes establecidos. Se censuran temas relacionados con la sexualidad, la violencia, la emancipación de las comunidades y su reivindicación del destino propio, y la explicación de los conflictos sociales y políticos del país. Es sobresaliente la tendencia a neutralizar las opiniones que despierta un texto. Se busca en la mayoría de los casos finalizar una sesión de lectura en voz alta u otra actividad de 96 96 Informatio 26(2), 2021, pp. retorno 87-111 Informatio ISSN: 2301-1378 lectura haciendo conclusiones generales y uniformes que delimitan la interpretación y que no invitan a la conversación para la construcción conjunta de sentido. Nos propusimos, a partir de la identificación y la socialización de estas tendencias, promover en comunidades de maestros una discusión sobre diferentes obras de literatura y prácticas de promoción de lectura en las que los significados de los textos y el contenido de las conversaciones propiciaran experiencias para el reconocimiento y la observación de la complejidad humana en diálogo con la realidad de sus comunidades y de nuestro país. Tal fue el caso de los seminarios realizados sobre nueve obras de literatura infantil colombiana ―«Literatura infantil y juvenil: nueve historias colombianas, nueve miradas a una misma realidad»―, en los que se retrataron situaciones y experiencias de niños, niñas y jóvenes en el conflicto armado o la vulnerabilidad social. En los seminarios se buscó explorar junto con los maestros la manera en que esta literatura podía contribuir a la construcción de sentido sobre asuntos que afectan la vida cotidiana de gran parte de niños, niñas y jóvenes colombianos. Partiendo de la lectura en voz alta de fragmentos de las obras y de textos teóricos para luego ampliar el espectro de discusión, reflexionamos y conversamos con los maestros y mediadores sobre el sentido que tenían dichas problemáticas en sus vidas y compartimos relatos sobre las experiencias de diferentes estudiantes que asisten a la escuela, cuyas realidades a menudo son difíciles de exteriorizar, tanto para ellos como para sus familias y sus profesores. La perspectiva ética de esta propuesta de mediación de lectura y escritura se manifiesta en el desarrollo de las conversaciones entre lectores y textos. En función de una reflexión que trasciende lo individual y se propone enunciar, comprender y hacer memoria, se consigue poner en el centro del encuentro la experiencia de personas cuyas vivencias del mundo y de los conflictos humanos tienen que ser escuchadas y acogidas desde una mirada ética ―empática― para participar de la construcción de sentido del mundo y la realidad. Así, la lectura y la escritura se asumen como mediaciones éticas para la afirmación de la vida y el intercambio de experiencias y pensamiento. 97 97 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 6. La ética y la atención a poblaciones vulnerables En Colombia, por ejemplo, al hablar de ética habría que cuidarse de no dogmatizar los discursos, de no hacer de cuestiones como el desplazamiento, la violencia y el reclutamiento etiquetas vacías que solo designan una realidad corporal[3]. Hasta ahora se hace evidente que la atención a las personas en situaciones de crisis se limita a buscar sin orden un bienestar en los planos material y corporal, lo que por regla general deriva en asistencialismos y altruismos insustanciales que no permiten trascender la realidad vital de las víctimas, marcada por la barbarie. No nos convoca a la responsabilidad de escuchar la voz que habita el cuerpo de la víctima. Solo la escuchamos a través de los índices convencionales a los que la reducimos en su victimización (la pobreza, el hambre, la suciedad, el desamparo), no como sujeto dotado de pensamiento y actuar propios. En el proceso adelantado para la incorporación de grupos de lectura y discusión en los programas de atención a poblaciones en situación de desplazamiento y desmovilización ―durante el desarrollo de los grupos de lectura con niños y niñas en las unidades para atención integral a poblaciones desplazadas―, con frecuencia pudimos observar cómo, dentro del protocolo oficial para la atención a las víctimas del desplazamiento, la palabra de estas personas solo tiene valor en la medida en que dé cuenta de actos violentos, es decir, en la medida en que sus relatos sirvan como testimonios dentro del contexto de la indagatoria, la burocracia judicial o el efectismo mediático. Con el reconocimiento del problema y el interés en transformar esta tendencia, avanzaríamos, como plantea Alain Badiou (2004), de la piedad a la justicia. Una política justa es la consecuencia de dos afirmaciones: todo cuerpo soporta un pensamiento; todo el mundo es igual a todo el mundo. Con el reconocimiento del problema y el interés en transformar esta tendencia, avanzaríamos, como plantea Alain Badiou (2004), de la piedad a la justicia. Una política justa es la consecuencia de dos afirmaciones: todo cuerpo soporta un pensamiento; todo el mundo es igual a todo el mundo. Badiou expresa: ¿Podemos fundar una idea de la justicia a partir del cuerpo como espectáculo? 6. La ética y la atención a poblaciones vulnerables Yo creo que hay que responder negativamente, ciertamente la piedad es un sentimiento importante, pero podemos ir directamente de la piedad a la justicia, porque para ir a la justicia, se hace necesario algo más que el cuerpo sufriente, se hace necesario una definición de la humanidad, más amplia que la víctima, es necesario que la víctima sea testimonio de algo más que la víctima (2004). ¿Podemos fundar una idea de la justicia a partir del cuerpo como espectáculo? Yo creo que hay que responder negativamente, ciertamente la piedad es un sentimiento importante, pero podemos ir directamente de la piedad a la justicia, porque para ir a la justicia, se hace necesario algo más que el cuerpo sufriente, se hace necesario una definición de la humanidad, más amplia que la víctima, es necesario que la víctima sea testimonio de algo más que la víctima (2004). 98 98 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 Así mismo, Badiou agrega que la víctima es cuerpo creador, cuerpo que porta ideas y está dotado de pensamiento. Así mismo, Badiou agrega que la víctima es cuerpo creador, cuerpo que porta ideas y está dotado de pensamiento. Resumiendo, de lo que se trata es de pensar a las víctimas, entre ellas a las personas en situación de vulnerabilidad, más allá de los hechos tangibles que las determinan como tales (la muerte, la violencia, el hambre, el abandono), para que las atenciones a su situación no sean solo de carácter material e inmediato. Se pretende, mediante prácticas pedagógicas y culturales, orientar acciones para conseguir, por un lado, la movilización social de una reflexión profunda que tenga como resultado reconocer a las víctimas y los condicionamientos de su situación y, por otro lado, condiciones pedagógicas, culturales y políticas para que las víctimas puedan expresar su palabra y las ideas que porta su cuerpo vulnerado en su condición de ser humano que piensa, siente, habla, sueña, juzga. Conseguir el propósito de este objetivo se dificulta en la medida en que, por lo general, las víctimas, en sus contextos naturales, también han sido excluidas del uso de la palabra e imposibilitadas para la transmisión de experiencias. En el caso de los grupos con población desmovilizada, no bastaba con la apertura de los espacios para la lectura en voz alta. 7. Leer y escribir: representar el mundo Las prácticas de lectura y escritura, en especial aquellas que involucran a la literatura, permiten poner en el terreno de los símbolos los actos más trascendentes de la vida humana y, por lo tanto, enriquecen y dotan de sublimación la transmisión de experiencia y lenguaje mencionados anteriormente. Es la capacidad de simbolizar lo que permite a los hombres compartir la condición humana, de acuerdo con Lluís Duch, «en el fondo, la aptitud simbólica, que es lo que real y radicalmente comparten todos los humanos, es la base más firme e incontestable para la afirmación de la única humanidad del hombre; cursivas añadidas» (2004, p. 28), en la medida en que los símbolos les permiten trascender la realidad a partir de mitos y rituales compartidos, universales y plurales (Roa, P. y Castrillón, S. 2012, pp.180-181). Al leer, al hacer un trabajo de simbolizar, los intérpretes asocian al signo representaciones e ideas a partir de las cuales es posible nombrar la realidad. Una obra literaria, de esta manera, es un conjunto de símbolos que al momento de ser escritos contienen la carga significativa que el autor impregnó en ellos y que al entrar en contacto con el lector adquieren sentido gracias a que este, a su vez, pone en juego su carga significativa. El lector, gracias al poder evocador de lo simbólico, les presta a las representaciones ideadas por el escritor el sentir que necesitan para adquirir vida, para existir, valorizando así el uso de la palabra y del lenguaje estético como formas de acceso a la humanidad de los otros. Al leer, al hacer un trabajo de simbolizar, los intérpretes asocian al signo representaciones e ideas a partir de las cuales es posible nombrar la realidad. Una obra literaria, de esta manera, es un conjunto de símbolos que al momento de ser escritos contienen la carga significativa que el autor impregnó en ellos y que al entrar en contacto con el lector adquieren sentido gracias a que este, a su vez, pone en juego su carga significativa. El lector, gracias al poder evocador de lo simbólico, les presta a las representaciones ideadas por el escritor el sentir que necesitan para adquirir vida, para existir, valorizando así el uso de la palabra y del lenguaje estético como formas de acceso a la humanidad de los otros. 6. La ética y la atención a poblaciones vulnerables Sus participantes se caracterizaban por expresiones de interés muy parcas, por el casi nulo uso de la palabra en público y, sobre todo, por la extrañeza o el rechazo a incorporar elementos de carácter emotivo o metafórico en su comunicación con otros. Así mismo, el acto de escuchar historias representaba una dificultad expresada por ellos en la incomodidad de detenerse en «cosas que no existen» o en expresiones poéticas muy «románticas» sobre la realidad. En estos casos predominaba la burla o la agresividad frente a los relatos y a las intervenciones del mediador. Para el equipo de trabajo de mediadores fue de mucha ayuda reflexionar y discutir acerca de los posibles motivos que determinan estas actitudes. Uno de ellos fue pensar en el escaso valor que tiene la palabra en los contextos de guerra, en los que, según los participantes de los grupos de lectura, la comunicación con los otros se limita a dar y recibir órdenes, o en los que el diálogo cuenta con tiempos y lugares limitados. Así mismo, en esos contextos la posibilidad de opinar es nula. Los grupos de lectura inauguraban una manera distinta de relacionarse mediante la argumentación, el juicio crítico y la socialización de ideas. 99 99 Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 7. Leer y escribir: representar el mundo Por ejemplo, leer libros y procurar una conversación sobre el amor o el viaje en una comunidad de jóvenes habitantes de la calle revela de manera significativa sentidos y formas de nombrar y representar estos fenómenos diferentes a lo que sucedía en una comunidad de jóvenes adultos en proceso de reinserción a la sociedad, o en un grupo de madres solteras entre los 12 y los 16 años, en cuanto al diálogo, la lectura y la socialización de ideas[4]. Aunque con frecuencia los actos de lo simbólico y lo argumentativo en la sociedad se asumen de forma implícita y sobre ellos no hay una reflexión y visibilización cotidianas, las prácticas de promoción de lectura son escenarios - siempre y cuando el maestro y el mediador hagan de ellas prácticas reflexionadas y trascendentes- en los que sus participantes sienten que son tomados en serio y que sus argumentos y los significados que le dan a lo que escuchan y perciben tienen valor y efecto sobre las percepciones de los demás. 7. Leer y escribir: representar el mundo La lectura en voz alta y la discusión de textos nos permitían llevar al escenario público estas representaciones e interpretaciones. Nos proponíamos un ejercicio de construcción compartida de significados. Confrontando el acto de lectura íntima ―en la que el diálogo se establece entre la obra y el lector― con la dinámica de los grupos de lectores en la que un tercer actor participa de la lectura, nos percatamos de la manera en que el acto de simbolizar e interpretar el sentido de las historias se ve influido y enriquecido por la conversación. Se comunica el significado propio de una lectura y, mediante el argumento y la persuasión, se van incorporando diferentes miradas sobre los textos y las historias que se narran a partir de ellos. Incluso aquellas personas que no participan de forma explícita acceden a un escenario dialógico de carácter público sobre el que pueden asumir posturas críticas frente a los intercambios discursivos que allí se dan. Esta posibilidad de visibilización y exteriorización de interpretaciones de la realidad, a partir del código literario, nos despertó interés por rastrear y contrastar 100 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 la diversidad de imaginarios y representaciones que sobre muchos temas tienen las comunidades y los individuos. Por ejemplo, leer libros y procurar una conversación sobre el amor o el viaje en una comunidad de jóvenes habitantes de la calle revela de manera significativa sentidos y formas de nombrar y representar estos fenómenos diferentes a lo que sucedía en una comunidad de jóvenes adultos en proceso de reinserción a la sociedad, o en un grupo de madres solteras entre los 12 y los 16 años, en cuanto al diálogo, la lectura y la socialización de ideas[4]. Aunque con frecuencia los actos de lo simbólico y lo argumentativo en la sociedad se asumen de forma implícita y sobre ellos no hay una reflexión y visibilización cotidianas, las prácticas de promoción de lectura son escenarios - siempre y cuando el maestro y el mediador hagan de ellas prácticas reflexionadas y trascendentes- en los que sus participantes sienten que son tomados en serio y que sus argumentos y los significados que le dan a lo que escuchan y perciben tienen valor y efecto sobre las percepciones de los demás. la diversidad de imaginarios y representaciones que sobre muchos temas tienen las comunidades y los individuos. 8. No se trata de consignas ni recetas No se trata de mitificar la lectura ni de pensar que el acto de leer en sí mismo constituye una garantía para mejorar las relaciones entre los seres humanos y para una toma de conciencia ética. Se trata de que la lectura y la literatura formen parte de las posibilidades con las que cuenta la escuela para echar a andar una pedagogía ética que dé lugar a la discusión y contribuya a la formación del juicio crítico sobre la reproducción de desigualdades y violencias que se dan en la escuela. Para decir con Bárcena: No quiero sostener que más lectura y, sobre todo, una lectura mejor, tiene como fin natural arreglar los problemas de la humanidad. (…) Lo que deseo decir es que el fin de la lectura es que el lector deje de leer y mire a quien tiene al lado. (…) Creo que la lectura nos ayuda a entender en qué consiste vivir en un «mundo narrado» repleto de múltiples historias y mitos (Bárcena, 2004, p. 144). Nos interesa resaltar que tanto las representaciones sobre la bondad de la lectura como las prácticas para su promoción demandan un permanente cuestionamiento, con el fin de que no caigan en la rutinización o en la consigna intrascendente. Las prácticas pedagógicas, aun las que se encuentren ya transformadas, tocadas o intervenidas por estas y otras reflexiones, no pueden estancarse en la repetición 101 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 acrítica y aislada de procedimientos. Ello solo es posible si estas representaciones y estas prácticas son sometidas al escrutinio periódico de los maestros y de otras personas relacionadas con la escuela y su entorno que se preocupen por estos problemas. 9. Sin la memoria jamás podríamos responder a la pregunta ¿quién soy?[5] Es el pasado común lo que permite a un individuo saberse perteneciente a un grupo. Un pasado que gracias a la palabra y a mediaciones culturales como la literatura y el arte permanece vivo e interactúa con el presente y el futuro dando movimiento y vitalidad a la interpretación de quienes fuimos, quienes somos y quienes queremos ser. Según Elizabeth Jelin, en su libro Los trabajos de la memoria (2002), la relación con el tiempo y el sentido de pertenencia a él es el núcleo de cualquier identidad individual o grupal: «Poder recordar y rememorar algo del propio pasado es lo que sostiene la identidad». Señala que este sentido de pertenencia al tiempo y al pasado hace referencia a la memoria: La memoria es un elemento constitutivo del sentimiento de identidad, tanto individual como colectivo, en la medida en que es un factor extremadamente importante del sentimiento de continuidad y de coherencia de una persona o de un grupo en su reconstrucción de sí mismo (Jelin, 2002). Durante el desarrollo del proyecto se asumió la memoria como un proceso comunitario continuo y ético por medio del cual es posible reconstruir con una distancia interpretativa las historias de los sujetos, poniendo en diálogo el pasado, el presente y el futuro. Nos interesaba abordar la categoría de la memoria teniendo en cuenta la situación por la que atraviesan Colombia y en particular los niños, las niñas y los jóvenes con quienes nos reuníamos a leer y a conversar. Porque se hace evidente la necesidad de que las personas, en especial las víctimas de diferentes tipos de agresiones, se apropien de sus relatos de vida, tomen distancia crítica de ellos incorporándolos en el lenguaje, dotándolos de sentido y vinculándolos a narraciones y formas universales de sentir y explicar. Se trata, así, de que se puedan elaborar los duelos, y de que las niñas, los niños y los jóvenes al narrar y confrontar sus historias sientan un dominio sobre lo que les acontece, 102 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 reivindicando su libertad y oponiéndose a ser víctimas pasivas de la acción de otros. La ética, vista desde este foco de continuidad y constitución del sujeto, encuentra una relación de semejanza con la memoria, semejanza que las vincula en retroalimentación. 9. Sin la memoria jamás podríamos responder a la pregunta ¿quién soy?[5] Cuando los sujetos hacen de su memoria una posibilidad de formación, se disponen, a su vez, en el ejercicio reflexivo e interpretativo de lo suyo a una escucha y una comprensión éticas frente a lo que a los otros les pasa. Si la memoria transita a través del pasado, el presente y el futuro para adquirir un significado completo, la ética necesita también hacer este recorrido a través de una mirada en retrospectiva de las relaciones con los otros; necesita de la reflexión constante sobre el intercambio con quienes se comparte la realidad inmediata -el presente- y de la proyección de las relaciones de acogida y confrontación de las diferencias con las que se convive en el futuro. Esto supone, entonces, un ejercicio constante de apertura e integración de la memoria y el sentido propios con los actos de memoria e interpretación que sobre sí hace el otro. Es una retroalimentación de relatos que, al convertirse en objeto de escucha atenta y al hacernos responsables de la experiencia que narran, permite el entendimiento de aquello que no se puede explicar de manera técnica (las diferencias, el ataque, el sufrimiento) y nos ubica en una posición de responsabilidad respecto de lo que acontece a aquellos con quienes se interactúa. Esto nos convoca a la certeza de que lo que soy afecta la contigüidad de la condición humana y de que mi escucha y mi voz son manifestaciones del acontecer ajeno, incluso de aquellos que ya no están y cuya memoria adquiere vigencia cuando su voz es traída al presente y tenida en cuenta. Otro de los rasgos sobre los que nos interesaba reflexionar, en lo concerniente a la importancia de la literatura y la obra de arte para la construcción de memoria en función de la ética, tenía que ver con el sentimiento de continuidad y de referencia universal en el que se inscriben los actos individuales. Por tal razón, en los seminarios con los maestros incorporamos como temas de trabajo y reflexión los del arte y la literatura en tanto patrimonio cultural que les permite a los seres humanos reconocerse en culturas que los preceden mediante mitos, creencias e interpretaciones sobre los acontecimientos que siempre han afectado y asombrado al hombre. 103 Informatio 26(2), 2021, pp. Mircea Eliade se refiere así a esta cuestión: En el detalle de su comportamiento consciente (…), el hombre (…) no conoce ningún acto que no haya sido planteado y vivido anteriormente por otro (…). Lo que él hace, ya se hizo. Su vida es la repetición ininterrumpida de gestos inaugurados por otros. 9. Sin la memoria jamás podríamos responder a la pregunta ¿quién soy?[5] 87-111 Informatio ISSN: 2301-1378 En la obra de arte la humanidad encuentra un vehículo para transmitir a sus futuras generaciones las formas y los lenguajes con los que dieron explicación y sentido a sus experiencias. En ella se plasman las formas particulares con las que se ha ido representando el mundo según el repertorio simbólico de cada época o lugar. En este sentido, la riqueza de la obra literaria radica en que a la vez que se gesta en los mismos motivos, reiterando la pertenencia a una misma especie, también se plasma siempre en formas diferentes como seña de distinción entre los seres humanos. Mircea Eliade se refiere así a esta cuestión: 10. Las prácticas de lectura y escritura sobre las que hace énfasis este documento son aquellas en las que la conversación y la escucha[6] constituyen los actos fundamentales para la construcción de sentido. Son prácticas de lectura que demandan del maestro el acto pedagógico de la mediación. Esto implica el hecho de que el maestro, dentro de la pluralidad y la contingencia de los signos arrojados por las historias de un libro y las representaciones que sus alumnos hacen, posibilita un escenario para su encuentro e intercambio. En este intercambio se reconocen como participantes de la conversación las narraciones, las opiniones y los argumentos particulares manifestados a través de la palabra, de los gestos o del silencio. Es decir, aquello que se dice y la manera en que se dice es, además, plural y contingente; no corresponde siempre a una secuencia lógica, también se debe tener en cuenta como elemento constitutivo de los diálogos cotidianos. El acto pedagógico de la mediación se configura como acto, como acción ―para ponerlo en términos de la filósofa alemana Hannah Arendt―, en la medida en que se encuentre acompañado de la reflexión y del discurso[7]. La acción de una práctica de promoción de lectura incorpora en su planeación y desarrollo la reflexión constante del mediador acerca de las representaciones que se tienen sobre la lectura, sobre la escritura, sobre los lectores y sobre sí mismo, en interacción con las representaciones e imaginarios que los otros hacen y 104 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 manifiestan sobre los mismos u otros aspectos de carácter universal. Los espacios y las lecturas que se proponen a una comunidad de lectores están compuestos por esta carga significativa y tendrían que propiciar la transformación y creación de otras significaciones. Niños, niñas y jóvenes necesitan espacios en la escuela que den valor a sus narraciones de vida como soporte de sus aprendizajes. Necesitan una escuela en la que su palabra tenga sentido, y en la que, a la par que se les exige su atención y su disposición hacia los saberes impartidos, puedan percatarse de cómo sus maestros abren un espacio para que sus acontecimientos habiten la cotidianidad escolar. 10. Una escuela como la colombiana, en la que la mayoría de sus estudiantes sufren las consecuencias de la guerra y la crisis social, no puede darles la espalda a los miedos, al dolor y a la agresividad que portan los sujetos que todos los días asisten a los centros escolares. Tampoco puede abordar estas cargas identitarias y discursivas de manera metafórica, con iniciativas que en vez de invitar a la exteriorización solapen la consigna de que «a la escuela y al trabajo no se lleva la vida privada». No se trata solo de que la escuela cree espacios artificiales de tranquilidad y felicidad (en donde se ofrezcan desayunos, tecnologías, bellas edificaciones), o de disciplina y autoridad para reprimir las manifestaciones violentas. Se trata, también en el caso de la escuela, de que su cuerpo esté dotado de ideas, las ideas de todos, las historias de todos, por más duras que sean (Roa, P. y Castrillón, S. 2012, pp. 182). De ninguna manera se espera que este sea un llamado a regodearse en el dolor o hacer de la experiencia pedagógica una procesión de testimonios trágicos para lamentarse. No privilegiamos el dolor y el drama por encima de otras manifestaciones de la vida, de las que, por supuesto ―y esto es fundamental―, también la literatura se ocupa. En el acto pedagógico de la mediación, el maestro cuenta con la oportunidad y la responsabilidad de que con la lectura de la literatura se tome distancia frente a la tragedia, se la interpele, se la cuestione y se logre trascender la imagen trágica. Así como sucede con la alegría y la belleza ―sobre las cuales no se podrían establecer estrategias de receptividad edulcoradas en las que se perciba la felicidad como un fin en sí mismo―, lo doloroso por sí solo carece de significado y únicamente convoca a la piedad, a la indiferencia o a la resistencia. El dolor y las diversas emociones a las que se enfrentan los sujetos necesitan un reconocimiento en el ámbito público y también medios para poder ser expresados. Theodor Adorno (1966), al hablar del dolor y de su reconocimiento por parte de la educación, señala: 105 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 La persona dura consigo misma se arroga el derecho de ser dura también con los demás, y se venga en ellos del dolor cuyas emociones no puede manifestar, que debe reprimir. 10. Ha llegado el momento de hacer consciente este mecanismo y de promover una educación que ya no premie como antes el dolor y la capacidad de soportar los dolores. Con otras palabras, la educación debería tomar en serio una idea que de ningún modo es extraña a la filosofía: la angustia no debe reprimirse. Cuando la angustia no es reprimida, cuando el individuo se permite tener realmente tanta angustia como esta realidad merece, entonces desaparecerá probablemente gran parte del efecto destructor de la angustia inconsciente y desviada. 11. Conclusiones y posibilidades Dentro de las anteriores consideraciones, el contexto colombiano sugiere que han sido pocas las posibilidades para el diálogo entre generaciones, grupos étnicos, clases sociales, coterráneos… A la vez, este contexto debería sugerir la urgencia de acciones que se encaminen a un restablecimiento del decir, del contar, del trasmitir y de hacer memoria de los tiempos que corren. Para ello, las mediaciones pedagógicas y culturales que involucran la lectura y la escritura son fundamentales. Una de las satisfacciones que obtuvimos en el desarrollo de esta iniciativa de trabajo fue conocer por medio de los relatos de los maestros las particularidades de la vida en la escuela colombiana. Pudimos acercarnos -gracias a la voz que les dieron sus maestros en los espacios de formación- a las experiencias de muchos niños, niñas y jóvenes. Aunque en estos encuentros y seminarios solo participaron maestros y maestras, las palabras y las expresiones vitales de muchos otros tuvieron cabida a través de ellos. Junto con los maestros tratamos de tomar conciencia sobre la manera en que volvía a cobrar vigencia, vida y sentido la experiencia de los personajes de las historias leídas, la de los estudiantes que evocábamos a partir de ellas, y de otorgarle a esa experiencia un lugar destacado. En el caso de la lectura de los testimonios reales de víctimas de la violencia, procuramos reflexionar sobre la manera en la que, al leerlos, volvían al presente y mediante la palabra y la escucha tenían otra vez un lugar en el mundo. Con relación al encuentro con cerca de quinientos niños, niñas y jóvenes en diferentes situaciones de vulnerabilidad y victimización, en estas conclusiones encontramos importante resaltar el hecho de que fueron determinantes, tanto en lo concerniente a la práctica como a la exploración y reflexión teóricas, cada uno de 106 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 sus aportes en las sesiones de lectura. Cabe recordar que esos aportes nunca fueron medidos de forma exclusiva desde las intervenciones explícitas y orales, o en relación con su aprobación y participación «juiciosa» en los grupos de lectura, sino entendidos desde la generosidad de permitirnos participar de su cotidianidad y de su palabra, la disposición a la escucha y el pacto de asombro que conservan con las historias y la literatura. 11. Conclusiones y posibilidades Cuando nos cuestionábamos de manera crítica sobre el sentido de nuestro trabajo en estas comunidades, descubrimos que, a pesar de las experiencias dolorosas e injustas que les han tocado en suerte, los niños, las niñas y los jóvenes portan consigo la esperanza en la transformación del mundo, la noción sobre la importancia que tienen aquellos otros que los rodean y la necesidad imperiosa de construir y relatar sus historias. Así, nuestro trabajo tuvo como objetivo propiciar espacios para su visibilización significativa a partir de las prácticas de promoción de lectura y comunicar a la comunidad en general -sobre todo a la educativa- los hallazgos y reflexiones producto de esta experiencia. 12. De los maestros y su formación Hablar de la transformación de las prácticas pedagógicas en una país como Colombia requiere situarse, antes que en el terreno de la técnica y de las estrategias metodológicas, en el plano de la reflexión, el estudio y el análisis crítico de esas prácticas. Señalamos la urgente necesidad de que al momento de la toma de decisiones sobre las políticas educativas ―tanto en la administración como en el ámbito de la escuela― se asuma de manera colectiva una postura crítica y vital acerca de la sociedad en la que crecen niñas, niños y jóvenes y sobre el papel que juegan en su formación el arte ―dentro de él la literatura―, pero también la filosofía y las ciencias humanas. Los maestros deben tener a su alcance las posibilidades para oponer resistencia a los hechos que transcurren fuera de la escuela y que la afectan, aunque es verdad que no son los maestros quienes pueden cambiar las realidades particulares e inmediatas que vive cada niña, niño o joven (certeza que, por lo general, se manifiesta en una profunda frustración o en el escepticismo de los maestros). 107 Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 Pensar en una resistencia pedagógica es considerar la capacidad que tienen los maestros de brindar las condiciones para que sus estudiantes ―por medio del conocimiento, la imaginación, el lenguaje y una cotidianidad acogedora― puedan reflexionar, interpretar e interpelar lo que les pasa, comprender y resignificar la manera en la que se les presenta la realidad. Es decir, aplicar una resistencia pedagógica para que niños, niñas y jóvenes puedan resguardarse e inaugurar ―de la mano de los aprendizajes vitales y científicos― una relación con el mundo en la que prime la certeza sobre las posibilidades de transformación y de ser sujetos activos de un presente y de un futuro propios. Enseñar sería así mostrar el mundo y acompañar el descubrimiento y la transformación que de él hacen los recién llegados, usando un término de la filósofa Hannah Arendt (1993). Lo anterior significaría que la formación de maestros debe priorizar la reflexión permanente sobre estas cuestiones; debe ser una formación que contemple su palabra y su experiencia en consonancia con el flujo de acontecimientos externos que irrumpen en la escuela. 12. De los maestros y su formación La creación de espacios en los que los maestros lean, escriban, dialoguen y construyan conocimiento sobre su quehacer es un paso adelante para que la escuela colombiana reivindique su papel como lugar de la posibilidad y la acogida. 13. Referencias bibliográficas 13. Adorno, T. (1966). La educación después de Auschwitz. Conferencia emitida en Radio Hesse. Recuperado de https://www.equintanilla.com/documentos/articulo_adorno.pdf Asololectura. (12 de octubre de 2007). Clubes de lectores: una propuesta de promoción y reflexión. Clubes de lectura. Recuperado de http://clubesdelectura.blogspot.com/2007/10/introduccin.html Badiou, A. (2004). La ética. Barcelona: Herder. Bárcena, F. (2004). El delirio de las palabras. Barcelona: Herder. Bruneteau, B. (2006). El siglo de los genocidios. Madrid: Alianza. Duch, L. (2004). Estaciones del laberinto. Barcelona: Herder. 108 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 Gordimer, N. (2004). Escribir y ser. En Discursos Premios Nobel (Tomo 3). Bogotá: Común Presencia Editores. Jelin, E. (2002). Los trabajos de la memoria. Madrid/ Buenos Aires: Siglo XXI. Mate, R. (2003). Memoria de Auschwitz: actualidad moral y política. Barcelona: Trotta. Mèlich, J. C. (2002). Filosofía de la finitud. Barcelona: Herder. Mircea, E. (2009). El mito del eterno retorno: arquetipo y repetición. Alianza- Emecé. Roa, P., Castrillón, S. (2012). Narrar la violencia. Docta, 10(8), 175-182. Recuperado de http://apcweb.com.ar/wp- content/uploads/2014/10/Docta08-WEB.pdf Tafalla, M. (2003). Theodor W. Adorno: una filosofía de la memoria. Barcelona: Herder. Notas [1] Según Hannah Arendt, «el discurso corresponde al hecho de la distinción y es la realización de la condición humana de la pluralidad, es decir, de vivir como ser distinto y único ente iguales (1993)». [2] Nos permitimos tomar como derrotero fundamental de este documento el imperativo categórico de Theodor W. Adorno, que ―después de la Segunda Guerra Mundial― establece como exigencia de la educación evitar que Auschwitz se repita. Dicho derrotero de reflexión, con el que Asolectura ha planteado sus propuestas de trabajo durante los últimos tres años ―sobre todo en lo concerniente al proyecto IBBY para el trabajo de mediación de lectura en poblaciones de niños, niñas y jóvenes en situación de crisis―, se toma dentro del contexto colombiano guardadas todas las proporciones. Si bien nos permite establecer un espejo de confrontación importante y un marco conceptual definitivo, no tiene ningún interés ―y en esto ponemos especial énfasis― hacer una comparación entre ambas tragedias sobre las cuales nuestro alcance de estudio se ve limitado, además de no ser del interés de este documento. Sin embargo, con respecto al tema, nos acogemos a la tesis de Bernard Bruneteau (2006) expuesta en el libro El siglo de los genocidios, en el que señala que «comparar es 109 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 simplemente intentar dar un sentido histórico a los acontecimientos a menudo independientes entre sí, resaltando sus diferencias y similitudes sin pretender decir que lo uno “equivale” a lo otro». Con respecto al imperativo categórico adorniano, se recomienda explorar su bibliografía, y como texto de referencia importante, su conferencia de 1966 titulada «La educación después de Auschwitz». [3] Según Alain Badiou (2004), subordinar la idea de víctima al cuerpo que sufre, despojando al cuerpo de toda idea, funda la «esclavitud moderna»: La injusticia es un cuerpo sufriente visible, es el espectáculo de las personas sometidas a suplicios, muertos de hambre, heridos, torturados, y es cierto que en la gran muerte del espectáculo hay un sentimiento de la piedad. Notas Pero si la víctima es el espectáculo del sufrimiento, podemos decir que la cuestión de la justicia es solamente la cuestión del cuerpo, la cuestión del cuerpo sufriente (…) cuerpo visible y deviene cada vez más un cuerpo espectáculo, ya que nuestra época transforma cada vez más el sufrimiento en espectáculo, no solamente el espectáculo imaginario, en el cine de la tortura y la violencia, pero también es documento bruto, que nos muestra el cuerpo espectáculo, el cuerpo sufriente, en donde la humanidad es reconducida a la anormalidad. [4] La referencia a esta experiencia y observación concretas se amplía en el documento de investigación del Programa Clubes de Lectores para el período 2009. [4] La referencia a esta experiencia y observación concretas se amplía en el documento de investigación del Programa Clubes de Lectores para el período 2009. [5] Las ideas planteadas en este capítulo están basadas en las propuestas teóricas de los profesores y filósofos de la educación Joan-Carles Mèlich (2002) y Fernando Bárcena (2004). De la misma manera fue fundamental el acercamiento a Elizabeth Jelin (2002), cuyas reflexiones sobre la memoria en el contexto de las dictaduras de los países del Cono Sur permitieron hacernos a una idea más concreta del significado de la memoria en situaciones de violencia como las colombianas. [6] Se recomienda para ampliar el concepto de la escucha como acto pedagógico revisar el trabajo realizado por la profesora argentina Cecilia Bajour. [6] Se recomienda para ampliar el concepto de la escucha como acto pedagógico revisar el trabajo realizado por la profesora argentina Cecilia Bajour. [7] Hacemos referencia a este concepto para sugerir la exploración de las propuestas de Hannah Arendt, en especial en el texto La condición humana. Las obras de los filósofos Fernando Bárcena y Joan-Carles Mèlich son excelentes aproximaciones a la pedagogía de esta autora. Nota del editor El presente manuscrito fue aprobado para su publicación por Cristina Deberti. La corrección de estilo del presente texto fue realizada por Irene Busakre en el marco del convenio celebrado entre la FHCE (Tecnicatura Universitaria en Corrección de Estilo-Facultad de Humanidades y Ciencias de la Educación) y la FIC (Facultad de Información y Comunicación). Notas del autor 110 Informatio 26(2), 2021, pp. 87-111 Informatio 26(2), 2021, pp. 87-111 Informatio ISSN: 2301-1378 26(2), 2021, pp. 87-111 El presente artículo es producto de un proyecto financiado con el Fondo de IBBY para Niños en Crisis, y realizado por Asolectura ―Colombia―, con la colaboración del Banco del Libro ―Venezuela― en 2009. Ha sido revisado y actualizado para su publicación en esta oportunidad, mayo 2021. Nota de contribución autoral Las autoras elaboraron en partes iguales el presente manuscrito. 111 111
https://openalex.org/W4380872794	https://escholarship.org/content/qt6gf3b5pp/qt6gf3b5pp.pdf	English	null	Correction: Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons	The ISME journal	2,023	cc-by	338	Title Correction: Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons Permalink Correction: Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons Permalink https://escholarship.org/uc/item/6gf3b5pp Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ www.nature.com/ismej Published online: 15 June 2023 Powered by the California Digital Library University of California eScholarship.org CORRECTION OPEN Correction: Identiﬁcation of beneﬁcial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons Mingsheng Qi , Jeffrey C. Berry , Kira M. Veley , Lily O’Connor , Omri M. Finkel , Isai Salas-González , Molly Kuhs , Julietta Jupe, Emily Holcomb , Tijana Glavina del Rio, Cody Creech , Peng Liu , Susannah G. Tringe , Jeffery L. Dangl , Daniel P. Schachtman and Rebecca S. Bart © The Author(s) 2023 © The Author(s) 2023 The ISME Journal (2023) 17:1353; https://doi.org/10.1038/s41396-023-01442-9 Correction to: The ISME Journal https://doi.org/10.1038/s41396- 022-01245-4, published online 6 May 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons 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 this article the author name Kira M. Veley was incorrectly written as Kira W. Veley. The original article has been corrected. The original article has been corrected. Published online: 15 June 2023
https://openalex.org/W4379794881	https://www.preprints.org/manuscript/202304.0989/v1/download	English	null	An Emerging Animal Model for Querying the Role of Whole Genome Duplication in Development, Evolution, and Disease	Journal of developmental biology	2,023	cc-by	14,307	Review Not peer-reviewed version doi: 10.20944/preprints202304.0989.v1 Keywords: Whole genome duplication; tetraploidy; polyploidy; aneuploidy; Caenorhabditis; WGD model (List three to ten pertinent keywords specific to the article yet reasonably common within the subject discipline.) Preprints.org is a free multidiscipline platform providing preprint service that is dedicated to making early versions of research outputs permanently available and citable. Preprints posted at Preprints.org appear in Web of Science, Crossref, Google Scholar, Scilit, Europe PMC. Copyright: 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. An Emerging Animal Model for Querying the Role of Whole Genome Duplication in Development, Evolution, and Disease Mara Schvarzstein * , Fatema Alam , Muhammad Toure , Judith L Yanowitz Mara Schvarzstein * , Fatema Alam , Muhammad Toure , Judith L Yanowitz * Correspondence: mschvarzstein@brooklyn.cuny.edu; Tel.: 718-951-5000 x3582 * Correspondence: mschvarzstein@brooklyn.cuny.edu; Tel.: 718-951-5000 x3582 Abstract: Whole genome duplication (WGD) or polyploidization can occur at the cellular, tissue, and organismal levels. At the cellular level, tetraploidization has been proposed as a driver of aneuploidy and genome instability, and correlates strongly with cancer progression, metastasis, and development of drugs resistance [1–6]. WGD is also a key developmental strategy for regulating cell size, metabolism, and cellular function [1,7–10]. In specific tissues, WGD is involved in normal development (e.g. organogenesis), tissue homeostasis, wound healing, and regeneration [10–17]. At the organismal level, WGD propels evolutionary processes such as adaptation [18], speciation [19], and crop domestication [20]. An essential strategy to further our understanding of the mechanisms promoting WGD and its effects is to compare isogenic strains that differ only in their ploidy. Caenorhabditis elegans (C. elegans) is emerging as an animal model for these comparisons, in part because relatively stable and fertile tetraploid strains can be produced rapidly from nearly any diploid strain [7]. Here we review the use of Caenorhabditis polyploids as tools to understand important developmental processes (e.g. sex determination, dosage compensation, and allometric relationships) [21–27] and cellular processes (e.g. cell cycle regulation, chromosome dynamics during meiosis) [28–32]. We also discuss how the unique characteristics of the C. elegans WGD model will enable significant advances in our understanding of mechanisms of polyploidization and its role in development and disease. Keywords: Whole genome duplication; tetraploidy; polyploidy; aneuploidy; Caenorhabditis; WGD model (List three to ten pertinent keywords specific to the article yet reasonably common within the subject discipline.) Preprints.org is a free multidiscipline platform providing preprint service that is dedicated to making early versions of research outputs permanently available and citable. Preprints posted at Preprints.org appear in Web of Science, Crossref, Google Scholar, Scilit, Europe PMC. Copyright: 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. Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 doi:10.20944/preprints202304.0989.v1 Review An Emerging Animal Model for Querying the Role of Whole Genome Duplication in Development, Evolution, and Disease Mara Schvarzstein1,2,, Fatema Alam1, Muhammad Toure1 and Judith L. Yanowitz3,4 1 Brooklyn College at the City University of New York, Biology Department; mschvarzstein@brooklyn.cuny.edu 2 The Graduate Center at the City University of New York, Biology and Biochemistry Departments Affiliation 2 The Graduate Center at the City University of New York, Biology and Biochemistry Departments Affiliation 3 Magee-Womens Research Institute, 4 Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine 4 Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine Correspondence: mschvarzstein@brooklyn.cuny.edu; Tel.: 718-951-5000 x3582 1. Whole genome duplication in development, evolution, and disease Whole genome duplication (WGD) or polyploidization is a key step in vital biological processes including development [1,7–10], genome instability [1,2], adaptation [18], speciation [19], and crop domestication [20] (Figure 1). In nature, WGD exists at the cellular, tissue, organ, and organismal level. Polyploid cells normally exist in most multicellular diploid organisms [33,34]. In humans, certain cells from the placenta [35], mammary glands [36], liver [37], heart [38], skin [39], and bone marrow must become polyploid to perform specialized functions [40], for tissue homeostasis [11], or as part of the wound healing process [17]. For instance, during development, megakaryocytes undergo several rounds of endomitosis until they acquire 16 copies of the genome, and thus become giant cells that are hypermetabolic. This polyploidization is a basic requirement to produce platelets in the blood, as platelets are produced by fragmenting the cytoplasm of megakaryocytes [41–43]. Polyploid cells can arise by the fusion of cells resulting in a larger multinucleate cell, by endoreduplication (alternating cycles between G and S phases without undergoing mitosis or © Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 2 cytokinesis) resulting in a mononucleated cell, or by endomitosis (the cell enters mitosis but does not complete cell division) to generate either mononucleated giant cells or multinucleated cells [44]. cytokinesis) resulting in a mononucleated cell, or by endomitosis (the cell enters mitosis but does not complete cell division) to generate either mononucleated giant cells or multinucleated cells [44]. Figure 1. Roles of Whole Genome Duplication (WGD). Diagram depicting key examples of processes affected by polyploidization/WGD. Polyploidy can occur at the cellular, tissue or organismal level. It is a key step in important biological processes affecting cellular metabolism and function, normal development, tissue homeostasis, regeneration, wound healing, organismal adaptation to the environment, and speciation. In addition, polyploidization is can cause and/or prevent disease, repair injury, and promote tissue regeneration during aging, damage, and wound healing. Figure 1. Roles of Whole Genome Duplication (WGD). Diagram depicting key examples of processes affected by polyploidization/WGD. Polyploidy can occur at the cellular, tissue or organismal level. It is a key step in important biological processes affecting cellular metabolism and function, normal development, tissue homeostasis, regeneration, wound healing, organismal adaptation to the environment, and speciation. In addition, polyploidization is can cause and/or prevent disease, repair injury, and promote tissue regeneration during aging, damage, and wound healing. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 3 and gene expression [68]. Induction of WGD can reduce the levels of proteins involved in chromatin packaging resulting in a reorganization of the 3D DNA compartments and domains. Over time, loss of this domain structure predisposes the cell to additional cancer-promoting alterations and expression of oncogenic genes [68]. Polyploidization can also be protective, as in the liver where it both provides the basis for genetic adaptation to hepatotoxic stress and also protects from malignant transformations [14,42]. Organismal WGD is common in extant plants [19,61,70–72], and is also observed in protists [73], fungi [4,62,74], bacteria and archaebacteria [75,76], and in several animal clades [12,34,47,48,50,51,77,78]. Evidence for ancestral whole genome polyploidization events is found in the genomes of organisms of most clades, including vertebrates [48,79,80]. Polyploid metazoans arise due to errors during meiosis that result in the formation of diploid gametes that when fertilized give rise to polyploid organisms. Fertilization between diploid (2n) and haploid (n) gametes results in a triploid (3n) organism; fertilization between two diploid (2n) gametes results in a tetraploid (4n) [45,47]. When the diploid gametes of two related species fuse, the resulting organism is said to be allopolyploid, whereas fertilization of diploid gametes from the same species gives rise to autopolyploid [80]. Polyploidization from the cell to organismal level most often results in lethality [61,81]. This lethality may be caused by errors partitioning the additional chromosomes during cell division, by WGD driven increases in cell size that interfere with tissues or organ scaling, or by expression changes that affect dose-dependent processes and complexes [80]. A newly formed polyploid may very occasionally overcome these obstacles, e.g. by establishing balanced genome expression. Then, if polyploidy provides a competitive advantage—as in a changing environment—polyploid lineages may outgrow their diploid counterparts and become established [51,61,80]. The established polyploid population will continue to evolve and then may “diploidize” to become a new species that has an adaptative advantage compared to the parental diploid species [61]. 1.2. Laboratory multicellular organism models for WGD Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints20230 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 1.1. Causes and downstream effects for WGD Polyploidization and its effects have been observed in different biological contexts (e.g. in cells, tissues, and organisms) [10,33,45,46] and in multiple processes (e.g. during development, evolution, and disease) [1,3,5,11,47–51]. For instance, environmental stresses induce WGD in tissues (e.g. wounding or viral infection) [15,52,53] and organisms (e.g. large changes in temperature or water availability and salinity) [54–59]. The effects of WGD are diverse in nature—ranging from changes in gene expression and cell size, to epigenetic remodeling, and to altered responses to environmental stress [45,60,61]. Stress-induced WGD is a platform for genomic diversity and versatility that provides the means for rapid adaptability. In whole organisms, this can elicit a competitive advantage in a changing environment [61,62]; in cancer, it can elicit an adaptative advantage that promotes cancer development, progression or resistance to chemotherapeutic drugs [61,62]. In the pathogenic yeast Candida abdicans, WGD promotes formation of drug-resistant aneuploid progeny in response to antifungal drugs [18]. Therefore, queries about mechanisms that give rise to WGD and its effects in both sub-organismal and organismal systems will likely reveal additional commonalities across systems and processes. Polyploidy often is confused with aneuploidy, in part because in humans, both phenomena are hallmarks of cancer [1,3,50,63]. Observations from numerous studies identify WGD in about 30% of human cancers [6,64] and as a precursor for many malignancies in cancer evolution [3,65–69]. Although, WGD does not seem to require a preexisting cancer driver mutation, it most often emerges after oncogenic mutations, such as lesions in the TP53 genes [6,66]. WGD promotes diversification of copy number alterations (CNAs) and is permissive for other chromosomal aberrations and genome instability associated with poor cancer patient prognosis [3,68,69]. Interestingly, a recent study revealed a mechanism by which WGD enhances cancer-promoting alterations in DNA organization 1.2. Laboratory multicellular organism models for WGD Diagram of the published method for generating tetraploid strains from diploids. This protocol relies on the transient knockdown of the meiosis-specific cohesin rec-8 which results in the production of diploid instead of haploid gametes. Thus upon fertilization, tetraploid progeny (which are longer and bigger) are produced. The method works for both during selfing of the hermaphrodites (left) and during outcrossing (right). Crossing allows for the generation of tetraploids with two different genetic backgrounds. After two to three generations of exposure to rec-8 dsRNA, the larger hermaphrodites are individually plated on normal growth media. Strains that consistently sire large (Lon) hermaphrodites are then screened to confirm these the strains are tetraploid. B). Diploid hermaphrodites (left) produce haploid gametes. Haploid sperm are stored in the spermatheca. Diploid oocytes have 6 pairs of connected homologous chromosomes that can be visualized by DAPI in the diakinesis-stage oocytes (C, below) just adjacent to the spermatheca (-1, -2, -3 oocytes based on position). The oocyte moves through the spermatheca and is fertilized and completes the meiotic divisions to produce a haploid pronucleus. The tetraploid worms derived from exposure to rec-8 dsRNA, (right) produce diploid gametes with 12 DAPI bodies. C) Examples of fixed hermaphrodites stained with DAPI to screen for ploidy by counting chromosome pairs in the -1, -2 and -3 diakinesis oocytes. Figure 2. Method for rapid isolation of C. elegans tetraploids. A). Diagram of the published method for generating tetraploid strains from diploids. This protocol relies on the transient knockdown of the meiosis-specific cohesin rec-8 which results in the production of diploid instead of haploid gametes. Thus upon fertilization, tetraploid progeny (which are longer and bigger) are produced. The method works for both during selfing of the hermaphrodites (left) and during outcrossing (right). Crossing allows for the generation of tetraploids with two different genetic backgrounds. After two to three generations of exposure to rec-8 dsRNA, the larger hermaphrodites are individually plated on normal growth media. Strains that consistently sire large (Lon) hermaphrodites are then screened to confirm these the strains are tetraploid. B). Diploid hermaphrodites (left) produce haploid gametes. Haploid sperm are stored in the spermatheca. Diploid oocytes have 6 pairs of connected homologous chromosomes that can be visualized by DAPI in the diakinesis-stage oocytes (C, below) just adjacent to the spermatheca (-1, -2, -3 oocytes based on position). 1.2. Laboratory multicellular organism models for WGD The oocyte moves through the spermatheca and is fertilized and completes the meiotic divisions to produce a haploid pronucleus. The tetraploid worms derived from exposure to rec-8 dsRNA, (right) produce diploid gametes with 12 DAPI bodies. C) Examples of fixed hermaphrodites stained with DAPI to screen for ploidy by counting chromosome pairs in the -1, -2 and -3 diakinesis oocytes. 1.2. Laboratory multicellular organism models for WGD A major impediment to addressing WGD and its impacts on the physiology and function of cells, tissues and organs is the scarcity of laboratory models that permit comparison between isogenic organisms with differing ploidy [45]. Many laboratory models are sterile or embryonic lethal when polyploid [82–84]. Generation of autopolyploid laboratory model organisms that are not sterile (e.g. plants) frequently involves the use of chemicals such as colchicine, known to cause aneuploidy and genetic mutations. These polyploids, therefore, may not be truly isogenic with the parental diploid strains they were derived from [62,80]. The recently developed method for generating tetraploids in Caenorhabditis utilizes transient knock-down of a meiosis-specific cohesin (i.e. rec-8) by RNA interference to produce diploid gametes [7]. Therefore, the derived tetraploids are unlikely to differ significantly from the diploid strains from which they were derived (See Figure 2A to overview the method) [7]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 Figure 2. Method for rapid isolation of C. elegans tetraploids. A). Diagram of the published method for generating tetraploid strains from diploids. This protocol relies on the transient knockdown of the meiosis-specific cohesin rec-8 which results in the production of diploid instead of haploid gametes. Thus upon fertilization, tetraploid progeny (which are longer and bigger) are produced. The method works for both during selfing of the hermaphrodites (left) and during outcrossing (right). Crossing allows for the generation of tetraploids with two different genetic backgrounds. After two to three generations of exposure to rec-8 dsRNA, the larger hermaphrodites are individually plated on normal growth media. Strains that consistently sire large (Lon) hermaphrodites are then screened to confirm these the strains are tetraploid. B). Diploid hermaphrodites (left) produce haploid gametes. Haploid sperm are stored in the spermatheca. Diploid oocytes have 6 pairs of connected homologous chromosomes that can be visualized by DAPI in the diakinesis-stage oocytes (C, below) just adjacent to the spermatheca (-1, -2, -3 oocytes based on position). The oocyte moves through the spermatheca and is fertilized and completes the meiotic divisions to produce a haploid pronucleus. The tetraploid worms derived from exposure to rec-8 dsRNA, (right) produce diploid gametes with 12 DAPI bodies. C) Examples of fixed hermaphrodites stained with DAPI to screen for ploidy by counting chromosome pairs in the -1, -2 and -3 diakinesis oocytes. Figure 2. Method for rapid isolation of C. elegans tetraploids. A). 2.1. Polyploidy and aneuploidy in C. elegans In humans, all monosomies, except of the X chromosome are lethal, whereas trisomies 13, 18, 21, X and Y are viable. In C. elegans some aneuploidies are tolerated. These include X-chromosome trisomies (2A;3X = 13 chromosomes triplo-X hermaphrodites) and monosomies (2A;1X = 11 chromosomes XO males shown in Figure 3) [92]. Compared to diploid hermaphrodites, triplo-X animals have a Dumpy body morphology, lower fertility, and slower growth rate [92]. Trisomy of chromosome 4 (LGIV) has also been reported [93]. Although animals with this autosomal trisomy are viable and have a general morphological appearance that seems normal, they produce 50% fewer progeny than normal hermaphrodites [94]. Interestingly, large chromosomal fragments, often Mb is size, have been generated and preserved as free duplications and have been found for part of each of the chromosomes of C. elegans (reviewed in [22]). In nature, most triploids are inherently unstable, unless they reproduce asexually [95]. For instance, the parasitic nematodes of the Meloidogyne incognita group (MIG) are stable hypotriploids that reproduce by asexual parthenogenesis [96]. C. elegans triploid animals (3A;3X and 3A;2X shown in Figure 3) can be generated by crossing diploid and tetraploid animals [24]. Only 15% of the embryos sired by triploid animals’ hatch [24], and those that do hatch tend to be diploid or become diploid in the next generation [28,97,98]. Triploid progeny inviability is likely because the odd number of homologs results in abnormal meiotic pairing and recombination [28,30,31], yielding aneuploid gametes that produce embryos that are inviable. Victor Nigon reported the first induction of polyploid C. elegans—the first in any animal—in the laboratory in 1949 [97]. These tetraploid strains were generated by treating spermatogenic animals with either heat shock (several hours at 25°C) or exposure to colchicine. Both treatments were followed by screening for ‘larger than normal’ animals and cytological assessment of chromosome numbers in diakinesis-stage oocytes, when the chromosomes are highly condensed prior to the meiotic divisions (e.g. in Figure 2C). Newly formed tetraploids sired a range of phenotypes in offspring that included: progeny with variable body sizes; infertile hermaphrodites and males; intersexuality; and progeny that had reverted to diploidy [97,98]. The frequency of these aberrant phenotypes decreased with continuous passaging, but did not disappear, even after 20 generation of selection for tetraploids with high fecundity. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 5 study of genes in all of the processes that polyploidy has been reported to affect. The transparency of the worms allows visualization and direct comparison of physiology and function in the live animal. Combined, these characteristics make the C. elegans WGD model unique and provide an unprecedented opportunity to answer long-standing questions about organismal polyploidization and its effects in a multicellular animal. 2. Caenorhabditis elegans as an animal laboratory model for understanding WGD Several attributes of C. elegans nematodes make it an exceptional laboratory model for querying WGD: 1) It has a small number of chromosomes (five autosomes and a sex chromosome) allowing for easy visual evaluation of ploidy and differentiation between polyploidy and aneuploidy [85] (Figures 2B,C). 2) Despite C. elegans being a self-fertilizing hermaphroditic species, males arise from meiotic nondisjunction at a 0.2% frequency [86–88], allowing for genetic analysis [89]. 3) Each diploid hermaphrodite produces 200-300 offspring and has a lifecycle of three to four days from egg to egg, both of which facilitate multigenerational ecological and evolutionary studies. 4) C. elegans is normally diploid but like vertebrates (including humans), it has polyploid tissues or cells [90]. These include the intestine (20 cells with a total of 30-34 nuclei and a ploidy of 32C per nucleus) and the hyp7 cell (a syncytium of 139 nuclei with an average ploidy of 10.7C) [90,91]. Critically, the quick and reliable method to generate fertile and viable tetraploid Caenorhabditis strains from nearly any diploid strain [7] allows direct comparison of identical cells in isogenic organisms with differing ploidy (Figure 2). The availability of hundreds of mutant strains that impinge on different aspects of development, aging, stress, and reproduction, will facilitate directed 2.1. Polyploidy and aneuploidy in C. elegans Two major types of tetraploids were identified [24,99]: About 66% of the induced tetraploids sired very low frequencies of males (0.6%), compared to the ~2% incidence of males in diploids; the remainder sired an average of ~42% males. Interestingly, tetraploids of both classes could interconvert and give rise to a proportion of progeny of the other type, albeit at different frequencies [98]. After 30 generations the patterns of inheritance of high and low incidence of male progeny did not change. The tetraploid strains that sired high proportions of males are now known to correspond to a 4A;3X genotype and those that produced a low incidence of males to 4A;4X genotype (see Figure 3) [24]. Even in the most stable lines, tetraploid hermaphrodites had substantially fewer self-progeny (average of ~65 progeny) than diploid (250-300 offspring in wild type, N2), and reduced hatching (decrease of 13%) [24]. Nigon’s tetraploid strains were eventually all accidentally lost after 78 generations. Several other groups have isolated fertile and relatively stable tetraploid C. elegans and C. briggsae strains [7,21,23,24,31]. Like their predecessors, each of these tetraploid strains has been reported to produce reduced numbers of self- progeny and to sporadically diploidize. Since the diploids can rapidly overtake a population due to their higher fertility, C. elegans tetraploid strains need to be maintained by continuous selection for the largest animals—at least for the first tens of generations. Studies of the early events post- tetraploidization (e.g. in generations 1-10) therefore should shed light on how organisms acclimate to tetraploidy and become established. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 6 Figure 3. Sexual fate in C. elegans is determined by the X-chromosome/autosome ratio. C. elegans has six chromosomes, 5 autosomes and one X chromosome. Diploid males and hermaphrodites have 2 sets of autosomes and one or two X chromosomes, respectively. Triploid males and hermaphrodites have three sets of autosomes and one or two X chromosomes making 17 or 18 total chromosomes, respectively. Tetraploid males have 22 chromosomes: four sets of autosomes and two X chromosomes. By contrast, tetraploid hermaphrodites have four sets of autosomes and either three or four X chromosomes. . Figure 3. Sexual fate in C. elegans is determined by the X-chromosome/autosome ratio. C. elegans has six chromosomes, 5 autosomes and one X chromosome. 3. Utilizing polyploid C. elegans as tools to investigate developmental processes In the last ~50 years, comparisons between C. elegans strains of differing ploidy have been utilized as tools to query developmental processes including meiosis, embryonic divisions, in addition to sex determination and dosage compensation. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 7 2.2. Polyploid and aneuploid animals uncover C. elegans modes of sex determination and dosage compensation 2.2. Polyploid and aneuploid animals uncover C. elegans modes of sex determination and dosage compensation One of the most critical developmental decisions that a heterogametic organism must make early in life is whether to develop as male or female and, concomitantly, whether to activate dosage compensation. From studies in C. elegans with different ploidies, it became apparent that sex is assessed by the ratio of sex chromosomes to autosomes (X/A) and that induction of hermaphrodite fate activates dosage compensation, which is achieved by reducing expression from the pair of X chromosomes [87,100]. Male phenotypes arose from animals with 2A;1X, 3A:2X, and 4A;2X, whereas 2A;2X, 3A;3X, 4A;4X, and 4A;3X gave rise to hermaphrodite phenotypes [24] (Table 1 and Figure 3). Table 1. X/A ratio determines sex. Karyotype X/A ratio Sexual Fate 2A;2X 1 female 2A;1X 0.5 male 3A;3X 1 female 3A;2X 0.67* male 4A;4X 1 female 4A;3X 0.75* female 4A;2X 0.5 male X/A limits for female and male development. An X/A ratio ≥ 0.75 yields female fates and a X/A ratio ≤0.67 promotes male fates. X/A limits for female and male development. An X/A ratio ≥ 0.75 yields female fates and a X/A ratio ≤0.67 promotes male fates. *X/A limits for female and male development. An X/A ratio ≥ 0.75 yields female fates and a X/A ratio ≤0.67 promotes male fates. Together, these studies led to the conclusion that an X:A ratio > 0.74 induces female/hermaphrodite identity and < 0.67 promotes male development. Further support for these conclusions came from manipulation of the X/A ratio using X-linked gene duplications in the 2X;3A triploids to show at least 3 loci were involved in determining sex [23]. We now know the molecular details about the genes and processes involved in both assessing the X/A ratio and in executing male and hermaphrodite fates and dosage compensation [101]. Nevertheless, questions remain about how allotetraploids generated by hybridization of different tetraploid Caenorhabditis species would assess the X/A ratio, and whether manipulation or modification of this system could be used to stabilize polyploid organisms. 2.1. Polyploidy and aneuploidy in C. elegans Diploid males and hermaphrodites have 2 sets of autosomes and one or two X chromosomes, respectively. Triploid males and hermaphrodites have three sets of autosomes and one or two X chromosomes making 17 or 18 total chromosomes, respectively. Tetraploid males have 22 chromosomes: four sets of autosomes and two X chromosomes. By contrast, tetraploid hermaphrodites have four sets of autosomes and either three or four X chromosomes. . Figure 3. Sexual fate in C. elegans is determined by the X-chromosome/autosome ratio. C. elegans has six chromosomes, 5 autosomes and one X chromosome. Diploid males and hermaphrodites have 2 sets of autosomes and one or two X chromosomes, respectively. Triploid males and hermaphrodites have three sets of autosomes and one or two X chromosomes making 17 or 18 total chromosomes, respectively. Tetraploid males have 22 chromosomes: four sets of autosomes and two X chromosomes. By contrast, tetraploid hermaphrodites have four sets of autosomes and either three or four X chromosomes. . 3.2. Understanding meiotic and early embryonic cell divisions 3.2. Understanding meiotic and early embryonic cell divisions Female meiotic divisions are asymmetric. Each division gives rise to a large oocyte and a tiny polar body. The resulting egg contains a haploid set of chromosomes, as half of the chromosomes in each division are discarded into the polar bodies. Therefore, C. elegans hermaphrodites trisomic for the X chromosome (2A;3X) would be expected to produce equal proportions of eggs that carry either one or two X chromosomes (one sister from the paired homologues +/- one sister from the self- synapsed chromosome). However, the observed X:XX ratio in these eggs is 2:1 [92]. Live and high- resolution visualization of the oocyte cell division in these trisomic worms showed that the self- synapsed chromosomes were preferentially partitioned into the polar body during oogenesis [28,32]. In addition, in triploid animals that have one set of homologous chromosome pairs (bivalents) and a set of unpaired chromosomes (univalents); univalents were most often discarded into the polar body of the first oocyte division [32]. In these triploids, preferential segregation of the single sister chromatids into the polar body could also be observed in the second division. These results provide mechanistic insights into why triploid animals rapidly diploidize. Several seminal studies elucidating mechanism of embryonic cell division have utilized C. elegans tetraploid and haploid (1A;1X) embryos [26,104–107]. One of the first questions this work addressed was how the paternally-inherited centrosomes duplicate and become associated with the nucleus. In the zygote, the migration of the maternal and paternal pronucleus towards one another and their subsequent fusion is important to ensure the fidelity of the first division of the embryo. In addition to contributing a haploid genome, the sperm also contributes a pair of centrioles to the zygote. These centrioles remain in the proximity of the male pronucleus and, as they gather pericentriolar proteins (PCM) from the maternal cytosol, they duplicate to form the first pair of centrosomes of the zygote [108]. Concurrently, the male pronucleus grows as DNA decondenses and the centrosomes then become attached to its surface [109]. Comparison of zygotes with different ploidies suggests that centrosome attachment to the nuclear membrane depends on the size of the male pronucleus [104]. Live imaging showed that diploid embryos with mutations in nuclear envelope genes have aberrantly small male pronuclei with only one attached centrosome; the second centrosome only became attached as the male pronucleus surface increases [104]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 8 8 homologous chromosomes form in tetraploids [31]. This pairwise synapsis also takes place in germ lines of mutants (i.e. spo-11) that are deficient in early steps of recombination, as in diploids. In triploid germ lines, one pair of homologs synapse and the remaining homolog eventually folds onto itself, in a process known as self-synapse. By contrast to diploids, the pairwise synapsis is disrupted in the presence of the recombination-deficient mutations, suggesting that in C. elegans, as in other organisms, homologous recombination plays a role in promoting synapsis [31]. Polyploid animals heterozygous for translocation chromosomes (e.g. nT1(IV;V)) were utilized to test whether pairwise synapsis is dictated by full sequence homology along the chromosome length. Synapsis was normal in diploid translocation heterozygotes (i.e. nT1/+ or mln1/+), as long as they contained the pairing center (PC) sequence that is required for homologous chromosomes to initially find each other in the nucleus [103]. In tetraploids (nT1/nT1/+/+), there was no preference for a synapsis partner as translocation chromosomes paired with normal chromosomes with the same frequency as with themselves. In contrast, associations and synapsis between pairs of homologous chromosomes was biased towards homologs with identical sequences in the triploids [31]. These findings support the proposed model in which the initiation of synapsis depends on PC-mediated associations of homologs but completion of synapsis along the homologous pair is promoted by recombination [31]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints2023 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 3.1. Understanding early events of meiotic prophase I During meiosis, recombination between the duplicated maternal and paternal homologous chromosomes transiently connects the homologs to orderly partition them in the first division, while maintaining sister chromatids together until the second division. To recombine, homologous chromosomes must first find one another in the nucleus (i.e. pair), become aligned along their lengths, and then stabilize their association by assembling the synaptonemal complex (i.e. synapse) [102]. Observations of the effects of having additional chromosomes in C. elegans triploid and tetraploid animals have helped to uncover rules underpinning homologous chromosome dynamics during meiosis [30,31]. Additional chromosomes did not significantly alter the initial steps of pairing into groups of three or four homologs in triploids and tetraploids, respectively. However, full alignment was often perturbed and synapsis was frequently incomplete, resulting in lengthening of the period in which synapsis normally takes place [30]. The imperfectly synapsed chromosomes became marked by di-methylation (H3K9me2), which has been proposed to prevent the synapsis defects from being detected by the quality control system [30]. Further analysis revealed that two pairs of synapsed Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 9 A second major question that was addressed used animals of differing ploidy was whether nuclear to cytosol ratio regulates cell cycle timing which had been hypothesized from work in other organisms [111–113]. The roles of nuclear size, its ratio to cytosolic volume, DNA content, and overall cell size in cell cycle timing of the C. elegans embryo were evaluated by comparing diploid (N2 Bristol), tetraploid (strain SP343), and haploid embryos (generated by extracting the pronucleus from the fertilized egg before the zygote formed) [105]. In these embryos, different nuclear/cytosolic volume ratios were examined by extruding nuclei with little to no cytosol, some cytosol, or increased cytosolic volume, the latter created by laser-induced cell fusion followed by extrusion of one nucleus. Live imaging revealed that cell cycle timing was neither affected significantly by altering cell size, nor by nuclear to cytosol ratio, nor by DNA content [105]. Instead, cell fusion experiments revealed that cell cycle periods could be altered by the cytosol of another cell in a different cell cycle period. Together with other findings, these early results are consistent with studies that posited models for the existence of “cytoplasmic factor(s)” that controls cell cycle timing [114–116]. The cytoplasmic factors that regulate many aspects of the cell cycle and its timing have now been identified [117]. g y p y g Alterations of ploidy in embryos was also utilized to assess scaling of the microtubule-based spindle [26]. Accurate chromosome partitioning relies on the assembly of a properly shaped spindle during cell division. The metaphase spindle forms a diamond shape, with two opposing corners at each centrosome and two at the ends of the metaphase plate. The ratio between the spindle length (from centrosome to centrosome) and width (of the metaphase plate at the equator) was constant in in vitro Xenopus extract studies that either molecularly or mechanically perturbed the dimensions of the metaphase spindles [118,119]. In vivo studies in C. elegans have been instrumental to our understanding of spindle shape. Initial studies showed that spindle length is affected by cell size as spindle length and width decreased as embryogenesis progressed [26,114]. Interestingly, reduction of the spindle width was observed when the spindle length was decreased (e.g. by RNAi depletion of tpxl-1 (the C. elegans homolog of Xenopus TPX2 involved in spindle assembly) or spd-2 (involved on multiple aspects of centrosome biogenesis and thus spindle formation)) [120]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 Conversely, the opposite was not the case: changing the width of the spindle did not affect the spindle length. In haploid embryos the spindle width is reduced and in polyploid embryos [26] increased; yet in neither case was embryo size or spindle length affected [121–125]. Correlation analysis between spindle length and width in haploid, diploid and polyploid embryos revealed a simple equation that can predict spindle width based on the spindle length and ploidy of the embryo [26]. Another important correlation that was observed with the reduction in cell size during embryogenesis is related to chromosome condensation, a chromosome feature that is important for accurate chromosome inheritance [120,126–129]. Haploid and tetraploid embryos produced by diploid klp-18(RNAi) mothers show differential condensation of chromosomes compared to diploid control embryos [120]. Chromosome condensation decreased in haploid embryos in comparison to the diploid embryos, whereas condensation increased in tetraploid embryos. Differential condensation was independent of cell size, the speed of spindle elongation, and spindle length [26,120]. Therefore, an inverse correlation between nuclear DNA density and chromosome condensation was proposed and tested by observing chromosome condensation in dividing diploid and klp-18(RNAi) haploid embryos, during divisions as the size of the nucleus decreases during development [120]. The relationship between the DNA density in the nucleus and chromosome length (a measure of chromosome condensation) fit a regression line for the dividing diploid embryo. Importantly, this regression line also fitted well the measurements of the dividing haploid embryo, as well as, when the nuclear size was manipulated by reducing the transcripts by RNAi of ima-3 (nuclear pore import protein), ran-3 (ortholog of the human RCC1 regulator of chromosome condensation) and C27D9.1 [120], suspected or known to affect nucleus size [130]. These analyses revealed an allometric relationship between chromosome length/condensation and the relative ratio of DNA amount per nucleus size that is consistent with the hypothesis that chromosome condensation is regulated to adapt to fit optimally the amount of DNA in the physical space within the nucleus. 3.2. Understanding meiotic and early embryonic cell divisions The detached centrosome phenotype was suppressed in zygotes from tetraploid animals that had larger pronuclei. Conversely, the detached centrosome phenotype was enhanced in haploid zygotes with a smaller pronuclei. It has been speculated that dynein motor accumulation on the pronuclear surface area is the limiting factor for centrosome attachment with a threshold of dynein required for both centrosomes to attach [106,110]. Of note, the haploid embryos in this study were generated utilizing conditional mutant sperm that lack nuclear DNA (i.e. emb-27(g48ts)) [106]. 4.1. Polyploid tissues in C. elegans At least two tissues are polyploid in C. elegans: the intestine and hypodermis (part of the nematode epidermis) [90]. The intestine is a tube responsible for digestion and makes up nearly a third of the animal’s body. All intestinal cells are derived from a single blastomere (E) starting at the eight-cell embryo stage [131,132]. The intestine of a newly hatched embryo has twenty diploid cells. When the L1 larvae hatch, they have 20 intestinal cells that have 20 diploid nuclei [90]. Of these nuclei six do not divide nor replicate their DNA, four nuclei may or may not replicate their DNA or divide, and 10 nuclei replicate and divide by endomitosis cells with two diploid nuclei each. Therefore, by the end of the L1 stage the 20 cells of the intestine have 30-34 diploid nuclei. Prior to the transition molt to the L2 stage all nuclei of the intestine endoreduplicate their DNA (4C each nucleus). Nuclei continue to endoreduplicate prior to the molts into the L2 (8C each nucleus), the L3 (16C), and finally the L4 (32C) larval stages. Thus, the adult intestine is composed of 20 cells with a total of 30-34 nuclei, each with 32C ploidy [90]. The hypodermis has many roles in development including establishment of the basic body plan in the embryo such as body shape and size, regulation of cell fate specification, and guidance of migrating axons and cells [133–135]. The hypodermal cell hyp7 is a multi-nucleated syncytium that encases most of the adult animal body (the tail and head of the animal are covered by smaller hypodermal cells) [90]. The hyp7 syncytium contains 139 nuclei in the adult and arises from many cell fusions (during embryogenesis 23 cells fuse to hyp7 and post-embryonically 116 cells also fuse to it) [90,136]. The embryonically-derived nuclei in hyp7 remain diploid, whereas the ~98 cells generated post-embryonically endoreduplicate to become tetraploid [90,136]. Most of these nuclei undergo up to 2 more endoreduplication cycles that result in the hypodermal nuclei having an average 10.7C ploidy by the fully grown adult stage [91]. The ploidy of the hyp7 cell is key for adult animal size regulation, (see section on allometric studies, below) [137]. In diploid cells, coordination of the centrosome and cell cycle ensures a single duplication of the centrosome per mitotic cycle, which is crucial to prevent supernumerary centrosomes accumulation that can lead to genomic instability [138,139]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 10 In addition to being utilized as tools to study development, C. elegans polyploids have also been used to study the process of polyploidization itself and its immediate after-effects. 4.2. Biological size and scaling (allometry) Regulation of cell size and scaling of tissues and organs are critical for development and influence homeostasis, metabolism, and function [45]. Polyploidization has been generally associated with increases in cell and organism size. For example, genome size and ploidy correlate linearly with cell volume in yeast [144–146]. However, effects of polyploidy are cell-type specific in many multicellular organisms, with different cell types having a nonlinear relationship between ploidy and cell size [147]. For instance, in plants there is a strong correlation between ploidy and cell volume in epidermal pavement cells, but the volume of palisade mesophyll cells remains at a constant size despite changes in ploidy [148]. These and other observations [45,149] make it apparent that the DNA mass does not fully explain the effect of ploidy on cell (and nuclear) size [45,149–152]. In most polyploidization models, including plants, interpreting the direct effect of ploidy is complex because the increase in cell size in tissues and organs is balanced by a reduction in cell number [45,150,153– 155]. The invariant cell lineage in C. elegans simplifies the identification of mechanisms underpinning correlations between ploidy and scaling of cells, tissues and organs. p y g g Allometry studies related to the effects of ploidy in C. elegans have been performed for cells and animals [25,26,91,98,120,156–159]. Nigon (1949) [99] reported that, compared to the 1300 µm long diploid hermaphrodites, 4A;3X hermaphrodites were on average 1360 µm and the 4A;4X hermaphrodites were 1560 µm long. The more profound increase in 4A;4X hermaphrodites containing 24 chromosomes (See Figure 3) compared to the 4A;3X hermaphrodites (23 chromosomes) is surprising and suggests that gene expression from the X chromosome may contribute to animal size significantly more than gene expression from autosomes. The hypodermis secretes the cuticle forming the exoskeleton that houses the nematode’s internal organs and nervous system. There is a proportional relationship between the size of the C. elegans body and the size of the hypodermal cells (seam cells and the syncytium hyp7) [156,160,161]. Comparison of 12 nematode species from the order Rhabditida revealed a weak correlation between the number of nuclei and hypodermal volume (hyp7 in C. elegans), suggesting that body size was at least partly independent of cell number [91]. A significant correlation between animal size and the product of the number of nuclei and the ploidy was observed across species. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.2 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 11 mononucleated cells of identical ploidy using auxin- induced degradation [143] of either a regulator of mitotic entry (e.i. CDK-1) or a kinetochore protein required for chromosome partitioning in mitosis (i.e. KNL-1) to block endomitosis [49]. Whereas absence of CDK-1 prevents entry into mitosis and essentially converts the endomitosis into an endocycle, degradation of KNL-1 allows for entry into mitosis but prevents chromosome partitioning into two nuclei [49]. Neither knock-down affects cell size or morphology, but the mononucleated intestinal cells have reduced nuclear to cytoplasmic ratio given that their nuclei more than double in size [49]. Transcriptome analysis revealed that all of the vitellogenin genes (vit-1 to vit-6) are downregulated in mononucleated intestines. Vitellogenins are yolk proteins that are required for lipid transport and lipid loading onto oocytes and embryos and promote progeny fitness. Accordingly, the embryos from mothers with mononucleated intestines have reduced survival. Similarly, decreasing vitellogenin expression in binucleated gut cells phenocopies the impact on progeny fitness and increasing vitellogenin expression, by overexpressing their transcriptional activators, results in mothers siring progeny with similar fitness regardless of whether they had mono- or bi-nucleated intestines [49]. Importantly, many of the genes that are downregulated in animals with mononucleated intestines normally increase in expression during the L4 to adult transiition, implicating a major role for binucleation at this developmental stage [49]. 4.1. Polyploid tissues in C. elegans Thus, the regulation of the centrosome in polyploid tissues is equally important for maintaining genome stability [140]. The endopolyploidy of the intestine and hypodermis of developing C. elegans allowed for comparative studies of how tissues respond to this challenge. In both the polyploid intestine and the hypodermis V1 cell nuclear lineages of the hyp7 syncytium, loss of PCM correlates with an apparent lack of centriole duplication resulting from uncoupling of the centrosome cycle in the first endo-cycle followed by progressive centriole elimination [141]. The SPD-2 protein with a normal dynamic localization to the PCM and the centrioles is key in for centriole duplication. An spd-2 phosphomimetic mutant, SPD-2S545E that can be phosphorylated in vitro by CDK1 and CDK2 [141] leads to supernumerary centrosomes in the intestinal cells [141]. This supports the hypothesis that the putative CDK phosphorylation site at S545 is a part of the molecular mechanism preventing centriole duplication during the endo-S phases of the intestine [141]. Since the SPD-2S545E mutation does not affect centriole number in the hypodermal cells, this regulation appears to be specific to intestinal cells. A different putative phosphomimetic SPD-2S375E mutation, affects a Polo Like Kinase-1 (PLK-1 in C. elegans) consensus phosphorylation site, and results in the persistence of SPD-2 on centrioles at the L2 stage that agrees with the observed delay in centriole elimination in some of the intestinal nuclei of this mutant. Surprisingly however, PLK-1 does not seem to regulate SPD-2 stabilization nor centriole elimination [141]. Instead, knockdown of the proteasome b-subunit (pbs-3) results in both SPD-2 persistence indicative of a lack of centriole elimination in many of the intestinal cells. This finding implicates ubiquitin-mediated proteolysis in the uncoupling of the endocycle and centriole cycle in the intestine [141]. One question that arose when looking at polyploid tissues is whether there is a biologically- significant functional difference between mononucleated polyploidy (restricted to a single nucleus) and multinucleate polyploidy (in more than one nucleus) [12,142]. The developing intestine in C. elegans was used to address this question by converting the normally binucleated intestinal cells into Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 12 the original, but these were not examined. Multiple independent tetraploid strains from nearly any diploid strain provides the possibility of conducting this comparison more rigorously. Levels of endoreduplication in the hyp of C. elegans was found to be important in regulation of the adult animal size. When C. elegans young adults were exposed to the DNA replication inhibitor, hydroxyurea (HU), endoreduplication of the hyp7 nuclei was inhibited and a reduction in body size was observed [25]. Cyclin E is required for endoreduplication in mice and Drosophila [162,163]. Null mutants carrying mutations the C. elegans homolog of Cyclin E, cye-1, have an ~1.8-fold reduction in size of the hyp7 syncytium and are 35-54% the size of wild-type worms [25]. yp y y yp Many signaling pathways and genes affecting diploid animal size have been characterized in C. elegans. These include pathways that only regulate embryo size, that regulate multiple developmental stages, and that influence adult body size. The TGF฀/BMP signaling pathway has a major role in the response to ploidy as it regulates both adult size and hyp7 ploidy [25,137,160,161,164,165]. The TGF฀ signaling pathway that regulates body size in C. elegans includes the DAF-4 type two receptor, the DBL-1 ligand, and the downstream SMADs (SMA-2, SMA-3, SMA-4, and SMA-6) [166]. DBL-1, the human ortholog of human BMP7, is required for post-embryonic growth animal [91,159,167]. Whereas DBL-1 overexpression results in longer-than-normal animals (Lon phenotype) [164], mutations in the dbl-1 gene cause a dwarf phenotype (Sma) and reduced hypodermal ploidy [25,164]. Interestingly, as with the cye-1 mutant, exposure of dbl-1 mutants to hydroxyurea neither further reduced hyp7 ploidy nor affected its body size, suggesting that the DBL-1 normally promotes hyp7 endoreduplication [25]. Together these and other findings [25,161] suggest that DBL-1 regulates adult growth in a dose dependent manner [158,164] by promoting endoreduplication in the adult hypodermis (hyp7) [161], likely via the CYE-1 cyclin [25]. The regulation of body size by DBL-1 pathway is complex. Many of the molecular components in the DBL-1 pathway and other pathways affecting endoreduplication in the hypodermis have been identified, and their localization and molecular or genetic interactions described. For instance, overexpression of LON-1, a member of the conserved PR-protein superfamily [157,168], causes hypo- endoreduplication and absence of LON-1 protein causes hyper-endoreduplication in the hypodermis [157]. Specifically, the levels of lon-1 transcript depend on the dosage of the dbl-1 gene and TGF฀ signaling. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 Other genes and processes affecting diploid animal size are also likely to play important roles in this process. These include cuticle collagens, ß-H Spectrin, TOR kinase, MAPK signaling, Hippo-Warts signaling, and the insulin signaling pathway [159,169–178]. These genes are expected to show altered expression in polyploid animals [45,51,179,180]. Global metabolic genes, such as rRNA or tRNA genes, are likely also involved in regulating biological scaling in polyploid animals [45,181]. Whether these pathways contribute equally to growth regulation in polyploids remains an open question. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints2023 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 4.2. Biological size and scaling (allometry) Therefore, unlike most organisms where the evolution of body size relies in changes in cell number, in nematodes body size evolution was likely driven by the size of the hypodermal syncytium [91]. Comparison of a tetraploid strain and its diploid revertant revealed a 39% increase in the adult body size of tetraploids [25]. The unexpected 1.4-fold increase in volume (instead of the expected 2-fold) in tetraploids is at least in part explained by the lower-than-expected ploidy in the hyp7 cell (16.7C instead of 22C) and the small reduction in the number of cells (by less than two nuclei) [25]. However, one should be careful in making conclusions from this comparison because it is likely that the reverted diploid differed from Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 Author Contributions: All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NIGMS, grant number R01GN104007 to JLY; PSC-CUNY award, 65508- 00 53 and the CUNY-Research Foundation, FFPP award to MS; Tow mentoring and research program to MT and MS; Brooklyn College’s, Samuel and Rose Goldstein award to MT. Acknowledgments: In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints202304.0989.v1 13 multi-subunit complexes (ribosomes, polymerases, etc.) tend to be retained [186]. Previous observations in plants showed that they undergo loss of repetitive sequences, mostly transposable elements, by recombination [188–190]. This can reduce the genome by up to 25% within the first few generations of newly formed autopolyploid [188]. Synthetic auto- and allo-tetrapolyploid C. elegans will allow both short term and longitudinal studies of the process of genome downsizing given the nematodes’ short life cycle and the ability to manipulate its environment. Additionally, these animals would facilitate study of tissue-specific differences and mitotic stability of ploidy during development. Of particular interest will be comparative analyses of the endopolyploid hyp7 and intestinal cells to determine how their DNA size/mass scales with ploidy. Gene expression studies have been done to assess differential gene expression between in allopolyploid plants, often comparing diploids to an ancestral hybrid species [45]. An important mystery remaining is the stoichiometry of the changes in whole transcriptomes of similar cells from animals with differing ploidy [182,189]. Two competing hypotheses regarding the effect of organism polyploidization on global gene expression have been proposed; 1) each duplicated genome retains diploid transcription levels, or 2) all gene loci are affected by dosage compensation to maintain gene dose similar to that of diploid. Although very few studies have been done to compare the effect of differing ploidy on transcriptome size, neither global suppression nor doubling of expression is uniquely likely since allopolyploids studies showed an intermediate transcriptome size. Importantly, single-cell transcriptomic studies showed a high variability or noise in gene expression within a cell type and between different cell types. Specific regulatory genes (e.g. RNA polymerase 2 and 5.8S rRNA) have been found to differentially increase in expression with increasing ploidy [190]. Global and specific gene expression can also be affected by chromatin remodeling (e.g. chromatin and histone modifications) and specific histone modification, particularly histone H3K56 acetylation, are thought to buffer increases in mRNA synthesis due to increased number of gene copies [191]. The ability to create tetraploids with any strain facilitates longitudinal studies of the tetraploid. For example, one can now create recombinant inbred strains of auto- and allotetraploids to address how the genome changes over 10s -100s of generations. Combining these studies with exposures to environmental stressors, such as disease, temperature, infection and DNA damage, has the potential to shed light on genomic changes that provide flexibility and drive adaptation. References 1. Davoli, T.; Lange, T. de The Causes and Consequences of Polyploidy in Normal Development and Cancer. http://dx.doi.org/10.1146/annurev-cellbio-092910-154234 2011, doi:10.1146/annurev-cellbio-092910-154234. 3. Storchova, Z.; Pellman, D. From Polyploidy to Aneuploidy, Genome Instability and Cancer. Nat Rev Mol Cell Bio 2004, 5, 45–54, doi:10.1038/nrm1276. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 doi:10.20944/preprints20230 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 26 April 2023 5. Potential future queries utilizing C. elegans polyploids The ease of creating synthetic autotetraploids from any strain utilizing RNAi is shepherding in a new era. We can now expand the use of C. elegans polyploids to study cellular and developmental processes (e.g. allometric relationships of different cell types, cell division dynamics) and disease (e.g. genome instability, pathological cell divisions, cancer) by comparing equivalent organisms, tissues, organs and cells from isogenic diploid, triploid, and tetraploid animals. This method allows for the first time in C. elegans the ability to query the direct impact of organismal polyploidization on individual tissues across time. In addition, this polyploid model's unique features will be of particular benefit for advancing our understanding of WGD and its effect on genome structure, transcriptome size (total RNA per unit of DNA mass) [182], specific-gene expression, regulation of gene expression by chromatin modification, and importantly how these changes affect the physiology, metabolism and developmental character of the organism. In the course of evolution, parts of the genomes of polyploids are lost [183–185], a process known as fractionation [186]. This can occur because the purifying selection – the drive to remove mutated genes – is relaxed on duplicated genes, leading to both gene and regulatory element loss [186,187]. In allotetraploids, allele dominance can facilitate gene retention/fractionation, but genes involved in 1. 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https://openalex.org/W2028996033	https://tc.copernicus.org/articles/9/1129/2015/tc-9-1129-2015.pdf	English	null	Air temperature variability over three glaciers in the Ortles–Cevedale (Italian Alps): effects of glacier fragmentation, comparison of calculation methods, and impacts on mass balance modeling	The cryosphere	2,015	cc-by	13,757	The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ doi:10.5194/tc-9-1129-2015 © Author(s) 2015. CC Attribution 3.0 License. The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ doi:10.5194/tc-9-1129-2015 © Author(s) 2015. CC Attribution 3.0 License. Air temperature variability over three glaciers in the Ortles–Cevedale (Italian Alps): effects of glacier fragmentation, comparison of calculation methods, and impacts on mass balance modeling L. Carturan1, F. Cazorzi2, F. De Blasi1, and G. Dalla Fontana1 1Department of Land, Environment, Agriculture and Forestry, University of Padova, Viale dell’Università 16, 35020 Legnaro, Padova, Italy 2Department of Agriculture and Environmental Sciences, University of Udine, via delle Scienze 208, 33100, Udine, Italy Correspondence to: L. Carturan (luca.carturan@unipd.it) Received: 20 November 2014 – Published in The Cryosphere Discuss.: 15 December 2014 Revised: 22 April 2015 – Accepted: 23 April 2015 – Published: 27 May 2015 Received: 20 November 2014 – Published in The Cryosphere Discuss.: 15 December 2014 Revised: 22 April 2015 – Accepted: 23 April 2015 – Published: 27 May 2015 Abstract. Glacier mass balance models rely on accurate spa- tial calculation of input data, in particular air temperature. Lower temperatures (the so-called glacier cooling effect) and lower temperature variability (the so-called glacier damping effect) generally occur over glaciers compared to ambient conditions. These effects, which depend on the geometric characteristics of glaciers and display a high spatial and tem- poral variability, have been mostly investigated on medium to large glaciers so far, while observations on smaller ice bodies ( < 0.5 km2) are scarce. Using a data set from eight on-glacier and four off-glacier weather stations, collected in the summers of 2010 and 2011, we analyzed the air tempera- ture variability and wind regime over three different glaciers in the Ortles–Cevedale. The magnitude of the cooling ef- fect and the occurrence of katabatic boundary layer (KBL) processes showed remarkable differences among the three ice bodies, suggesting the likely existence of important re- inforcing mechanisms during glacier decay and fragmenta- tion. The methods proposed by Greuell and Böhm (1998) and Shea and Moore (2010) for calculating on-glacier tem- perature from off-glacier data did not fully reproduce our ob- servations. Among them, the more physically based proce- dure of Greuell and Böhm (1998) provided the best overall results where the KBL prevails, but it was not effective else- where (i.e., on smaller ice bodies and close to the glacier mar- gins). The accuracy of air temperature estimations strongly impacted the results from a mass balance model which was applied to the three investigated glaciers. Most importantly, even small temperature deviations caused distortions in pa- rameter calibration, thus compromising the model generaliz- ability. 1 Introduction and background Air temperature exerts a crucial control on the energy and mass exchanges occurring at the glacier surface. It regulates the accumulation processes via the snowfall elevation limit and the snowpack metamorphism (which affect redistribution phenomena) and regulates the ablation processes via turbu- lent ﬂuxes and long-wave radiation. It is also closely related to important feedbacks such as albedo, the mass balance– elevation feedback, and the glacier cooling effect, which changes as glaciers adjust their size in response to climatic ﬂuctuations (Khodakov, 1975; Klok and Oerlemans, 2004; Paul et al., 2005; Raymond and Neumann, 2005; Haeberli et al., 2007; Fischer, 2010; Paul, 2010; Carturan et al., 2013). Distributed models of different complexity have been pro- posed for calculating the mass balance of glaciers under different climatic scenarios at a variety of spatial scales and with different purposes. The current concern about sea level rise and future availability of water resources stored in glaciers, under projected global warming scenarios, has led to increased efforts to develop models able to account for (i) direct effects of climate change and (ii) reinforcing L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1130 mechanisms which control glacier decay (Hock, 2005; Barry, 2006). cesses has a twofold effect, consisting of lower on-glacier temperatures (the so-called glacier cooling effect) and lower temperature variability (the so-called glacier damping effect, also referred to as reduced climate sensitivity) compared to ambient conditions (Braithwaite, 1980; Greuell and Böhm, 1998; Braithwaite et al., 2002; Gardner et al., 2009). As a re- sult, on-glacier lapse rates generally differ from average en- vironmental lapse rates (i.e., −0.0065 ◦Cm−1). Cooling and damping effects are not homogeneous over glacial surfaces and mainly depend on the size and geometric characteristics, in particular the slope, of single glaciers and on the speciﬁc position along the glacier. Generally, they are directly related to the size of glaciers and the fetch distance along the ﬂow line and inversely related to the slope of glaciers. The lat- ter controls the prevalence of the cooling due to turbulent exchanges over the adiabatic heating of air forced to move downward by katabatic winds. These models rely on accurate spatial calculation of in- put data, in particular air temperature, which affects not only their ﬁnal performance but also the calibration of parame- ters and model generalizability. Indeed, wrong temperature estimates lead to wrong calibration and/or distortion of pa- rameters, possibly hampering the applicability of models to ungauged catchments, despite the good knowledge achieved for individual processes (Savenije, 2001; Sivapalan, 2006). p ( j , ; p , ) Charbonneau et al. (1981), for example, highlighted that issues in extrapolating meteorological input data are much more crucial than the possible choice between different ap- proaches for modeling snow yields from a well-equipped catchment in the French Alps. Similarly, intercomparison projects of runoff models by the World Meteorological Or- ganization (e.g., WMO, 1986) revealed that simple mod- els provided results comparable to more sophisticated mod- els, given the difﬁculties of assigning proper model param- eters and meteorological input data to each catchment ele- ment. Machguth et al. (2008), analyzing model uncertainty with Monte Carlo simulations at one point on the tongue of Morteratsch Glacier in Switzerland, concluded that the out- put of well-calibrated models, when applied to extrapolate in time and space, is subject to considerable uncertainties due to the quality of input data. According to Carturan et al. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale (2012a), who compared three melt algorithms in a 6-year application of an enhanced temperature-index model over two Italian glaciers, uncertainties in extrapolating tempera- ture measurements from off-site data partly mask the pecu- liar behavior of each algorithm and do not allow deﬁnitive conclusions to be drawn. Few methods have been proposed in the literature to model these processes, mainly due to the scarcity of glaciers in- strumented for distributed measurements of air temperature. Among the ﬁrst authors who measured the glacier cooling effect, deﬁned as the temperature difference between an on- glacier and an off-glacier site with the same altitude, were Schytt (1955) and Eriksson (1958), who detected tempera- ture depressions ranging from 1.1 to 2.2 ◦C on Storglaciären (Sweden) and 3–4 ◦C on Skagastøl Glacier (Norway), re- spectively. Havens (1964) measured an average cooling ef- fect ranging from 1.5 to 2.7 ◦C at a weather station lo- cated 1 km up-glacier from the terminus of White Glacier (Canada), recognizing maximum values during warm and sunny weather and minimum values during overcast and un- settled weather. Two main issues affect the correct estimation of air tem- perature distribution over glacial surfaces: (i) the absence of on-site weather stations in most operational model applica- tions and (ii) the development of a katabatic boundary layer (KBL) over the typically inclined glacier surfaces (van den Broeke, 1997). Several experiments with automatic weather stations (AWSs) deployed over glaciers demonstrated that general assumptions in extrapolating air temperature, based on the application of ﬁxed lapse rates which account for the linear dependency of ambient (i.e., off-glacier) temperature on altitude, have serious limitations (e.g., Greuell et al., 1997; Strasser et al., 2004; Petersen and Pellicciotti, 2011). To our knowledge, the ﬁrst attempt to parameterize the mean summer cooling effect at the ﬁrn line altitude was made by Khodakov (1975), who proposed a relationship with glacier length based on temperature data obtained from mountain glaciers and ice sheets. Analyzing direct observa- tions from glaciers in Caucasus, Pamir, Scandinavia, Tian Shan, and Altay, Davidovich and Ananicheva (1996) pro- vided a simple relationship for calculating the mean summer temperature at the equilibrium line altitude (ELA) in function of the mean off-glacier summer temperature at the same al- titude. The same authors suggested that the cooling effect is maximal at the ELA and decreases towards both the terminus and up-glacier. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale (Carturan et al., 2012a). The highest summit is Mount Cevedale (3769 m a.s.l.), while the basin outlet is located at 1950 m a.s.l. The catchment lies in the southern part of the Ortles–Cevedale massif, the largest glacierized mountain group in the Italian Alps. The Careser Diga weather station (2607 m a.s.l.) has been operating since the 1930s, record- ing daily 2 m air temperature, precipitation, snow depth, and fresh-snow height. In the 1990s, an automatic weather sta- tion replaced the old manual instruments. At this site, the mean 1979–2009 annual precipitation (corrected for gauge errors) was 1233 mm and the mean annual air temperature in the same period was −0.5 ◦C. dian Arctic glaciers (Sverdrup and White), similar to that proposed by Davidovich and Ananicheva (1996) but applied to monthly temperatures. Shea and Moore (2010) suggested empirical relationships based on piecewise linear regressions of on-glacier vs. ambient temperatures collected in British Columbia (Canada) between 2006 and 2008 for calculating (i) the threshold temperature triggering KBL development and (ii) the glacier damping effect as a function of eleva- tion and ﬂow path length (i.e., the “average ﬂow distance to a given point starting from an upslope limit or ridge”). At present these methods have rarely been used by other authors, and they have not been compared using independent test sites. Petersen et al. (2013) tested the Greuell and Böhm (1998) model using a data set of air temperature measure- ments from Haut Glacier d’Arolla, Switzerland, concluding that results of spatial extrapolations along the glacier are only a little better than using a constant linear lapse rate calcu- lated between on-glacier data points, attributing this result to the spatial variability of the thickness of the glacier boundary layer. The investigated glaciers are very different. Careser Glacier (2870–3279 m a.s.l.) is ﬂat and mainly exposed to the south. In 2005 it spread in two parts: Careser Orien- tale (2.13 km2 in 2006) and Careser Occidentale (0.27 km2 in 2006). La Mare Glacier (2650–3769 m a.s.l., 3.79 km2 in 2006) faces to the east and is steeper. On all glaciers, topographic shading is of minor importance. The Careser glaciers have no accumulation area and exhibit down-wasting and fragmentation in smaller units (Carturan et al., 2013), while La Mare Glacier still has an accumulation area and shows “active” retreat towards higher altitudes (Zanon, 1982; Small, 1995; Carturan et al., 2009, 2014). 3.1 Experimental setup An AWS has been operating since July 2007 on the abla- tion area of La Mare Glacier (2973 m a.s.l.), measuring air temperature and relative humidity, wind speed and direction, incoming and outgoing shortwave and long-wave radiation, and snow depth. The thermo-hygrometric probe is housed in a ventilated radiation shield. Data are sampled every 60 s, with 15 min means stored in a Campbell Scientiﬁc CR1000 data logger; the AWS is powered by a 25 W solar panel. Data were periodically downloaded with a portable laptop until July 2011. Since August 2011, a satellite modem has automatically transmitted data at 3-day intervals (Abbate et al., 2013). L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Long-term moni- toring programs started in 1967 on Careser and in 2003 on La Mare. In the last 10 years, the glaciers have been the subject of investigations on snow accumulation, snow and ice abla- tion, point energy balance, and runoff generation (Carturan, 2010). The transferability of the proposed methods remains to be tested. In addition, it should be noted that many of them have been developed using temperature data collected from medium (from 0.5 to 10 km2) to large (larger than 10 km2) glaciers. As the glacier cooling effect and the damping effect depend on the size of glaciers, it is opportune to investigate the thermal effects of ice bodies smaller than 0.5 km2, which are widespread and increasing in number in mid-latitude mountain regions as a result of glacier shrinking and frag- mentation. In this work we present the results of a glacial– meteorological experiment, carried out in summer 2010 and 2011, deploying several automatic weather stations over three neighboring glaciers in the Ortles–Cevedale mountain group (Italian Alps). The study was focused on the variability of air temperature over the three glaciers which differ in size, geometric characteristics, and reaction to climatic changes (Carturan et al., 2014). In this paper, we analyze the tempo- ral and spatial behavior of air temperature and glacier cooling effect in the study area, testing existing methods for calculat- ing on-glacier temperatures from off-site data and evaluating their impact in mass balance simulations using a distributed enhanced temperature-index model. 2 Study area The investigated glaciers are located in the Alta Val de la Mare (AVDM), eastern Italian Alps (Fig. 1). This 36 km2 ex- perimental watershed is the subject of detailed studies con- cerning the impacts of climate change on the cryosphere and hydrology. The area has previously been selected for studying the behavior of meteorological variables at high altitude (Carturan et al., 2012b) and for developing an enhanced temperature-index glacier mass balance model On 3 July 2010 three Vantage Pro Plus (VPP) weather sta- tions, manufactured by Davis Instruments, were placed along a longitudinal proﬁle on La Mare Glacier at elevations rang- ing from 2709 m, close to the terminus, to 3438 m, near to the upper divide. Davis VPP stations are low-cost, commer- cial weather stations, characterized by a compact design and low weight, that can be moved rather easily along glaciers by few persons. Their thermo-hygrometric probe is shielded L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale In particular, these assumptions do not apply when kata- batic ﬂows and the KBL form, that is, during the ablation sea- son on melting mid-latitude glacial surfaces, when the ambi- ent temperature is higher than the surface temperature, which cannot exceed 0 ◦C. Katabatic winds are gravity winds orig- inated by the cooling of the near-surface air layers, result- ing in density gradients that force a downward movement of the air under the effect of gravity. The two main processes affecting the temperature of the air during this downslope movement are the cooling due to the exchange of sensible heat and the adiabatic heating. The interplay of these pro- The ﬁrst comprehensive glacial–meteorological experi- ment providing distributed temperature measurements was carried out in summer 1994 on Pasterze Glacier, Austria, and comprised ﬁve automatic weather stations placed along a ﬂow line. From this experiment, Greuell and Böhm (1998) developed a thermodynamic model for calculating air tem- perature in function of slope and distance along the ﬂow line, accounting for sensible heat exchanges and adiabatic heating. Braithwaite et al. (2002) used an empirical approach and a formulation derived from data gathered in two Cana- The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ 1131 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1132 Figure 1. Geographic setting of Alta Val de La Mare and location of the automatic weather stations. Figure 1. Geographic setting of Alta Val de La Mare and location of the automatic weather stations. by a ventilated screen, which is important for air temperature measurements in high-radiation and/or low-wind-speed con- ditions on glaciers (Georges and Kaser, 2002). Hourly mean data are stored in a Davis data logger. During the experiment, the data were downloaded with a portable laptop every 2 weeks. The three VPP stations were removed on 23 Septem- ber 2010. who applied a mass balance model using the standard envi- ronmental lapse rate for extrapolating air temperature from an off-glacier weather station, as commonly used in most model applications where on-glacier data are not available. The four VPP stations were removed on 12 September 2011. Table 1 reports the conﬁguration of the weather stations operated on Careser and La Mare glaciers, locations of which are shown in Fig. 1. Four off-glacier weather sta- tions (Table 1) were also used in this study for the calcu- lation of the glacier cooling effect in comparison to ambi- ent temperature and for testing two methods of calculation of on-glacier temperatures from off-site data. Two of them are part of the regional weather station networks (Bel_3328, at Cima Beltovo, 3328 m a.s.l.; Cog_1202, at Cogolo Pont, who applied a mass balance model using the standard envi- ronmental lapse rate for extrapolating air temperature from an off-glacier weather station, as commonly used in most model applications where on-glacier data are not available. The four VPP stations were removed on 12 September 2011. Table 1 reports the conﬁguration of the weather stations operated on Careser and La Mare glaciers, locations of which are shown in Fig. 1. Four off-glacier weather sta- tions (Table 1) were also used in this study for the calcu- lation of the glacier cooling effect in comparison to ambi- ent temperature and for testing two methods of calculation of on-glacier temperatures from off-site data. Two of them are part of the regional weather station networks (Bel_3328, at Cima Beltovo, 3328 m a.s.l.; Cog_1202, at Cogolo Pont, On 7 July 2011 four VPP stations were deployed, two on Careser Glacier and two on La Mare Glacier. www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale One weather station was re-positioned at 3438 m on La Mare Glacier be- cause instrumentation failure occurred at that place in 2010 due to lightning damage. The other three weather stations were placed in areas where systematic errors in mass bal- ance simulations were recognized by Carturan et al. (2012a), The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Table 1. Location, ﬂow path length (FPL), period of observation, and used variables for glacier and ambient weather stations∗. T with common records are 3 July–23 September 2010 and 7 July–12 September 2011. Weather station Easting Northing Elevation FPL Period of observation Used variables (m) (m) (m a.s.l.) (m) Summer Summer 2010 2011 La Mare Glacier Mar-gl_2709 626 692 5 143 668 2709 2896 x T , W Mar-gl_2973 625 960 5 143 483 2973 2132 x x T , W Mar-gl_3215 625 205 5 143 101 3215 1278 x T , W Mar-gl_3140 625 290 5 143 523 3140 805 x T , W Mar-gl_3438 624 199 5 142 924 3438 40 damaged x T , W Careser Glacier Car-gl_3082 632 283 5 145 512 3082 313 x T , W Car-gl_3144 629 690 5 145 375 3144 354 x T , W Ambient weather stations Cog_1202 629 915 5 135 988 1202 \ x x T Car_2607 630 570 5 142 410 2607 \ x x T , P Car_3051 630 799 5 145 553 3051 \ x x T Bel_3328 624 957 5 151 212 3328 \ x x T ∗T : air temperature; W: wind speed and direction; P: precipitation. On-glacier sites are in bold type. The “x” indicates the periods of observations for each station. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1133 ∗T : air temperature; W: wind speed and direction; P: precipitation. On-glacier sites are in bold type. The “x” indicates the periods of observations for each station. tations are common in mountain regions and imposed com- parable or even lower densities of AWSs, as well as the use of different types of sensors with different radiation shields, in most similar studies on glaciers (e.g., Shea and Moore, 2010; Petersen and Pellicciotti, 2011; Petersen et al., 2013). 1202 m a.s.l., Fig. 1). L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale The other two weather stations consist of HOBO Pro data loggers (Onset Computer Corporation) in- stalled at Careser Diga (Car_2607, at 2607 m a.s.l.) and close to Careser Glacier (Car_3051, at 3051 m a.s.l.). All these sta- tions are far enough from the thermal inﬂuence of glaciers (minimum distance of 300 m from Car_3051 to the margin of Careser Glacier) and equipped with temperature probes housed in naturally ventilated radiation shields. Possible is- sues related to the use of different types of temperature sen- sors and radiation shields are addressed in the following sec- tion. Intercomparison tests have been carried out in order to assess the impact of using different sensors and radiation shields for this study. The four VPP weather stations were run for some days within a 10 m radius, both before and after the glacial–meteorological experiment, conﬁrming the almost identical readings of air temperature, wind speed, and wind direction. Mean differences in air temperature data during the tests were lower than 0.20 ◦C (maximum STD = 0.16 ◦C). For comparison purposes, one VPP station was run close to the AWS of La Mare Glacier in summer 2009, revealing mean differences in air temperature readings of 0.10 ◦C (STD = 0.12 ◦C). A further comparison was car- ried out in the summers of 2007 and 2008, running a VPP station close to the HOBO Pro data logger and close to a temperature sensor of the regional weather service installed at Careser Diga. These two instruments, which have natu- ral ventilation systems, showed mean differences of 0.10 ◦C (STD = 0.40 ◦C) and 0.23 ◦C (STD = 0.66 ◦C), respectively, compared to the aspirated VPP station. Based on these re- sults, no corrections were applied to the measured air tem- peratures. 3.2 Data processing and accuracy assessment For our analyses, hourly means were calculated from sub- hourly meteorological data. After being synchronized with local solar time, the data were checked for possible gaps, outliers, and inhomogeneities. The major gap concerned a few days in summer 2011 in the precipitation data at Careser Diga, which was ﬁlled using the manual observa- tions recorded by the personnel of the local hydropower com- pany. Other gaps of 1–2 h occurred during the maintenance of weather stations and were ﬁlled by linear interpolation. The spatial density and type of weather stations used in this study were decided based on (i) the pre-existing net- work of regional AWSs and (ii) the logistic constraints af- fecting the access to the glaciers and limiting the number of research-grade AWSs which could be deployed. These limi- The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 3.4 Calculation of on-glacier temperature from off-site data where T0 (◦C) is the temperature at x = 0, Teq (◦C) is deﬁned as the “equilibrium temperature”, x0 and z0 (m) are the loca- tion and elevation where the air enters the glacier-wind layer, Tcs (◦C) and zcs(m) are the temperature and the elevation at the off-glacier weather station, γ (◦Cm−1) is the ambient lapse rate, H (m) is the height of the glacier wind layer, α (◦) is the glacier slope, CH is the bulk transfer coefﬁcient for heat, and 0d is the dry adiabatic lapse rate (−0.0098 ◦Cm−1). The potential temperature is converted into temperature by means of The measured on-glacier temperatures served for testing the procedures suggested by Shea and Moore (2010) and Greuell and Böhm (1998) (from now on “S&M” and “G&B”, respec- tively) for calculating the air temperature distribution over glacierized surfaces. The empirical methods by Khodakov (1975), Davidovich and Ananicheva (1996), and Braithwaite et al. (2002) were not tested because they are more empiri- cal, the coefﬁcients were calculated in very different environ- ments, and they do not take into account the temporal vari- ability of the cooling effect. (10) T (x,z) = 2(x) −0d [z(x = 0) −z(x)], (10) S&M suggested the use of a piecewise regression model: where z(x) is the surface proﬁle of the glacier. Tg (x,t) = T1 + k2 (Ta −T ∗), Ta ≥T ∗ T1 −k1 (T ∗−Ta), Ta < T ∗, (1) (1) For both methods, the original formulations and param- eters were tested unchanged against our experimental data, evaluating also possible modiﬁcations as detailed in Sect. 4. The efﬁciency was evaluated by means of three different statistics: (i) the mean error (ME), (ii) the root mean square error (RMSE), and (iii) the efﬁciency criterion by Nash and Sutcliffe (N&S, 1970). The topographic information required to apply these methods was extracted from a 2m×2m DEM surveyed by LiDAR in late summer of 2006. A map of the FPL was calculated from this DEM, using algorithms de- veloped for drainage area calculations (Fig. 2, Tarboton et al., 1991). where Tg(x,t) (◦C) is the on-glacier temperature for site x at time t, T ∗(◦C) represents a threshold ambient temper- ature for KBL effects on Tg, T1 (◦C) is the corresponding on-glacier threshold temperature, and k2 (k1) is the so-called sensitivity of on-glacier temperature to ambient temperature (Ta, ◦C) changes when Ta is above (below) T ∗. 3.3 Analysis of ﬁeld data with T0 = Tcs −γ (zcs −z0), (6) Teq = bLR, (7) LR = H cos(α) CH , (8) b = 0d tan(α), (9) (8) (9) (8) (9) L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale elevation model (DEM): www.the-cryosphere.net/9/1129/2015/ www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1134 3.3 Analysis of ﬁeld data T ∗= β1 + β2Z, (2) k1 = β3 exp(β4FPL), (3) k2 = β5 + β6 exp(β7FPL), (4) T ∗= β1 + β2Z, (2) k1 = β3 exp(β4FPL) (3) T ∗= β1 + β2Z, (2) (2) (3) (4) T ∗= β1 + β2Z, (2) k1 = β3 exp(β4FPL), (3) The meteorological data collected by the weather stations were ﬁrstly analyzed by calculating descriptive statistics for each of the two summers 2010 and 2011 and focusing on vertical lapse rates. Afterwards, the data were analyzed at hourly resolution focusing on the calculation of ambient (i.e., off-glacier) temperature, which is crucial for estimat- ing on-glacier near-surface temperatures and is required by all methods proposed in the literature for this purpose. More- over, the correct estimation of the ambient temperature is an essential prerequisite for quantifying the site-speciﬁc cool- ing effect on glaciers, which is deﬁned as “the difference between screen-level temperatures over glaciers compared to equivalent-altitude temperatures in the free atmosphere” (Braithwaite, 1980). Different combinations of lapse rates (i.e., ﬁxed standard or hourly variable obtained by linear re- gression of temperature vs. elevation) and subsets of weather stations were tested (see details in Sect. 4.2). T β1 + β2Z, (2) k1 = β3 exp(β4FPL), (3) k1 = β3 exp(β4FPL), (3) k2 = β5 + β6 exp(β7FPL), (4) (4) where βi are the coefﬁcients of the transfer functions, Z (m) is the elevation, and FPL (m) is the ﬂow path length, deﬁned as “the average ﬂow distance to a given point starting from an upslope summit or ridge” (Shea and Moore, 2010). T1 is calculated as T ∗· k1. The G&B model assumes the presence of a katabatic wind, and therefore it applies when the ambient temperature is higher than the surface temperature. In these conditions the potential temperature 2 (◦C) at the distance x along the ﬂow line (x = 0 at the top of the ﬂow line) is calculated as 2(x) = T0 −Teq exp −x −x0 LR −b(x + x0) + Teq, (5) (5) The spatial and temporal variability of the cooling effect were then investigated, plotting the average diurnal cycle of the cooling effect vs. average cycles of wind speed and direc- tion and drawing charts of the daily average cooling effect vs. daily temperature and precipitation recorded at Careser Diga, in order to assess the role of different weather types in the glacial temperature regimes. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1135 Figure 2. Map of the ﬂow path length calculated for Careser and La Mare glaciers. in EISModel. In the present work we use the additive melt al- gorithm, which explicitly separates the thermal and radiative components: MLTX,t = TMF · TX,t + RMF · CSRX,t 1 −αX,t , (11) (11) where TMF and RMF are empirical coefﬁcients called the temperature melt factor (mmh−1 ◦C−1) and the radiation melt factor (mmh−1 W−1 m2), TX,t (◦C) is the air temper- ature at pixel X in hour t, CSRX,t (Wm−2) is the clear sky shortwave radiation, and αX,t is the surface albedo (spa- tially variable for ice and spatially and temporally variable for snow). For a detailed description of the model, we refer the reader to the work of Carturan et al. (2012a). where TMF and RMF are empirical coefﬁcients called the temperature melt factor (mmh−1 ◦C−1) and the radiation melt factor (mmh−1 W−1 m2), TX,t (◦C) is the air temper- ature at pixel X in hour t, CSRX,t (Wm−2) is the clear sky shortwave radiation, and αX,t is the surface albedo (spa- tially variable for ice and spatially and temporally variable for snow). For a detailed description of the model, we refer the reader to the work of Carturan et al. (2012a). The cumulated mass balance measured at ablation stakes drilled in close proximity to the glacial weather stations (AWS and VPP) served for model calibration and valida- tion. We used alternatively each of the two summer sea- sons of 2010 and 2011 as an independent data set for cali- bration/validation. Point-based EISModel calculations at the weather stations were run, using four temperature series: (i) measured data, (ii) calculated temperature from Careser Diga via the standard ambient lapse rate (−6.5 ◦Ckm−1), (iii) calculated temperature according to the S&M method, and (iv) calculated temperature according to the G&B method. Option (ii) is commonly used in the absence of tem- perature data from glaciers (e.g., Gardner and Sharp, 2009; Michlmayr et al., 2008; Nolin et al., 2010). Figure 2. Map of the ﬂow path length calculated for Careser and La Mare glaciers. 4.1 Seasonal characteristics of temperature data A close dependency on altitude has been detected for mean summer air temperature, both outside the glaciers and, remarkably, over them (Table 2, Fig. 3). Because of thermal inversions occurring at the lowermost weather station (Cog_1202) during the night and early morning, the vertical lapse rate was much steeper above Car_2607 (−8.0 ◦Ckm−1 in 2010 and −8.3 ◦Ckm−1 in 2011) than be- low (−5.3 ◦Ckm−1 in 2010 and −5.2 ◦Ckm−1 in 2011). At a given altitude, the on-glacier air temperature was systemati- cally lower than ambient temperature, the difference decreas- ing with altitude. Lapse rates were also lower on the glaciers (−7.2 ◦Ckm−1 in 2010 and −6.7 ◦Ckm−1 in 2011), com- pared to high-altitude off-glacier weather stations, and close to the standard ambient lapse rate (−6.5 ◦Ckm−1). Much shallower on-glacier lapse rates and fewer dependency of air temperature on elevation were found by earlier works (e.g., Greuell and Böhm, 1998; Strasser et al., 2004; Petersen et al., 2013). As reported in Table 2, the average daily temper- ature range and the average standard deviation are largest at the valley ﬂoor and both decrease with altitude, reach- ing their minima over the glaciers as previously reported, for example, by Oerlemans (2001). Hourly temperatures among www.the-cryosphere.net/9/1129/2015/ 3.4 Calculation of on-glacier temperature from off-site data Empirical transfer functions were obtained by S&M, relating the ﬁtted coefﬁcients (T ∗, k1, and k2) for each weather station used in their work to topographic attributes extracted from a digital www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Lines indicate linear regressions of temperature vs. altitude for subsets of weather stations. LR = vertical lapse rates. Table 2. Descriptive statistics for air temperature data recorded by the weather stations. On-glacier sites are in bold type. Weather Minimum Maximum Mean Standard Mean station deviation daily range Summer 2010 Mar-gl_2709 −1.9 14.2 5.9 3.3 2.2 Mar-gl_2973 −4.4 11.6 3.8 3.1 2.5 Mar-gl_3215 −6.6 10.6 2.2 3.4 2.9 Cog_1202 2.3 29.8 14.8 5.5 10.2 Car_2607 −2.4 18.4 7.3 4.1 4.6 Car_3051 −5.6 14.1 3.9 4.0 2.8 Bel_3328 −10.5 13.9 1.5 4.5 3.6 Summer 2011 Mar-gl_2973 −4.8 12.0 4.3 2.7 2.6 Mar-gl_3140 −6.2 9.7 3.3 2.8 2.1 Mar-gl_3438 −7.9 9.5 1.1 3.1 3.2 Car-gl_3082 −6.0 10.8 3.3 2.9 2.6 Car-gl_3144 −6.1 10.9 3.5 3.1 2.3 Cog_1202 4.0 29.8 15.4 4.9 10.5 Car_2607 −0.9 19.5 8.1 3.6 4.9 Car_3051 −5.3 13.7 4.6 3.5 2.8 Bel_3328 −8.2 13.5 2.1 3.8 3.5 Table 2. Descriptive statistics for air temperature data recorded by the weather stations. On-glacier sites are in bold type. 4.2 Ambient temperature calculation For the calculation of ambient temperature at the altitude of glaciers, which is crucial for the quantiﬁcation of the glacier cooling effect, we tested the following methods: (i) use of a ﬁxed standard ambient lapse rate (−6.5 ◦Ckm−1), (ii) use of a ﬁxed calibrated lapse rate (seasonal mean value), and (iii) use of an hourly variable lapse rate. Methods (ii) and (iii) were implemented using different combinations of off-glacier weather stations, calculating linear regressions of hourly temperature vs. altitude. The methods were tested re- moving alternatively Car_3051 or Bel_3328 from linear re- gressions and using them for validation. The results, dis- played in Table 3, show that regardless of the method used, the inclusion of the lowermost weather station gives poorer results. At Car_3051, the method (iii) applied to Car_2607 and Bel_3328 works best, indicating that in our case hourly variable lapse rates are the most appropriate solution while interpolating temperatures between two weather stations. Conversely, method (ii) applied to Car_2607 and Car_3051 provides the best results at Bel_3328, which suggests that a ﬁxed calibrated lapse rate should be used while extrapolating above the uppermost station, although uncertainty persists in these cases. Figure 3. Mean temperature vs. altitude: (a) from 3 July to 23 Figure 3. Mean temperature vs. altitude: (a) from 3 July to 23 September, 2010, and (b) from 7 July to 12 September, 2011. Lines indicate linear regressions of temperature vs. altitude for subsets of weather stations. LR = vertical lapse rates. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1136 Table 2. Descriptive statistics for air temperature data recorded by the weather stations. On-glacier sites are in bold type. Weather Minimum Maximum Mean Standard Mean station deviation daily range Summer 2010 Mar-gl_2709 −1.9 14.2 5.9 3.3 2.2 Mar-gl_2973 −4.4 11.6 3.8 3.1 2.5 Mar-gl_3215 −6.6 10.6 2.2 3.4 2.9 Cog_1202 2.3 29.8 14.8 5.5 10.2 Car_2607 −2.4 18.4 7.3 4.1 4.6 Car_3051 −5.6 14.1 3.9 4.0 2.8 Bel_3328 −10.5 13.9 1.5 4.5 3.6 Summer 2011 Mar-gl_2973 −4.8 12.0 4.3 2.7 2.6 Mar-gl_3140 −6.2 9.7 3.3 2.8 2.1 Mar-gl_3438 −7.9 9.5 1.1 3.1 3.2 Car-gl_3082 −6.0 10.8 3.3 2.9 2.6 Car-gl_3144 −6.1 10.9 3.5 3.1 2.3 Cog_1202 4.0 29.8 15.4 4.9 10.5 Car_2607 −0.9 19.5 8.1 3.6 4.9 Car_3051 −5.3 13.7 4.6 3.5 2.8 Bel_3328 −8.2 13.5 2.1 3.8 3.5 Figure 3. Mean temperature vs. altitude: (a) from 3 July to 23 September, 2010, and (b) from 7 July to 12 September, 2011. Lines indicate linear regressions of temperature vs. altitude for subsets of weather stations. LR = vertical lapse rates. different weather stations in Val de La Mare were highly cor- related (r > 0.9, signiﬁcant at the 0.001 level), with the re- markable exception of Cog_1202, at the valley ﬂoor, whose correlation with the other weather stations ranged from 0.65 to 0.75, peaking at 0.84 with Car_2607. Table 2. Descriptive statistics for air temperature data recorded by the weather stations. On-glacier sites are in bold type. Weather Minimum Maximum Mean Standard Mean station deviation daily range Summer 2010 Mar-gl_2709 −1.9 14.2 5.9 3.3 2.2 Mar-gl_2973 −4.4 11.6 3.8 3.1 2.5 Mar-gl_3215 −6.6 10.6 2.2 3.4 2.9 Cog_1202 2.3 29.8 14.8 5.5 10.2 Car_2607 −2.4 18.4 7.3 4.1 4.6 Car_3051 −5.6 14.1 3.9 4.0 2.8 Bel_3328 −10.5 13.9 1.5 4.5 3.6 Summer 2011 Mar-gl_2973 −4.8 12.0 4.3 2.7 2.6 Mar-gl_3140 −6.2 9.7 3.3 2.8 2.1 Mar-gl_3438 −7.9 9.5 1.1 3.1 3.2 Car-gl_3082 −6.0 10.8 3.3 2.9 2.6 Car-gl_3144 −6.1 10.9 3.5 3.1 2.3 Cog_1202 4.0 29.8 15.4 4.9 10.5 Car_2607 −0.9 19.5 8.1 3.6 4.9 Car_3051 −5.3 13.7 4.6 3.5 2.8 Bel_3328 −8.2 13.5 2.1 3.8 3.5 Figure 3. Mean temperature vs. altitude: (a) from 3 July to 23 September, 2010, and (b) from 7 July to 12 September, 2011. 3.5 Mass balance modeling The impact that the calculation of on-glacier temperatures ac- cording to different methods has on mass balance modeling was assessed using EISModel (Cazorzi and Dalla Fontana, 1996), which was already applied to Careser and La Mare glaciers by Carturan et al. (2012a). EISModel employs an enhanced temperature-index approach for computing melt, using the clear-sky shortwave radiation calculated from the DEM as a distributed morpho-energetic index. The model, which is suitable for applications on glaciers with limited data availability, does not require incoming shortwave radia- tion measurements, which are less commonly available than air temperature and precipitation. We tested the effect of in- cluding daily cloudiness in our mass balance calculations, computing it from incoming shortwave radiation measured at Mar-gl_2973, as detailed in Pellicciotti et al. (2005). As no signiﬁcant changes were obtained in the efﬁciency statis- tics, we assumed that the daily cloudiness could be omitted in mass balance calculations. Three melt algorithms (multiplicative, additive, and ex- tended) have been implemented and can be used alternatively www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 4.3 The glacier cooling effect The cooling effect at each on-glacier weather station was cal- culated as the difference between the measured temperature and the ambient temperature at the same elevation, computed on the basis of the results described in Sect. 4.2 (i.e., hourly variable lapse rate below Bel_3328 and ﬁxed calibrated lapse rate above it). The average seasonal cooling effect (Table 4) was maximal at Car-gl_3082 (−1.01 ◦C in 2011) and at Mar- gl_2973 (−0.74 ◦C in 2010 and −0.90 ◦C in 2011). Null or negligible cooling was detected at Mar-gl_3438, close to the top of La Mare Glacier, and at Car-gl_3144 on the small Careser Occidentale Glacier. Minor cooling occurred at Mar-gl_3215 (−0.27 ◦C in 2010), which was close to the balanced-budged ELA of the glacier, and at Mar-gl_3140 (−0.47 ◦C in 2011), in the upper ablation area. Notably, the narrow and steep terminus of La Mare Glacier experienced a signiﬁcant cooling effect in 2010 (−0.65 ◦C). Figure 4 reports the mean daily cycles of the cooling ef- fect and wind regime. A common pattern emerges, with min- imum cooling at night and maximum cooling around noon or in the afternoon, coherent with the diurnal cycle of am- bient air temperature and deriving temperature differences from the glacier surface. For ﬁve out of the seven moni- Figure 3. Mean temperature vs. altitude: (a) from 3 July to 23 September, 2010, and (b) from 7 July to 12 September, 2011. Lines indicate linear regressions of temperature vs. altitude for subsets of weather stations. LR = vertical lapse rates. The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Table 3. Validation statistics for ambient temperature calculations (global data set including summer 2010 and 2011)∗. 4.3 The glacier cooling effect Lapse rate Used weather Calculation of air temperature Calculation of air temperature (◦Cm−1) stations at Car_3051 at Bel_3328 Mean error RMSE N&S Mean error RMSE N&S (◦C) (◦C) index (◦C) (◦C) index Moist adiabatic lapse rate −0.0065 1 −1.14 3.81 −0.019 −0.51 3.59 0.276 −0.0065 2 0.59 1.32 0.878 1.22 2.02 0.771 −0.0065 3 \ \ \ 0.63 1.46 0.880 −0.0065 4 −0.63 1.46 0.851 \ \ \ Fixed calibrated lapse rate −0.0053 1, 2 1.13 1.64 0.812 2.11 2.65 0.605 −0.0059 1, 3 \ \ \ 0.81 1.54 0.866 −0.0063 1, 4 −0.70 1.49 0.845 \ \ \ −0.0078 2, 3 \ \ \ 0.27 1.34 0.899 −0.0082 2, 4 −0.17 1.32 0.877 \ \ \ −0.0057 1, 2, 3 \ \ \ 0.85 1.56 0.863 −0.0061 1, 2, 4 −0.74 1.51 0.841 \ \ \ Hourly variable lapse rate Hourly variable 1, 2 1.13 1.55 0.831 2.11 2.89 0.529 Hourly variable 1, 3 \ \ \ 0.81 1.74 0.830 Hourly variable 1, 4 −0.70 1.51 0.840 \ \ \ Hourly variable 2, 3 \ \ \ 0.27 1.64 0.849 Hourly variable 2, 4 −0.17 1.01 0.929 \ \ \ Hourly variable 1, 2, 3 \ \ \ 0.85 1.76 0.826 Hourly variable 1, 2, 4 −0.74 1.55 0.831 \ \ \ ∗Weather stations: 1 = Cog_1202, 2 = Car_2607, 3 = Car_3051, 4 = Bel_3328. N&S index is the efﬁciency criterion according to Nash and Sutcliffe (1970). Bold type indicates the best results for each tested method. 1137 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale ∗Weather stations: 1 = Cog_1202, 2 = Car_2607, 3 = Car_3051, 4 = Bel_3328. N&S index is the efﬁciency criterion according to Nash and Sutcliffe (1970). Bold type indicates the best results for each tested method. Table 4. Mean values of cooling effect, wind speed, and wind di- rection recorded at the on-glacier weather stations. Table 4. Mean values of cooling effect, wind speed, and wind di- rection recorded at the on-glacier weather stations. cooling. Down-glacier winds dominated on La Mare Glacier, with higher speeds compared to Careser Occidentale and Ori- entale glaciers where up-glacier winds prevailed. The wind speed was at its maximum at night on La Mare, especially in 2010, while it was at its maximum in the afternoon on the two Careser glaciers. 4.3 The glacier cooling effect A peculiar behavior was found at the terminus of La Mare Glacier (Mar-gl_2709), where down- glacier winds dominated at night, without a cooling effect, and were replaced by up-glacier winds from mid-morning to late afternoon, when the cooling effect increased sharply. Wind data were not available at Mar-gl_3438 due to instru- mentation failure, but we can argue that katabatic winds were not prevalent at this site, which is close to the crest, based on results published for similar locations in previous works (e.g., Greuell et al., 1997; Strasser et al., 2004). Weather station Mean cooling Mean wind effect speed direction (◦C) (ms−1) (◦) Summer 2010 Mar-gl_2709 −0.65 2.00 247 Mar-gl_2973 −0.74 3.13 230 Mar-gl_3215 −0.27 3.47 258 Summer 2011 Mar-gl_2973 −0.90 2.82 224 Mar-gl_3140 −0.47 3.00 239 Mar-gl_3438 0.06 \ \ Car-gl_3082 −1.01 2.40 249 Car-gl_3144 −0.18 1.98 90 Different weather conditions led to a considerable tempo- ral variability of the glacier cooling effect during the two summer seasons of 2010 and 2011 (Fig. 5). Cooling was maximal during warm anticyclonic periods and nearly absent during cold unsettled weather. Differences among sites in- creased with warmer temperatures, whereas they nearly dis- appeared during cold and unstable periods. The highest vari- tored sites, the cooling occurred almost exclusively during daytime. Nighttime cooling took place only at Mar-gl_2973 and Car-gl_3082, which are the two sites with higher mean tored sites, the cooling occurred almost exclusively during daytime. Nighttime cooling took place only at Mar-gl_2973 and Car-gl_3082, which are the two sites with higher mean The Cryosphere, 9, 1129–1146, 2015 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Ceveda 1138 Figure 4. Mean daily cycle of the glacier cooling effect (δT ), wind direction, and wind speed at the eight on-glacier weather stations. The operation period of each station is indicated in parentheses. Down-glacier and up-glacier wind directions are indicated with straight lines marked with “D” and “U”. Mar-gl_3438 lacks wind data because of anemometer failure. Figure 4. Mean daily cycle of the glacier cooling effect (δT ), wind direction, and wind speed at the eight on-glacier weather stations. Th operation period of each station is indicated in parentheses Down glacier and up glacier wind directions are indicated with straight line Figure 4. Mean daily cycle of the glacier cooling effect (δT ), wind direction, and wind speed at the eight on-glacier weather stations. The operation period of each station is indicated in parentheses. Down-glacier and up-glacier wind directions are indicated with straight lines marked with “D” and “U”. Mar-gl_3438 lacks wind data because of anemometer failure. which uses the FPL (m) rather than elevation as a predic- tor, thus being potentially more generalizable. Neither the outlier already excluded by S&M nor Mag-gl_2709 was included in our calculation of Eq. (12) due to undersam- pling at below-zero temperatures. Figure 6 shows data points, transfer functions, and parameters. Calculated vs. measured temperature is shown in Fig. 7 along with related statis- tics. Four out of the ﬁve sites where the method works sat- isfactorily (ME < 0.5 ◦C in absolute value and N&S index > 0.87) have prevailing katabatic winds. Contrarily, lower performance affects sites close to the glacier margin (Mar- gl_3438 and, in particular, Mar-gl_2709), where katabatic winds are disrupted by valley winds or synoptic winds, and Car-gl_3082, where up-glacier winds prevail. The efﬁciency statistics for all sites are ME = −0.06 ◦C, RMSE = 0.73 ◦C, and N&S = 0.692. ations occurred at Mar-gl_2973, Mar-gl_3215, Mar-gl_3140, and Car-gl_3082, while at Mar-gl_3438 and Car-gl_3144 there was a smaller temporal variability. A warming, rather than cooling, effect was observed on some days, mainly at the upper weather stations of La Mare Glacier. A close check on the wind and temperature data revealed that this was ascrib- able to local föhn conditions, that is, forced adiabatic heating brought by strong northerly winds. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1139 . Ca tu a et a .: te pe atu e va ab ty ove t ee g p y g Figure 5. Mean daily cooling effect at the on-glacier weather sta- tions and corresponding daily precipitation and mean temperature at Careser Diga (Car_2607). Figure 6. Transfer functions for the coefﬁcients K1, K2, and T ∗of the Shea and Moore (2010) method. CMBC is the S&M study area; AVDM is our study area. Outliers due to undersampling at freezing temperatures have been removed (as in the S&M work). β3 to β7 fﬁi f S&M (J M Sh l i i Figure 6. Transfer functions for the coefﬁcients K1, K2, and T ∗of the Shea and Moore (2010) method. CMBC is the S&M study area; AVDM is our study area. Outliers due to undersampling at freezing temperatures have been removed (as in the S&M work). β3 to β7 are coefﬁcients from S&M (J. M. Shea, personal communication, 2014), while the transfer function and coefﬁcients for T ∗are new results from the present work. Figure 6. Transfer functions for the coefﬁcients K1, K2, and T ∗of the Shea and Moore (2010) method. CMBC is the S&M study area; AVDM is our study area. Outliers due to undersampling at freezing temperatures have been removed (as in the S&M work) β3 to β7 Figure 6. Transfer functions for the coefﬁcients K1, K2, and T ∗of the Shea and Moore (2010) method. CMBC is the S&M study area; AVDM is our study area. Outliers due to undersampling at freezing temperatures have been removed (as in the S&M work). β3 to β7 are coefﬁcients from S&M (J. M. Shea, personal communication, 2014), while the transfer function and coefﬁcients for T ∗are new results from the present work. Figure 5. Mean daily cooling effect at the on-glacier weather sta- tions and corresponding daily precipitation and mean temperature at Careser Diga (Car_2607). displayed in Fig. 5 of the Greuell and Böhm (1998) paper. The results are shown in Fig. 8 and indicate a fairly good alignment of our data with the other glaciers’ data and with the best ﬁt calculated by G&B for the Pasterze weather sta- tions. It therefore seemed appropriate to use the values of x0 and LR calculated by those authors, that is, 1440 and 8340 m, respectively. 4.4 Calculation of on-glacier temperature from off-site data According to the S&M method, piecewise linear regressions of on-glacier hourly temperature vs. ambient temperature at the same elevation have been calculated for each glacial weather station. The values of the parameters k1 and k2 (i.e., temperature sensitivities for ambient temperatures below and above the threshold temperature T ∗, respectively) were well aligned with the transfer functions proposed by S&M, using the FPL as predictor (Fig. 6). However, the transfer function for T ∗suggested by S&M, using station elevation as a pre- dictor, could not be used in AVDM given the different geo- graphic and climatic setting of the two study areas. We there- fore propose to substitute Eq. (2) with the following function: According to the G&B method, the location x0 where the air enters the glacier wind layer and the length scale LR can be calculated by an exponential function which expresses the “climatic sensitivity” in function of the distance x along the ﬂow line: dT (x) dTcs = exp −x + x0 LR . (13) (13) Climatic sensitivities were calculated, comparing daily mean temperature at our on-glacier sites to daily mean temperature at Car_3051 and have been added for comparison to the data T ∗= a · FPL b + FPL, (12) T ∗= a · FPL b + FPL, T ∗= a · FPL b + FPL, (12) The Cryosphere, 9, 1129–1146, 2015 The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale According to the G&B procedure, the hourly temperature above the freezing level was set equal to the am- bient temperature (Sect. 4.2). Below the freezing level, the glacier-wind model of G&B was applied, setting (i) x0 = 0 when the freezing level was below the top of the ﬂow line and (ii) x0 = 1440 m when it was above this point in order to take into account a climate sensitivity < 1 at the top of the ﬂow line. z0 was set equal to the freezing level in case (i) and equal to the altitude of the top of the ﬂow line in case (ii). These settings are the same as those used in the G&B paper. Nevertheless, no corrections were applied to the computed temperatures, as was done by G&B, who applied a ﬁxed off- set of −0.74 ◦C. Figure 9 displays the results of the G&B method. Calcu- lated temperatures matched the measured temperatures fairly 4.5 Mass balance modeling EISModel applications using measured temperature data sets resulted in RMSE values well below the mass balance mea- surement error from ablation stakes readings (∼200 mm w.e., Thibert et al., 2008; Huss et al., 2009), thus conﬁrming the good skill of the modeling tool. However, the RMSE was nearly double when calculated temperature data sets were used as input, and considerable differences also exist in the calibration parameters (Table 5). The spatial distribution of modeling errors using temper- ature extrapolations from Car_2607 via the standard lapse rate (Fig. 10, scatterplots b1–b4) replicated the ﬁndings of Carturan et al. (2012a) for the 6 previous years (2004–2009). In particular, the modeled vertical gradient of mass balance on La Mare Glacier in summer 2010 was lower than the ob- served one, in both calibration and validation runs, due to un- even errors in estimating air temperature (+0.77, +1.17, and +1.14 ◦C at Mar-gl_2709, Mar-gl_2973, and Mar-gl_3215, respectively). This data set of overestimated temperatures led to signiﬁcantly lower calibration parameters compared to the measured temperature data set. Moreover, including critical points close to the lower margin of the glacier (Mar-gl_2709 in summer 2010) led to wrong calibration at the other two points, which are likely to have a higher spatial representa- tiveness given the larger distance from the glacier margin. Figure 8. Sensitivity of on-glacier temperature to temperature out- side the thermal inﬂuence of glaciers and best ﬁt of Eq. (13) to Pasterze data. Redrawn ﬁgure from Greuell and Böhm (1998). Val- ues measured on Careser and La Mare glaciers (AVDM) have been added for comparison. Mar-gl_2973: two overlapping points (sum- mer 2010 and 2011 have identical sensitivity). Figure 8. Sensitivity of on-glacier temperature to temperature out- side the thermal inﬂuence of glaciers and best ﬁt of Eq. (13) to Pasterze data. Redrawn ﬁgure from Greuell and Böhm (1998). Val- ues measured on Careser and La Mare glaciers (AVDM) have been added for comparison. Mar-gl_2973: two overlapping points (sum- mer 2010 and 2011 have identical sensitivity). well and the efﬁciency statistics for all sites were better than for the S&M method: ME = −0.27 ◦C, RMSE = 0.40 ◦C, N&S = 0.908. Improvements were observed, in particular, at Mar-gl_2709, Car-gl_3082, and Mar-gl_3438, even if these sites lack predominant katabatic winds. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1140 Figure 7. On-glacier temperature calculated with the Shea and Moore (2010) method vs. observed temperature. Figure 7. On-glacier temperature calculated with the Shea and Moore (2010) method vs. observed temperature. Figure 8. Sensitivity of on-glacier temperature to temperature out- side the thermal inﬂuence of glaciers and best ﬁt of Eq. (13) to Pasterze data. Redrawn ﬁgure from Greuell and Böhm (1998). Val- ues measured on Careser and La Mare glaciers (AVDM) have been added for comparison. Mar-gl_2973: two overlapping points (sum- mer 2010 and 2011 have identical sensitivity). 4.5 Mass balance modeling A clear step is ob- servable at Mar-gl_2709 and, slightly less obvious, at Mar- gl_2973 in both summer 2010 and 2011, attributable to the jump of x0 from 0 to 1440 m when the freezing level exceeds the top of the ﬂow line. The calibration parameters obtained with the G&B tem- perature data set were closer to those obtained with the mea- sured temperature data set, as could be expected given the smaller errors in temperature estimations (Fig. 9). In sum- mer 2010, modeling results with the G&B temperature data set were also the best among the three tested methods for air temperature calculation, in both calibration and validation runs. The same cannot be stated for summer 2011 due to the larger temperature underestimation at Mar-gl_3140 and Car- www.the-cryosphere.net/9/1129/2015/ www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 The Cryosphere, 9, 1129–1146, 2015 The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Figure 9. On-glacier temperature calculated with the G&B method vs. observed temperature. 1141 Figure 9. On-glacier temperature calculated with the G&B method vs. observed temperature. of weaker local winds and more relevant entrainment of syn- optic winds have been hypothesized, for example, by Ayala et al. (2015), for glaciers without a well-deﬁned tongue. On the other hand, although katabatic ﬂows were generally ab- sent, this site was the coldest in summer 2011, exhibiting a mean depression of 1 ◦C compared to the ambient tempera- ture (Table 4). In addition, during warm anticyclonic periods it displayed a cooling effect similar to Mar-gl_2973 and Mar- gl_3140, located in the middle part of La Mare Glacier. This is unusual for locations close to the top of glacier ﬂow lines, which normally display a low cooling effect and high tem- perature sensitivity (e.g., Greuell and Böhm, 1998; Shea and Moore, 2010; Petersen et al., 2013). The efﬁcient cooling at Car-gl_3082 could have been caused by the combination of adiabatic cooling of ascending air and cooling by loss of sen- sible heat due to the rather long fetch (780 m from the lower edge of the glacier), whereas in katabatic ﬂows the loss of sensible heat is to some extent compensated by the adiabatic heating of descending air (Greuell and Böhm, 1998). gl_3144. Similar errors occurring at Mar-gl_3438 did not im- pact mass balance estimations because they mainly happened at below-zero temperatures (Fig. 9). The S&M temperature data set led to the worst results in summer 2010 due to the strong underestimation of air tem- perature at Mar-gl_2709 (−1.6 ◦C). Calibrated parameters in 2010 were thus overestimated and led to mass balances that were too negative, on average, in 2011. On the contrary, when used for calibration, the data of 2011 led to parameters much closer to the measured temperature data set, leading to cor- rect mass balance estimations in summer 2010 with the ex- ception of the already mentioned Mar-gl_2709. 5 Discussion The temperature distribution and wind regime were found to be remarkably different for the three investigated glaciers (Tables 2 and 4, Fig. 4). The most signiﬁcant differences were detected between La Mare Glacier, where the KBL and the cooling effect were clearly recognizable, and Careser Oc- cidentale Glacier, where the air temperature was not signiﬁ- cantly different from the ambient temperature and where pre- vailing up-glacier winds (i.e., valley winds) dominated. Dif- ferences were even more prominent during warm and stable weather (Fig. 5), brought by persistent anticyclonic systems (as detected by inspection of reanalysis weather charts from www.wetterzentrale.de, last access: 31 October 2014). The behavior of the two weather stations on Careser Oc- cidentale and Orientale glaciers provides evidence of the re- duced effectiveness of small glaciers (deriving from the frag- mentation of larger glaciers) in cooling the air above com- pared to wider glaciers or wider portions of the same parent glacier. This is suggested by the fact that these two weather stations (Car-gl_3082 and Car-gl_3144), despite being at al- most the same ﬂow path distance from the upper glacier margin (Table 1, Fig. 2), have very different cooling effects (Table 4, Fig. 4) which largely explain errors in modeled ab- lation rates (Fig. 10; Fig. 8 from Carturan et al., 2012a). The Car-gl_3082 site, on Careser Orientale Glacier, also displayed peculiar conditions compared to most weather sta- tions operated on La Mare Glacier. On the one hand, a pre- vailing up-glacier wind was recognized, but it cannot be at- tributed unequivocally to valley winds because the direc- tion roughly corresponds to prevailing synoptic winds in the Ortles–Cevedale area (Gabrieli et al., 2011). The occurrence In consideration of the high number and contribution to the world’s total ice volume of smaller glaciers (Haeberli et al., 1989; Paul et al., 2004; Zemp et al., 2008; Bahr and Radi´c, 2012), and given the absence of previous experimental data from such small ice bodies these results provide a ﬁrst L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1142 L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Figure 10. Measured vs. modeled mass balance at the eight glacial weather stations, using EISModel with four different air temperature inputs: (a1–a4) measured, (b1–b4) extrapolated from Car_2607 via the standard lapse rate (−6.5 ◦Ckm−1), (c1–c4) calculated via the G&B method, and (d1–d4) calculated via the S&M method. Corresponding statistics are reported in Table 5. Figure 10. Measured vs. modeled mass balance at the eight glacial weather stations, using EISModel with four different air temperature inputs: (a1–a4) measured, (b1–b4) extrapolated from Car_2607 via the standard lapse rate (−6.5 ◦Ckm−1), (c1–c4) calculated via the G&B method, and (d1–d4) calculated via the S&M method. Corresponding statistics are reported in Table 5. quantiﬁcation for an important reinforcing mechanism dur- ing glacier decay, that is, the disintegration of parent glaciers into smaller units, which have reduced effectiveness in cool- ing the air above and in triggering katabatic ﬂows. Clearly, these results are not conclusive and require further experi- mental data to assess their generalizability and to develop generalized strategies for calculating air temperature over glaciers with similar characteristics, to be implemented in distributed mass balance models. weather station. An even more unusual behavior was mea- sured at Mar-gl_2709, close to the terminus of the glacier. Here the cooling effect was detected only during daytime, with valley winds prevailing over katabatic winds, while at night the adiabatic heating of the air descending the steep tongue prevailed over the cooling due to turbulent exchanges. Besides the physical characteristics of the glacier, however, the steep lapse rates might also have been inﬂuenced by the steep lapse rate measured outside the thermal inﬂuence of glaciers. A clear dependency of air temperature on elevation was found on La Mare Glacier, where the weather stations were placed along a longitudinal proﬁle, exploring a large range of elevations (Fig. 3). The on-glacier lapse rate was steeper than the standard ambient lapse rate, unlike in previous works which mostly report shallower values ranging from −2.8 to −8.1 ◦Ckm−1 and averaging −4.9 ◦Ckm−1 (Petersen and Pellicciotti, 2011, and references cited therein; Petersen et al., 2013). The steep lapse rate measured on La Mare Glacier is likely due to its physical characteristics and to the spe- ciﬁc location of weather stations. www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1143 lower mean errors at Mar-gl_2709, Mar-gl_3438 and Car- gl_3082, compared to the S&M method, are coincidental be- cause at these sites the KBL was almost absent or not pre- vailing. ﬂoor sites, which are prone to thermal inversions and subject to high temperature oscillations during the day. The good alignment of our data points with the transfer functions of Shea and Moore (2010), which can be seen in Fig. 6, is remarkable given the different characteristics of glaciers and geographic setting of the two study areas. This result points to a good generalizability of the S&M method, which we have tried to improve by implementing a trans- fer function for T ∗based on the FPL rather than on eleva- tion. The S&M method was fairly successful at sites where the KBL was detected (Mar-gl_3140, Mar-gl_3215), that is, for the conditions under which the method has been imple- mented. Nevertheless, at Mar-gl_2973 it signiﬁcantly under- estimated the temperature, probably because it does not ac- count for gradients upslope of the weather station, which causes a local prevalence of adiabatic heating. A larger er- ror occurred at Mar-gl_2709, which is, however, inﬂuenced by valley winds and thermal emission from the surround- ing bare rocks, determining high temperature sensitivity and unusual T ∗at such a long FPL (2896 m, Fig. 6). With this method it was not possible to reproduce the temperature differences between Car-gl_3082 and Car-gl_3144, as ex- pected, because they have similar values of down-glacier FPL (313 and 354 m, respectively). g Other combinations of parameters x0 and LR have been tested to evaluate whether they are valid alternatives, for ex- ample for eliminating the artiﬁcial step in calculated vs. ob- served temperature at Mar-gl_2973 and Mar-gl_2709 (Fig. 9) caused by the jump of x0 from 0 to 1440 m when the freez- ing level exceeds the top of the ﬂow line. The tested com- binations were (i) x0 = 0 m (constant) and LR = 8340 m, (ii) x0 = 1440 m (constant) and LR = 8340 m, and (iii) x0 = 1835 m (constant) and LR = 12 682 m. The last combination results from the best ﬁt to AVDM data in Fig. 8, excluding the outlier Mar-gl_2709. We also tested the calculation us- ing the unmodiﬁed ambient temperature. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale For example, Mar-gl_2973, which is located 2.13 km downslope from the upper mar- gin of the glacier, displayed only a moderate cooling effect (−0.74 ◦C in 2010 and −0.90 ◦C in 2011) due to the presence of a steep slope causing adiabatic heating right above the g The speciﬁc reasons for the steepness of the high-altitude ambient lapse rates are not easy to identify. According to Marshall et al. (2007) and Minder et al. (2010), for exam- ple, they could have been caused by the prevailing synop- tic circulation, local energy balance regime, persistence of snow cover, or geographic position (windward or leeward with respect to the prevailing synoptic wind). Apart from these considerations, it has to be noted that the interpolation and extrapolation of ambient temperature at high altitudes, as a starting point for the computation of the on-glacier temper- ature ﬁelds, are strongly dependent on the availability and/or selection of suitable weather stations. As already suggested, e.g., by Oerlemans (2001), measurements from high-altitude weather stations are preferable to measurements from valley- The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale 1143 Table 5. Calibration parameters and mass balance statistics from EISModel applications with four different data sets of air temperature∗. Temperature Calibrated Calibration run Validation run data set parameters (summer 2010) (summer 2011) TMF RMF ME RMSE N&S ME RMSE N&S (mmh−1 ◦C−1) (mmh−1 W−1 m2) (m w.e.) (m w.e.) (m w.e.) (m w.e.) Measured temperature 0.246 0.00117 −0.027 0.080 0.992 +0.052 0.156 0.888 Standard lapse rate 0.202 0.00100 −0.049 0.252 0.918 −0.160 0.261 0.686 G&B method 0.251 0.00109 −0.006 0.113 0.984 +0.156 0.314 0.545 S&M method 0.291 0.00128 −0.049 0.359 0.832 −0.282 0.366 0.381 Calibrated Calibration run Validation run parameters (summer 2011) (summer 2010) TMF RMF ME RMSE N&S ME RMSE N&S (mmh−1 ◦C−1) (mmh−1 W−1 m2) (m w.e.) (m w.e.) (m w.e.) (m w.e.) Measured temperature 0.246 0.00138 +0.006 0.152 0.893 −0.095 0.119 0.982 Standard lapse rate 0.175 0.00111 −0.008 0.210 0.796 +0.178 0.346 0.844 G&B method 0.265 0.00141 +0.045 0.288 0.618 −0.172 0.226 0.934 S&M method 0.236 0.00129 −0.018 0.241 0.732 +0.315 0.522 0.647 ∗Calibration in 2010 and validation in 2011 in the upper table, vice versa in the lower table. Measured vs. modeled values are displayed in Fig. 10. L. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Tests indicate that at sites with almost no cooling effect (Mar-gl_3438 and Car- gl_3144) the unmodiﬁed ambient temperature or the com- bination (i) (x0 = 0) provide the best results (mean errors < 0.2 ◦C in absolute value). At the four sites with prevailing KBL the best overall solution was (iii), but this combination is speciﬁc for the AVDM and not generalizable due to the rather small size of our glaciers. At Mar-gl_2973, options (ii) and (iii) completely removed the step and provided the best statistics. At Mar-gl_3215, option (iii) provided almost iden- tical results to a variable x0, while options (i) and (ii) led to excessive overestimations and underestimations, respec- tively. At Mar-gl_3140, the best option was (iii). The G&B method provided the best overall results. Among sites with prevailing katabatic winds, the improve- ment was clearest at Mar-gl_2973, where the method was able to account for the combined effect of adiabatic heat- ing and turbulent exchanges, which were regulated by the slope variations along the upstream ﬂow line. However, it was worse than the S&M method at distinguishing between the two Careser glaciers, and the better results in terms of These ﬁndings highlight site-speciﬁc and glacier-speciﬁc conditions which still need investigation in order to gener- alize the G&B procedure, possibly by including smaller or disintegrating glaciers in the data sets used for the general- 6 Concluding remarks Abbate, S., Avvenuti, M., Carturan, L., and Cesarini, D.: Deploy- ing a communicating automatic weather station on an Alpine Glacier, Procedia Computer Science, 19, 1190–1195, 2013. The results of this work have interesting implications for the knowledge of glacier’s reactions to climatic changes and for their modeling. The main conclusions from this study are the following: Ayala, A., Pellicciotti, F., and Shea, J. M.: Modeling 2 m air temper- atures over mountain glaciers: Exploring the inﬂuence of kata- batic cooling and external warming, J. Geophys. Res. Atmos., 120, doi:10.1002/2015JD023137, online ﬁrst, 2015. 1. Our ﬁndings provide a ﬁrst experimental evidence for the reduced effectiveness of small glaciers (< 0.5 km2) in cooling the air above and in triggering katabatic ﬂows. This represents an important reinforcing mech- anism during glacier decay and fragmentation. Bahr, D. B. and Radi´c, V.: Signiﬁcant contribution to total mass from very small glaciers, The Cryosphere, 6, 763–770, doi:10.5194/tc-6-763-2012, 2012. Barry, R. G.: The status of research on glaciers and global glacier recession: a review, Prog. Phys. Geog., 30, 285–306, 2006. Braithwaite, R. J.: Regional modelling of ablation in West Green- land, Grønlands geologiske undersøgelse, 98, 20 pp., 1980. 2. A good match between our temperature measurements and the parameterizations proposed by Shea and Moore (2010) and, best of all, Greuell and Böhm (1998) was found, at least for the on-glacier weather stations where katabatic ﬂows prevail. This represents a step forward for the generalization of these methods, which still need reﬁnements in particular for areas close to the margins (e.g., the front) and for the smaller units resulting from glacier fragmentation. Braithwaite, R. J., Zhang, Y., and Raper, S. C. B.: Temperature sen- sitivity of the mass balance of mountain glaciers and icecaps as a climatological characteristic, Zeitschrift fur Gletscherkunde und Glazialgeologie, 38, 35–61, 2002. Carturan, L.: Climate change effects on the cryosphere and hydrol- ogy of a high-altitude watershed, PhD thesis, TeSAF – University of Padova, Padova, Italy, 2010. Carturan, L., Dalla Fontana, G., and Cazorzi, F.: The mass balance of La Mare Glacier (Ortles-Cevedale, Italian Alps) from 2003 to 2008, in: Epitome – Geoitalia 2009, Settimo Forum Italiano di Scienze della Terra, Rimini, Italy, 9–11 September 2009, 3, p. 296, 2009. 3. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale Radic and to two anonymous reviewers, whose comments were helpful for ﬁnalizing the paper. Edited by: V. Radic L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale ization. Sites where the KBL no longer exists and is replaced by prevailing valley winds and/or synoptic winds also need to be included as they reveal important controlling mechanisms during glacier shrinking, which require modiﬁcations to the main G&B algorithms in order to be taken into account. Author contributions. L. Carturan, F. Cazorzi, and G. Dalla Fontana designed the glacial–meteorological experiment and car- ried it out. L. Carturan and F. De Blasi processed and analyzed the experimental data. F. Cazorzi and L. Carturan developed the EISModel and performed the glacier mass balance simulations. L. Carturan prepared the manuscript with contributions from all co- authors. g The results of EISModel applications underline the im- portance of correct on-glacier air temperature estimation for reliable mass balance calculations (Table 5, Fig. 10). Even small estimation errors induce signiﬁcant distortions in cal- ibration parameters and compromise model generalizability. The 2010 data set on La Mare Glacier clearly demonstrates how single points, especially if they are displaced along alti- tudinal proﬁles, can affect the calibration of the model and its capability to account for the vertical gradients of the mass balance. This problem is clearly emphasized in our case study, with only three weather stations along the ﬂow line of La Mare Glacier in 2010. The spatial representative- ness of Mar-gl_2973 and Mar-gl_3215 is likely much higher than that of Mar-gl_2709 at the glacier terminus, which re- ﬂects the conditions close to the lower edge of glaciers. How- ever, mass balance models should be improved in order to account for the decreased thermal offset in these areas and in smaller glacier units resulting from the fragmentation of larger glaciers, because they represent important processes involved in the response of glaciers to climatic changes. Acknowledgements. The data and the mass balance model used in this study can be made available upon request to the authors. This study was founded by the Italian MIUR project (PRIN 2010-11) “Response of morphoclimatic system dynamics to global changes and related geomorphological hazards” (local and national coordi- nators G. Dalla Fontana and C. Baroni). The authors acknowledge the autonomous province of Trento and Enel SpA for providing the meteorological and topographic data. Special thanks to Vinicio Carraro for the help in the setup of automatic weather stations and to the students, colleagues, and alpine guides who have contributed to the ﬁeld surveys. Finally, thanks to the scientiﬁc editor V. L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale o longer exists and is replaced or synoptic winds also need to ortant controlling mechanisms Author contributions. L. Carturan, F. Cazorzi, and G. Dalla Fontana designed the glacial–meteorological experiment and car- ried it out. L. Carturan and F. De Blasi processed and analyzed 1144 www.the-cryosphere.net/9/1129/2015/ The Cryosphere, 9, 1129–1146, 2015 www.the-cryosphere.net/9/1129/2015/ L. Carturan et al.: Air temperature variability over three glaciers in the Ortles–Cevedale D.: Prediction of possi- ble changes in glacio-hydrological characteristics under global warming: Southeastern Alaska, USA, J. Glaciol., 42, 407–412, 1996. 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https://openalex.org/W4206711885	http://jurnal.stokbinaguna.ac.id/index.php/JPHR/article/download/521/335	Indonesian	null	PERKEMBANGAN E-SPORT PADA PELAJAR REMAJA USIA 13-16 TAHUN PADA MASA PANDEMI COVID-19	Journal Physical Health Recreation	2,021	cc-by	2,132	PERKEMBANGAN E-SPORT PADA PELAJAR REMAJA USIA 13-16 TAHUN PADA MASA PANDEMI COVID-19 PERKEMBANGAN E-SPORT PADA PELAJAR REMAJA USIA 13-16 TAHUN PADA MASA PANDEMI COVID-19 Ayub Tatya Admaja1, Yulis Agung Saputro2 1Universitas Mercu Buana Yogyakarta Jl. Raya Wates-Jogjakarta, Karanglo, Argomulyo, Daerah Istimewa Yogyakarta Email : ayub@mercubuana-yogya.ac.id Ayub Tatya Admaja1, Yulis Agung Saputro2 1Universitas Mercu Buana Yogyakarta Jl. Raya Wates-Jogjakarta, Karanglo, Argomulyo, Daerah Istimewa Yogyakarta Email : ayub@mercubuana-yogya.ac.id Ayub Tatya Admaja1, Yulis Agung Saputro2 1Universitas Mercu Buana Yogyakarta Jl. Raya Wates-Jogjakarta, Karanglo, Argomulyo, Daerah Istimewa Yogyakarta Email : ayub@mercubuana-yogya.ac.id Keywords: E-Sport, Sports Technology, Covid-19 Pandemic ABSTRACT E-Sport is part of a sport that has grown rapidly in the last 5 years and is a sport that is supported by the government in developing rapidly. The dilemma that occurs is that a student's psychic and physical changes are due to reduced physical activity and social contact due to more work from home or study at home. Researchers try to explore the consequences of student activities that are required to study at home due to the corona virus. The positive and negative impacts or pros and cons in the COVID-19 pandemic can be a blessing or a disaster depending on the perspective of the mind. Researchers try to examine the development of e-sports which is now starting to become popular and even an addiction for all ages from children to adults, and is supported by many athletes who have achieved world-class achievements in FIFA or Pro Evolution Soccer, PUBG, and other online games. The problem from the research is the basis why researchers are interested in researching by using a survey method to the point of view of students and the point of view of parents and the point of view of teachers / lecturers about the development of e-sports which is very popular today. This research data is on a regional scale such as the province of Yogyakarta and if possible will be distributed via google form to all corners of the country so that the data obtained is more diverse according to the point of view of each region according to the culture and circumstances of each region. This research will be very interesting because the issue taken is a national issue in the world of sports today. From this study, it can be concluded that children aged 13-16 years like to play online games, because: 1) Repel boredom 50%, 2) Gathering with friends 30%, 3) Get 20% achievement. Judging from the data, it is clear that the goals of E-Sport have not been fully achieved in Indonesia. Because it should be clear with Esports to hone intelligence thinking quickly and precisely by playing some games. Keywords: E-Sport, Sports Technology, Covid-19 Pandemic ABSTRAK Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 e-ISSN : 2747- 013X e-ISSN : 2747- 013X ABSTRAK ABSTRAK 69 Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 e-ISSN : 2747- 013X e-ISSN : 2747- 013X e-ISSN : 2747- 013X E-Sport merupakan bagian dari cabang olahraga yang berkembang pesat dalam kurun 5 tahun terakhir dan merupakan cabang olahraga yang didukung oleh pemerintah dalam berkembang secara cepat. Dilema yang terjadi adalah psikis dan fisik seorang pelajar menjadi berubah karena berkurangnya aktifitas fisik dan kontak sosial karena lebih banyak work from home atau study at home. Peneliti mencoba mengupas akibat dari aktifitas pelajar yang diharuskan belajar di rumah akibat virus corona. Dampak positif dan negatif ataupun pro dan kontra dalam pandemi covid 19 menjadi berkah ataupun menjadi bencana tergantung dari perspektif sudut pandang pemikiran. Peneiliti mencoba mengkaji tentang perkembangan e-sport yang sekarang ini mulai digemari dan bahkan menjadi candu bagi semua kalangan anak-anak hingga dewasa, dan didukung banyak atlet yang berprestasi hingga tingkat dunia dalam game FIFA atau Pro Evolution Soccer, PUBG, dan game online lainnya. Masalah dari peneilitian menjadi dasar mengapa peneliti tertarik dalam meneliti dengan menggunakan metode survey kepada susudt pandang pelajar dan sudut pandang orang tua dan sudut pandang guru/dosen tentang perkembangan e-sport yang sangat digemari saat ini. Data penelitian ini berskala daerah seperti provinsi Yogyakarta dan apabila memungkinkan akan dibagikan melalui google form ke seluruh penjuru tanah air sehingga Data yang didapat lebih beragam sesuai sudut pandang didaerah masing sesuai dengan budaya dan keadaan dari setiap daerah. Penelitian ini akan sangat menarik karena isu yang diambil adalah isu nasional dalam dunia olahraga saat ini. Dari penelitian ini dapat disimpulkan bahwa anak usia 13-16 tahun gemar bermain game online, karena : 1) Mengusir kebosanan 50%, 2) Ajang berkumpul dengan teman 30%, 3) Mendapatkan prestasi 20%. Ditinjau dari data tersebut sudah jelas tujuan dari E-Sport belum tercapai sepenuhnya di Indonesia. Karena harusnya dengan Esport sudah jelas untuk mengasah kecerdasan berpikir secara cepat dan tepat dengan memainkan beberapa game. e-ISSN : 2747- 013X yang dipertandingkan (Summerley, 2020). Permasalahan tersebut dipicu oleh perkembangan olahraga nasional yang semua terhenti dan tidak mendapat izin diadakannya pertandingan ataupun kejuaraan dan berlaku disemua cabang olahraga kecuali E-Sport, karena cabang olahraga ini bisa dilakukan dalam jarak yang jauh karena pertandingan dan kejuaraan menggunakan internet dan dipertandingkan secara online (J. Kim & Kim, 2020). Pengguna game online semakin tertantang untuk berkembang dan berusaha selalau mengembangkan potensi dalam bermain game online (Bascón-Seda & Ramírez-Macías, 2020). didapatkannya pun berhasil mematahkan stigma buruk bermain game online, terutama untuk anak-anak. Perjalanan E-Sport di Indonesia bahkan di level ASEAN menjadi sangat menarik untuk diperhatikan. Multi event ASEAN seperti SEA Games 2019, dan Piala Presiden 2019 E-Sport dipertandingkan secara resmi. Kemudian banyak sekali ajang kompetisi E-Sport di skala dunia seperti The 2016 Call of Duty World League Championship, The Dota 2 Asia Championship , The 2018 League of Legends World Championship, The International 8, dan masih banyak lagi. yang dipertandingkan (Summerley, 2020). yang dipertandingkan (Summerley, 2020). Permasalahan tersebut dipicu oleh perkembangan olahraga nasional yang semua terhenti dan tidak mendapat izin diadakannya pertandingan ataupun kejuaraan dan berlaku disemua cabang olahraga kecuali E-Sport, Berdasarkan pro dan kontra akan hadirnya E- Sport menjadi perhatian peneliti untuk mengupas lebih dalam akan pandangan dari berbagai sudut perspektif seseorang. Sudut pandang perkembangan esport akan digali melalui pelajar, guru, bahkan orang tua. Peneliti akan menggunakan kuisioner dalam bentuk google form yang akan dibagikan secara random ke seluruh masyarakat Daerah Istimewa Yogyakarta. Dengan data ini akan sangat membantu memberi informasi apa sebenarnya pendapat bahkan pandangan E- Sport dari berbagai sudut pandang. Perkembangan olahraga nasional di masa pandemi covid 19 mengalami peningkatan yang signifikan secara prestasi dan intensitas pengguna game online (Urbaniak et al., 2020). Sebagian orang berpendapat bahwa game sudah bukan lagi sebagai penghilang kepenatan dikala banyak kejenuhan dalam pekerjaan atau belajar di sekolah melainkan sebagai profesi yang bisa dijamin akan penghasilan dan bisa menjadi modal dalam kehidupan. Atlet game online mulai berlatih dengan gadget atau joystick mereka setiap hari demi mencapai juara yang mereka inginkan. Sebut saja game yang marak dimainkan disemua kalangan tua maupun muda yaitu Pro Evolution Soccer (PES) dan FIFA, mobile legend, PUBG, Free Fire, DOTA, COC, dan masih banyak game online lainnya yang sekarang ini menjadi sangat popular (Heere, 2018). PENDAHULUAN dalam latihan, juga terkena imbas dalam wabah ini. Pembelajaran dan latihan secara online dianggap bisa membantu dalam proses pembelajaran selama wabah virus corona belum hilang di dunia. Pandangan perspektif tersebut dirasa kurang bisa diterima oleh masyarakat luas khususnya bagi orang tua, guru ataupun pelatih. Karena ancaman di era ini adalah permainan game online yang mengalihkan segala sesuatu kewajiban yang harus dilakukan oleh seorang pelajar hanya untuk bermain gadget ataupun consol game. Olahraga juga mulai beralih ke kegiatan non fisik yaitu game online yang sekarang sudah dimasukan sebagai salah satu cabang resmi Pandemi covid 19 atau bisa disebut pandemi virus corona menjadi bencana dunia dengan wabah penyakit yang menyebar ke seluruh dunia, salah satunya negeri Indonesia (Y. H. Kim et al., 2020). Kegiatan belajar mengajar dan bekerja menjadi terganggu senhingga diberlakukannya bekerja di rumah saja, untuk memutus tali penyebaran virus corona. Dampaknya begitu terasa ketika pelajar belajar di rumah, banyak pelajaran dan tugas yang terbengkalai dan guru atau pekerja tidak dapat melakukan kegiatan dengan maksimal. Khususnya dalam pembelajaran olahraga ataupun kegiatan aktifitas fisik bagi atlet 70 yang dipert Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 e-ISSN : 2747- 013X KESIMPULAN Dari penelitian ini dapat disimpulkan bahwa anak usia 13-16 tahun gemar bermain game online, karena : 1) Mengusir kebosanan 50%, 2) Ajang berkumpul dengan teman 30%, 3) Mendapatkan prestasi 20%. Ditinjau dari data tersebut sudah jelas tujuan dari E-Sport belum tercapai sepenuhnya di Indonesia. Karena harusnya dengan Esport sudah jelas untuk mengasah kecerdasan berpikir secara cepat dan tepat dengan memainkan beberapa game. HASIL DAN PEMBAHASAN Tabel 1. Hasil Rekapitulasi Kuisioner Anak usia 13-16 tahun Pertanyaan PERSENTASE (%) DARI 100 ORAN YA TIDAK Apakah anda mengetahui E-Sport / Game Online pada laptop/komputer/ Smartphone (HP) ? 100 0 Apakah anda memainkan E-Sport / Game Online pada laptop/komputer/ Smartphone (HP) ? 80 20 Apakah berdampak positif memainkan E-Sport / Game Online pada laptop/komputer/ Smartphone (HP) bisagetahui Game Playstasion ? (dampak positif : berprestasi, mendapatkan uang, mengisi waktu luang untuk kreatif 80 20 Apakah berdampak negatif memainkan E-Sport / Game Online pada laptop/komputer/ Smartphone (HP) bisagetahui Game Playstasion ? (dampak negatif : mengganggu aktifitas belajar, pemalasm, memicu gangguan kesehatan 50 40 Jenis Permainan apa yang anda mainkan : 1. Smartphone 60 40 2. Laptop/Komputer 30 70 3. Playstation 50 50 Game yang dimainkan : 1. Mobile Legende 90 10 2. Free Fire 80 20 3. COC 10 90 4. FIFA/PES 50 50 5. PUBG 40 60 6. Game of Throne 10 90 7. Adventure Game 10 90 8. Arena Of Valor 10 90 9. League of legends 10 90 10. DOTA 10 90 Berdasarkan game diatas apakah anda yakin untuk berprestasi ? Apakah tujuan anda bermain game ? a. Mengisi waktu luang 80 20 b. Bergaul dengan teman dengan main bersama 80 20 c. berlatih agar berprestasi 10 90 Apakah aktifitas game anda mempengaruhi kesehatan anda ? 40 50 Apakah anda tidur larut malam ketika sedang bermain game ? 70 30 Tabel 1. Hasil Rekapitulasi Kuisioner Anak usia 13-16 tahun METODE PENELITIAN Metode penelitian menggunakan metode penelitian deskriptif kantitatif dengan menggunakan metode survey. Metode dirasa sangat cocok karena peneliti membutuhkan data bersifat jawaban dari responden agar dapat ditarik kesimpulan sesuai dengan tujuan penelitian. Peneliti menggunakan sampel penelitian yang terdiri dari guru, pelajar, dan orang tua yang akan mengisi Perkembangan dunia gaming saat ini telah berubah menjadi sesuatu yang punya potensi, menjanjikan dan lebih kompetitif berkat kehadiran E-Sport (Abanazir, 2019). Adanya E-Sport dengan segala benefit yang bisa 71 kuisioner Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 kuisioner dan wawancara secara online melalui google meet atau zoom meeting yang telah disipakan oleh peneliti. Teknik pengumpulan data menggunakan instrument google form yang akan disebar melalu media whatsapp, facebook, dan Instagram agar, data yang di dapat heterogen dan bisa menghasilkan data dari berbagai perspektif sudut pandang. ora me me pad set on me kec han jug on me Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 e-ISSN : 2747- 013X orang tua bersikap bahwa game online membuat anak menjadi pemalas dan akan mengganggu kesehatan. Data tersebut berada pada angka 70 persen dalam hal ketidak setujuan orang tua terhadap maraknya game online. Satu sisi pada orang tua yang sudah mengenal teknologi mengatakan bahwa kecerdasan dan prestasi seseorang tidak hanya berdasar hasil belajar melainkan bisa juga dari non akademik seperti halnya game online. Karena banyak juga yang mendapatkan uang dan prestasi gemilang melalui E-Sport, selain itu pemerintah dan beberapa organisasi KOI dan KONI juga sudah memasukkan E-Sport menjadi bagian olahraga yang dipertandingkan di PON PAPUA 2021. DAFTAR PUSTAKA Abanazir, C. (2019). Institutionalisation in E- Sports. Sport, Ethics and Philosophy, 13(2). https://doi.org/10.1080/17511321.2018.1453 538 Berdasarkan hasil data yang dikumpulkan oleh peneliti menunjukan bahwa E-Sport adalah permainan digital yang belum banyak dikenal oleh kalangan orang berusia 30 tahun Bascón-Seda, A., & Ramírez-Macías, G. (2020). Are E-sports a sport? The term ‘sport’ in checkmate. Movimento, 26(1). https://doi.org/10.22456/1982-8918.97363 keatas yaitu orang tua dan guru. Hasil wawancara dan kuisioner meunnjukan bahwa 72 Journal Physical Health Recreation Volume 2 Nomor 1 ; November 2021 e-ISSN : 2747- 013X e-ISSN : 2747- 013X Heere, B. (2018). Embracing the sportification of society: Defining e-sports through a polymorphic view on sport. In Sport Management Review (Vol. 21, Issue 1). https://doi.org/10.1016/j.smr.2017.07.002 Kim, J., & Kim, M. (2020). Spectator e-sport and well-being through live streaming services. Technology in Society, 63. https://doi.org/10.1016/j.techsoc.2020.10140 1 Kim, Y. H., Nauright, J., & Suveatwatanakul, C. (2020). The rise of E-Sports and potential for Post-COVID continued growth. Sport in Society, 23(11). https://doi.org/10.1080/17430437.2020.1819 695 Summerley, R. (2020). The Development of Sports: A Comparative Analysis of the Early Institutionalization of Traditional Sports and E-Sports. Games and Culture, 15(1). https://doi.org/10.1177/1555412019838094 Urbaniak, K., Watróbski, J., & Sałabun, W. (2020). Identification of players ranking in e- sport. Applied Sciences (Switzerland), 10(19). https://doi.org/10.3390/app10196768 73 73
https://openalex.org/W3123075443	https://eprints.gla.ac.uk/229127/2/229127.pdf	English	null	Invasive versus medically managed acute coronary syndromes with prior bypass (CABG-ACS): insights into the registry versus randomised trial populations	Open heart	2,021	cc-by	15,529	► ►Additional material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ openhrt-2020-001453). How might this impact on clinical practice?i ► ►The CABG-ACS pilot trial and registry fills in an evi- dence gap on the natural history and optimal treat- ment strategy for this comparatively large subgroup of patients. Furthermore, the CABG-ACS trial and registry may be helpful in the design of clinical trials in this patient group. ► ►The CABG-ACS pilot trial and registry fills in an evi- dence gap on the natural history and optimal treat- ment strategy for this comparatively large subgroup of patients. Furthermore, the CABG-ACS trial and registry may be helpful in the design of clinical trials in this patient group. Results Of 217 patients screened, 84 (39%) screenfailed, of whom 24 (29%) did not consent and 60 (71%) were ineligible. Of 133 (61%) eligible, 60 (mean±SD age, 71±9 years, 72% male) entered the trial and 73 (age, 72±10 years, 73% male) entered a registry (preferences: physician (79%), patient (38%), both (21%)). Results Of 217 patients screened, 84 (39%) screenfailed, of whom 24 (29%) did not consent and 60 (71%) were ineligible. Of 133 (61%) eligible, 60 (mean±SD age, 71±9 years, 72% male) entered the trial and 73 (age, 72±10 years, 73% male) entered a registry (preferences: physician (79%), patient (38%), both (21%)). Received 17 September 2020 Revised 23 November 2020 Accepted 22 January 2021 Received 17 September 2020 Revised 23 November 2020 Accepted 22 January 2021 Received 17 September 2020 Revised 23 November 2020 Accepted 22 January 2021 Received 17 September 2020 Revised 23 November 2020 Accepted 22 January 2021 Compared with trial participants, registry patients had more valve disease, lower haemoglobin, worse New York Heart Association class and higher frailty. Invasive versus medically managed acute coronary syndromes with prior bypass (CABG-ACS): insights into the registry versus randomised trial populations Matthew M Y Lee ‍ ‍ ,1,2,3,4,5 Mark C Petrie ‍ ‍ ,1,2,3 Paul Rocchiccioli ‍ ‍ ,2,3 Joanne Simpson,1,2 Colette E Jackson,1,2,4 David S Corcoran ‍ ‍ ,1,2 Kenneth Mangion ‍ ‍ ,1,2 Ammani Brown,4 Pio Cialdella,2 Novalia P Sidik,1,2 Margaret B McEntegart,1,2,4 Aadil Shaukat,2,3 Alan P Rae,1,3 Stuart H M Hood,1,2,5 Eileen E Peat,2,5 Iain N Findlay,5 Clare L Murphy,5 Alistair J Cormack,5 Nikolay B Bukov,6 Kanarath P Balachandran,6 Ian Ford ‍ ‍ ,7 Olivia Wu ‍ ‍ ,8 Alex McConnachie ‍ ‍ ,7 Sarah J E Barry ‍ ‍ ,9 Colin Berry ‍ ‍ ,1,2,4 On behalf of the CABG-ACS Investigators What does this study add? ► ►The CABG-ACS trial and registry provides novel, con- temporary insights into an understudied subgroup of ACS patients with a substantial health burden. Key questions Background Coronary artery bypass graft (CABG) patients are under-represented in acute coronary syndrome (ACS) trials. We compared characteristics and outcomes for patients who did and did not participate in a randomised trial of invasive versus non-invasive management (CABG- ACS). What is already known about this subject? What is already known about this subject? ► ►Pivotal clinical trials of invasive management versus non-invasive medical management in acute coro- nary syndromes (ACS) excluded patients with prior coronary artery bypass graft (CABG). ► ►Pivotal clinical trials of invasive management versus non-invasive medical management in acute coro- nary syndromes (ACS) excluded patients with prior coronary artery bypass graft (CABG). To cite: Lee MMY, Petrie MC, Rocchiccioli P, et al. Invasive versus medically managed acute coronary syndromes with prior bypass (CABG-ACS): insights into the registry versus randomised trial populations. Open Heart 2021;8:e001453. doi:10.1136/ openhrt-2020-001453 Methods ACS patients with prior CABG in four hospitals were randomised to invasive or non-invasive management. Non-randomised patients entered a registry. Primary efficacy (composite of all-cause mortality, rehospitalisation for refractory ischaemia/angina, myocardial infarction (MI), heart failure) and safety outcomes (composite of bleeding, stroke, procedure-related MI, worsening renal function) were independently adjudicated. Coronary artery bypass graft (CABG) surgery is standard of care for patients with obstructive coronary artery disease. However, occlusive disease of saphenous vein grafts is common within 10 years of surgery,1–3 meaning patients with prior CABG have a progressive longer- term risk of recurrent ischaemia, including angina and myocardial infarction (MI), heart failure (HF) and death. Given the large number of CABG recipients living globally, and their health complexities, including increasing age and multimorbidity, this represents an increasing healthcare challenge globally, © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. At baseline, invasive management was performed in 52% and 49% trial and registry patients, respectively, of whom 32% and 36% had percutaneous coronary intervention at baseline, respectively (p=0.800). After 2 years follow-up (694 (median, IQR 558–841) days), primary efficacy (43% trial vs 49% registry (HR 1.14, 95% CI 0.69 to 1.89)) and safety outcomes (28% trial vs 22% registry (HR 0.74, 95% CI 0.37 to 1.46)) were similar. EuroQol was lower in registry patients at 1 year. For numbered affiliations see end of article. To cite: Lee MMY, Petrie MC, Rocchiccioli P, et al. Invasive versus medically managed acute coronary syndromes with prior bypass (CABG-ACS): insights into the registry versus randomised trial populations. Open Heart 2021;8:e001453. doi:10.1136/ openhrt-2020-001453 Open access Open access Coronary artery disease © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Randomisation Patients fulfilling inclusion criteria without any exclusion criteria who consented to participate in the randomised trial were enrolled (figure 1). Randomisation was performed by the Trials Unit interactive voice recogni- tion system to one of two groups: initial medical manage- ment or initial invasive management (online supple- mental methods). Study population I l i i Figure 1 CONSORT diagram in CABG-ACS. CABG, coronary artery bypass graft; CONSORT, Consolidated Standards of Reporting Trials; NSTE-ACS, non-ST segment elevation acute coronary syndrome. Inclusion criteria were: (1) unstable angina or non-ST segment elevation MI; (2) stabilised symptoms without recurrent chest pain or intravenous therapy for 12 hours and (3) prior CABG. Exclusion criteria were: (1) refractory ischaemia (ie, recurrent angina with minimal exertion or at rest (ie, Canadian Cardiovascular Society (CCS) class III or IV) not controlled by medical therapy); (2) cardiogenic shock; (3) lack of informed consent and (4) unsuitable for invasive management. not least because of rehospitalisation due to recurrent ischaemia.4 5 Chest pain is the most common reason for hospital admission in the UK and about 1 in 10–15 patients admitted to hospital with an acute non-ST segment eleva- tion acute coronary syndrome (NSTE-ACS) have a prior CABG.6 Pivotal clinical trials comparing routine invasive manage- ment vs conservative non-invasive management in unstable coronary syndromes excluded patients with prior CABG (online supplemental table 1). Current guidelines recom- mend a routine early invasive strategy in higher risk NSTE-ACS patients.7–9 However, invasive management is performed less often in NSTE-ACS patients with prior CABG, probably because the risk:benefit balance is consid- ered to be less favourable in these patients compared with those without prior CABG.10–12 Furthermore, when inva- sive management is performed, percutaneous coronary intervention (PCI) is less likely in prior CABG patients,11–13 implying a lower likelihood of benefit with a routine inva- sive strategy. Real-world evidence implies clinical practice departs from the results of systematic reviews and guide- lines,14 15 indicating physician and patient preferences for treatment options may be relevant. Overall, evidence is lacking to inform the validity of current guideline recom- mendations,7–9 in NSTE-ACS patients with prior CABG. This important subgroup of ACS patients remains compar- atively understudied. Enrolment into randomised trials Correspondence to Correspondence to Professor Colin Berry, British Heart Foundation Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, Glasgow G12 8TA, United Kingdom; colin. berry@glasgow.ac.uk Conclusions Compared with trial participants, registry participants had excess morbidity, but longer-term outcomes were similar. Trial registration number NCT01895751. Trial registration number NCT01895751. 1 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 Figure 1 CONSORT diagram in CABG-ACS. CABG, coronary artery bypass graft; CONSORT, Consolidated Standards of Reporting Trials; NSTE-ACS, non-ST segment elevation acute coronary syndrome. can be challenging,16 particularly when the intervention disrupts standard care. Enrolment of elderly patients may be challenging, sometimes leading to premature trial discontinuation.17–19 We hypothesised that the clinical characteristics, treatment and health outcomes would differ between participants enrolled in a randomised controlled trial of routine invasive versus routine non-invasive management in NSTE-ACS patients with prior CABG, and participants enrolled in the registry due to physician and/or patient preference. We aimed to prospectively gather informa- tion on the trial and registry participants in order to gain contemporary information on the natural history and reasons for trial participation or not. on March 5, 2021 by guest. Protected by copyright. http://openheart.bmj.com/ hed as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from METHODS We undertook a randomised controlled trial of routine invasive management vs routine conservative manage- ment in NSTE-ACS patients with prior CABG. Concur- rently, patients who were ineligible for randomisation and who gave informed consent were entered into an observational registry. The study design,20 and results of the main trial have been published.21 on March 5, 2021 by guest. Protected by copyright. http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Coronary artery disease Table 1 Baseline clinical characteristics of the trial and registry participants Characteristics Statistic All N=133 Trial N=60 Registry N=73 P value Clinical Age, years Mean±SD 71±10 71±9 72±10 0.46 Female sex N (%) 37 (28) 17 (28) 20 (27) 1.00 Obese (body mass index >30 kg/m2) N (%) 37 (28) 22 (37) 15 (21) 0.05 Presentation type* Non-ST segment elevation MI N (%) 90 (68) 41 (68) 49 (67) 1.00 Unstable angina N (%) 43 (32) 19 (32) 24 (33) 1.00 Medical history Diabetes mellitus† N (%) 50 (38) 21 (35) 29 (40) 0.60 Previous MI N (%) 91 (68) 41 (68) 50 (68) 1.00 Cardiac arrhythmia N (%) 47 (36) 19 (32) 28 (39) 0.37 Treated hypertension N (%) 88 (66) 42 (70) 46 (63) 0.46 Renal impairment N (%) 38 (29) 13 (22) 25 (34) 0.13 Peripheral vascular disease N (%) 32 (24) 16 (27) 16 (22) 0.55 Cerebrovascular disease N (%) 30 (23) 13 (22) 17 (23) 0.84 Congestive HF N (%) 44 (33) 14 (23) 30 (41) 0.04 Anaemia N (%) 20 (15) 5 (8) 15 (21) 0.05 Valve disease N (%) 43 (32) 12 (20) 31 (42) 0.01 Pacemaker N (%) 11 (8) 5 (8) 6 (8) 1.00 History of smoking Current N (%) 27 (20) 12 (20) 15 (21) 0.35 Former (>3 months) N (%) 80 (60) 33 (55) 47 (64) Never N (%) 26 (20) 15 (25) 11 (15) Serum creatinine, μmol/L Median (IQR) 86 (71–114) 84 (68–101) 89 (73–131) 0.11 Haemoglobin, g/L Mean±SD 131±18 135±16 127±18 0.01 Charlson Comorbidity Index Median (IQR) 5 (3–7) 4 (3–6) 5 (3–7) 0.28 Health-related quality of life, EuroQol 5 Dimensions 5 Levels score Median (IQR) 0.674 (0.447–0.866) 0.748 (0.514–0.899) 0.658 (0.437–0.817) 0.11 ECG abnormalities at initial presentation ST-segment depression N (%) 67 (50) 28 (47) 39 (53) 0.49 ST-segment elevation N (%) 27 (20) 11 (18) 16 (22) 0.67 T-wave inversion N (%) 89 (67) 38 (63) 51 (70) 0.46 Q-waves N (%) 42 (32) 15 (25) 27 (37) 0.19 Left bundle branch block N (%) 11 (8) 5 (8) 6 (8) 1.00 AF or flutter N (%) 23 (17) 9 (15) 14 (19) 0.65 New ischaemic ECG changes‡ N (%) 66 (50) 30 (50) 36 (50) 1.00 Canadian Cardiovascular Society angina class§ 1 N (%) 4 (3) 2 (3) 2 (3) 0.83 2 N (%) 11 (8) 6 (10) 5 (7) 3 N (%) 26 (20) 10 (17) 16 (22) 4 N (%) 90 (69) 41 (69) 49 (68) New York Heart Association functional class Continued on March 5, 2021 by guest. Registry f Information was prospectively recorded in a registry for acute NSTE-ACS and prior CABG patients who were not randomised but consented to participate in the registry. Reasons for non-participation in the randomised trial were prospectively recorded: exclusion criteria present, unsuitability for either invasive or non-invasive manage- ment, physician preference, patient preference or a combination of these factors. Baseline and follow-up clinical information were obtained similarly to the trial patients. guest. Protected by copyright. Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 2 Coronary artery disease on March 5, 2021 by guest. Protected by copyright. http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Protected by co http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from on March 5, 2021 by guest. Protected by copyrigh http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Characteristics Statistic All N=133 Trial N=60 Registry N=73 P value I N (%) 47 (35) 30 (50) 17 (23) 0.01 II N (%) 47 (35) 18 (30) 29 (40) III N (%) 28 (21) 8 (13) 20 (27) IV N (%) 11 (8) 4 (7) 7 (10) Coronary artery bypass grafts Left internal mammary artery N (%) 109 (82) 50 (83) 59 (81) 0.82 No/unknown N (%) 24 (18) 10 (17) 14 (19) Saphenous vein graft 0 N (%) 10 (8) 3 (5) 7 (10) 0.74 1 N (%) 37 (29) 17 (29) 20 (29) 2 N (%) 54 (43) 25 (43) 29 (43) ≥3 N (%) 25 (20) 13 (22) 12 (18) Frailty index Fit or well (1,2,3) N (%) 60 (45) 35 (58) 25 (34) 0.03 Vulnerable (4) or mildly frail (5) N (%) 42 (32) 14 (23) 28 (38) Moderately frail (6) N (%) 29 (22) 10 (17) 19 (26) Severely frail (7) N (%) 2 (2) 1 (2) 1 (1) Medication prior to hospital admission Aspirin N (%) 113 (85) 52 (87) 61 (84) 0.81 Statin N (%) 113 (85) 55 (92) 58 (79) 0.06 Beta-blocker N (%) 94 (71) 42 (70) 52 (71) 1.00 Calcium channel blocker N (%) 133 (100) 60 (100) 73 (100) 1.00 Isosorbide mononitrate N (%) 50 (38) 20 (33) 30 (41) 0.38 Nicorandil N (%) 47 (35) 22 (37) 25 (34) 0.86 ACE inhibitor N (%) 101 (76) 50 (83) 51 (70) 0.10 Insulin N (%) 20 (15) 10 (17) 10 (14) 0.64 Oral antidiabetic therapy N (%) 28 (21) 10 (17) 18 (25) 0.29 Antidepressant therapy N (%) 24 (18) 13 (22) 11 (15) 0.37 Diuretic N (%) 52 (39) 18 (30) 34 (47) 0.07 Note that where there are missing values, the percentages are calculated out of the number of patients with data. Cardiac arrhythmia (2 missing from registry). New ischaemic ECG changes (1 missing from registry). Canadian Cardiovascular Society angina class (1 missing from trial, 1 missing from registry). Saphenous vein graft (2 missing from trial, 5 missing from registry). Mean ± SD or median (IQR) for normal and non-normally distributed data, respectively. on March 5, 2021 by guest. Protected by copyrigh http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from During the index hospitalisation, all patients in the randomised trial group had a type 1 MI, while seven patients in the registry group did not have a type 1 MI but had a type 2 MI (2 AF, 1 AF+HF, 1 severe aortic stenosis+HF, 1 anaemia, 1 AF+acute kidney injury, 1 HF+respiratory tract infection). †Diabetes mellitus was defined as a history of diet-controlled or treated diabetes. ‡Any previous episode with new ischaemic ECG changes. §The highest Canadian Cardiovascular Society value of any previous episode for each patient. ACE, Angiotensin-converting enyzme; AF, atrial fibrillation; HF, heart failure; MI, myocardial infarction. Table 1 Continued on March 5, 2021 by guest http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from o http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Table 1 Baseline clinical characteristics of the trial and registry participants uest. Protected by copyright. Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 3 In-hospital clinical course and invasive management 2. CCS angina class. More than twice as many patients in the registry versus the trial had a medication change for recurrent angina. Approximately three-quarters of registry patients had medication changes for standard optimisation of secondary prevention therapy compared with trial patients (table 2). 3. Hospitalisation for refractory ischaemia and/or angi- na. 4. Repeat invasive management during follow-up. 5. Freedom from coronary and/or bypass graft intervention. At baseline, invasive management (coronary angiog- raphy±PCI) was undertaken in 31 (52%) and 36 (49%) patients in the trial and registry groups, respectively, increasing during follow-up to 46 (77%) and 40 (55%), respectively (table 3). Of those who had invasive manage- ment at baseline and follow-up, PCI was performed in 10 (22%) and 13 (33%) of trial and registry patients at base- line, increasing to 21 (46%) and 18 (45%) patients during follow-up, respectively (table 3). For baseline procedures, the British Cardiovascular Intervention Society-1 Jeop- ardy Score was similar between trial (4±4) and registry (5±3; p=0.19) patients (table 3). At baseline and follow-up, compared with trial patients, registry patients had more urgent inpatient invasive procedures (39 (75%) vs 27 (47%)) and fewer outpatient invasive procedures (13 (25%) vs 30 (53%); p=0.004) (table 3). The culprit lesion was uncertain in 27 (47%) and 21 (40%) of procedures in the trial and registry groups, respectively (table 3). Primary efficacy outcome Defined as all-cause mortality, rehospitalisation for refrac- tory ischaemia/angina, MI or hospitalisation for HF. The endpoints were assessed during the study until the final randomised patient had completed 18 months follow-up. Primary outcome Defined as postrandomisation rate of major adverse events (coprimary composite outcome), including one composite outcome for efficacy and one composite outcome for safety. Secondary outcomes 1. Quality of life. Definitions of adverse events Refractory ischaemia, death, procedure-related MI, stroke, major bleeding and worsening renal function are defined in online supplemental methods. Refractory ischaemia, death, procedure-related MI, stroke, major bleeding and worsening renal function are defined in online supplemental methods. ch 5, 2021 by guest. Protected by copyright. Primary safety outcome Defined as bleeding (Bleeding Academic Research Consortium (BARC) types 2–4),22 stroke, procedure- related MI (type 4a, universal definition), worsening renal function or haemodialysis during the index hospi- talisation. Secondary outcomes Follow-Up An independent clinical event committee reviewed the primary efficacy and safety endpoints (online supple- mental methods). Follow-up (via telephone contact, clinic visits, letter) with completion of quality of life assessments (EuroQol Visual Analogue Scale (EQ-VAS) and EuroQol 5 Dimen- sions 5 Levels (EQ-5D-5L)) was maintained 6 monthly until ≥18 months follow-up was reached for the final recruited patient (online supplemental methods). 4 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 on March 5, 2021 by guest. Protected by copyright http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Coronary artery disease on March 5, 20 http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Table 2 Reasons for changing medical therapy during the index hospitalisation Reason All N=133 (%) Trial N=60 (%) Registry N=73 (%) P value Recurrent angina 47 (35) 13 (22) 34 (47) 0.003 Standard optimisation of secondary prevention therapy 103 (77) 54 (90) 49 (67) 0.002 Intolerance of therapy without adverse reaction 8 (6) 2 (3) 6 (8) 0.293 Adverse drug reaction 10 (8) 2 (3) 8 (11) 0.113 Other 7 (5) 3 (5) 4 (5) 1.000 Of 133 (61%) eligible patients who consented to either the trial or registry, 60 patients (mean±SD age, 71±9 years, 43 (72%) male) were randomised and 73 (mean±SD age, 72±10 years, 53 (73%) male) entered the registry. The decision for entering the registry included physician preference (58 (79%)), patient preference (28 (38%)) or both (15 (21%)). Baseline characteristics The characteristics of the trial and registry participants are described (table 1). Compared with trial participants, registry patients were twice as likely to have valve disease (31 (42%) vs 12 (20%); p=0.01), a lower haemoglobin (mean±SD 127±18 vs 135±16 g/L; p=0.01), worse New York Heart Association class (37% vs 20% in class III or IV; p=0.01) and higher frailty index (27% vs 18% moderately or severely frail and 38% vs 23% mildly frail; p=0.03). Fifty (83%) trial and 59 (81%) registry patients participants had a previous left internal mammary artery graft (p=0.82). Baseline EQ-VAS, EQ-5D-5L and medications were similar. Primary safety outcome on March http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Table 3 Invasive management (coronary angiography±PCI) in trial and registry patients at baseline (index admission) and follow-up (≥18 months) Table 3 Invasive management (coronary angiography±PCI) in trial and registry patients at baseline (index admission) and follow up (≥18months) ent (coronary angiography±PCI) in trial and registry patients at baseline (index admission) and Adoption of adjunctive techniques, for example, rotational atherectomy was low (table 3). Health outcomes During a median of 694 (IQR 558–841) days follow-up, the primary efficacy outcome occurred in 26 (43%) and 1.14, 95% CI 0.69 to 1.89) (table 4). The primary safety outcome occurred in 17 (28%) and 16 (22%) trial and registry participants, respectively (HR 0.74, 95% CI 0.37 to 1.46). on March http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from The observed proportion of registry patients experi- encing the efficacy outcome was slightly higher than in the trial group whereas the opposite occurred for the safety Table 3 Invasive management (coronary angiography±PCI) in trial and registry patients at baseline (index admission) and follow-up (≥18 months) Patients with procedures at baseline and follow-up Trial N=46 (%) Registry N=40 (%) P value Patients with one procedure 37 (80.4) 32 (80.0) 0.084 Patients with two procedures 8 (17.4) 4 (10.0) Patients with three procedures 0 (0) 4 (10.0) Patients with four procedures 1 (2.2) 0 (0) Patients with PCI at baseline 10 (21.7) 13 (32.5) 0.331 Patients with PCI at baseline and follow-up 21 (45.7) 18 (45.0) 1.000 Days from enrolment to patient’s first procedure 22.5 (6.5-64.75) 1.5 (0-7.25) <0.001 <30 days 25 (54.3) 36 (90.0) <0.001 30–59 days 9 (19.6) 0 (0) ≥60 days 12 (26.1) 4 (10.0) Procedures at baseline Trial n=31 Registry n=36 P-value BCIS Jeopardy Score (pre-PCI) at baseline 4.3±3.7 5.4±3.1 0.189 PCI at baseline 10 (32.3) 13 (36.1) 0.800 BCIS Jeopardy Score (post-PCI) at baseline 2.4±2.5 4.0±3.4 0.220 Procedures at baseline and follow-up Trial n=57 Registry n=52 P-value Urgent in-patient procedure 27 (47.4) 39 (75.0) 0.004 Outpatient procedure 30 (52.6) 13 (25.0) Hospitalisation† 28 (49.1) 41 (78.8) 0.002 Complications related to angiogram‡ 1 (1.8) 6 (11.5) 0.052 Culprit vessel uncertain 27 (47.4) 21 (40.4) 0.563 Culprit vessel identified 30 (52.6) 31 (59.6) Graft only 17 (56.7) 15 (48.4) 0.611 Native artery only 12 (40.0) 15 (48.4) 0.609 Both graft and native artery 1 (3.3) 1 (3.2) 1.000 Multiple culprit lesions 3 (10.0) 2 (6.5) 0.671 PCI at baseline and follow-up 24 (42.1) 25 (48.1) 0.567 Thrombus aspiration 1 (4.2) 0 (0) 0.490 Rotational atherectomy 4 (16.7) 0 (0) 0.050 Intravascular ultrasound 2 (8.3) 3 (12.0) 1.000 Distal protection device 3 (12.5) 2 (8.0) 0.667 Mean±SD and median (IQR) for non-normally distributed data. *BCIS Jeopardy score not available in one registry patient because of poor-quality angiogram and limited data for right coronary artery (only still frame and not a run). †Admission to hospital including at least one overnight stay. on March http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from ‡Complications in trial (N=1) was worsening renal function after angiography; complications in registry (N=6) were side branch abrupt closure, main branch distal embolisation into filter, no reflow, dissection post-angioplasty (+haematoma > 5 cm in same patient), pulmonary oedema on angiography table, side branch new/worsened thrombus. BCIS, British Cardiovascular Intervention Society; PCI, percutaneous coronary intervention. on March 5, 2021 by guest. Protected by cop http://openheart.bmj.com/ Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from guest. Protected by copyright. Adoption of adjunctive techniques, for example, rotational atherectomy was low (table 3). 1.14, 95% CI 0.69 to 1.89) (table 4). The primary safety outcome occurred in 17 (28%) and 16 (22%) trial and registry participants, respectively (HR 0.74, 95% CI 0.37 to 1.46). The observed proportion of registry patients experi- encing the efficacy outcome was slightly higher than in the trial group, whereas the opposite occurred for the safety RESULTS Two hundred and seventeen patients with prior CABG and an unplanned hospitalisation for a suspected NSTE-ACS were screened (figure 1). uest. Protected by copyright. Eighty-four (39%) participants were identified during screening but were deemed ineligible for progressing into the trial or registry, of whom 24 (29%) did not consent and 60 (71%) consented but were ineligible (≥1 reason): 42 (70%) not confirmed NSTE-ACS, 39 (65%) not stabi- lised symptoms, 4 (7%) no prior CABG, 5 (8%) refractory ischaemia, 3 (5%) unable to provide informed consent and 22 (37%) unsuitable for invasive management. 5 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 Coronary artery disease Coronary artery disease Table 4 Primary and secondary outcomes over follow-up period (≥18 months; median 694 (IQR 558–841) days) Outcome Trial N=60 Registry N=73 HR (registry vs trial) (95% CI) Primary efficacy outcome Composite of all-cause mortality, rehospitalisation for refractory ischaemia/angina, MI and HF 26 (43%) 36 (49%) 1.14 (0.69 to 1.89) Primary safety outcome Composite of bleeding (BARC≥2), stroke, procedure-related MI and worsening renal function during the index hospitalisation 17 (28%) 16 (22%) 0.74 (0.37 to 1.46) Experienced both primary efficacy and safety outcomes 10 (17%) 8 (11%) 0.64 (0.25 to 1.63) Experienced either primary efficacy or safety outcomes 33 (55%) 44 (60%) 1.05 (0.67 to 1.65) Components of primary efficacy outcome All-cause mortality 8 (13%) 17 (23%) Cardiovascular death 2 (3%) 10 (14%) Non-cardiovascular death 4 (7%) 5 (7%) Unknown cause of death 2 (3%) 2 (3%) Rehospitalisation for refractory ischaemia/angina 0 (0%) 0 (0%) Non-fatal MI 22 (37%) 18 (25%) HF 7 (12%) 11 (15%) Primary efficacy outcome at 12 months Composite of all-cause mortality, rehospitalisation for refractory ischaemia/angina, MI and HF at 12 months 20 (33%) 23 (32%) All-cause mortality at 12 months 5 (8%) 9 (12%) Components of primary safety outcome Bleeding (BARC 2–4) 11 (18%) 10 (14%) Stroke 0 (0%) 2 (3%) Procedure-related MI 0 (0%) 0 (0%) Worsening renal function during the index hospitalisation 8 (13%) 5 (7%) Primary safety outcome at 12 months Composite of bleeding (BARC≥2), stroke, procedure-related MI and worsening renal function during the index hospitalisation at 12 months 12 (20%) 14 (19%) Other secondary outcomes Total number of SAE 170 117 Number of patients with a SAE 40 (67%) 49 (67%) Number of SAEs per patient, median (IQR) 1(0, 2) 1(0, 2) Number of patients experiencing Rehospitalisation 39 (65%) 47 (64%) Invasive management (coronary angiography) 46 (77%) 40 (55%) PCI 21 (35%) 18 (25%) Redo CABG 0 (-) 1 (1%) Coronary revascularisation (PCI or CABG) 21 (35%) 18 (25%) Quality of life and angina EQ-VAS health status, 6 months, median (IQR) 75 (60-80) 50 (40-75) EQ-5D-5L score, 6 months, median (IQR) 0.82 (0.53-0.94) 0.61 (0.29-0.82) CCS angina class, 6 months, median (IQR) 3.0 (1.0-3.0) 3.0 (2.0-4.0) EQ-VAS health status, 12 months, median (IQR) 70 (50-80) 58 (40-75) EQ-5D-5L score, 12 months, median (IQR) 0.82 (0.62-0.95) 0.78 (0.50-0.89) CCS angina class, 12 months, median (IQR) 3.0 (3.0-4.0) 3.0 (2.0-4.0) HRs and corresponding 95% CI from an unadjusted Cox model are given for the time from study entry to occurrence of primary outcomes only, comparing the registry to the trial group. Health outcomes During a median of 694 (IQR 558–841) days follow-up, the primary efficacy outcome occurred in 26 (43%) and 36 (49%) trial and registry participants, respectively (HR Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 6 Coronary artery disease Median (IQRs) are used for non-normally distributed data. Follow-up period was over median 694 (IQR 558–841) days. BARC, Bleeding Academic Research Consortium; CABG, coronary artery bypass graft; CCS, Canadian Cardiovascular Society; EQ-5D-5L, EuroQol 5 Dimensions 5 Levels; EQ-VAS, EuroQol Visual Analogue Scale; HF, heart failure; MI, myocardial infarction; PCI, percutaneous coronary intervention; SAE, serious adverse event. on March 5, 2021 by guest. Protected by http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from on March 5, 2021 by guest. Protect http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from HRs and corresponding 95% CI from an unadjusted Cox model are given for the time from study entry to occurrence of primary outcomes only, comparing the registry to the trial group. Median (IQRs) are used for non-normally distributed data. Follow-up period was over median 694 (IQR 558–841) days. BARC, Bleeding Academic Research Consortium; CABG, coronary artery bypass graft; CCS, Canadian Cardiovascular Society; EQ-5D-5L, EuroQol 5 Dimensions 5 Levels; EQ-VAS, EuroQol Visual Analogue Scale; HF, heart failure; MI, myocardial infarction; PCI, percutaneous coronary intervention; SAE, serious adverse event. HRs and corresponding 95% CI from an unadjusted Cox model are given for the time from study entry to occurrence of primary outcomes only, comparing the registry to the trial group. Median (IQRs) are used for non-normally distributed data. Follow-up period was over median 694 (IQR 558–841) days. BARC, Bleeding Academic Research Consortium; CABG, coronary artery bypass graft; CCS, Canadian Cardiovascular Society; EQ-5D-5L, EuroQol 5 Dimensions 5 Levels; EQ-VAS, EuroQol Visual Analogue Scale; HF, heart failure; MI, myocardial infarction; PCI, percutaneous coronary intervention; SAE, serious adverse event. 7 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 on March 5, 2021 by guest. http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Figure 2 Kaplan-Meier survival curves: during a median of 694 (IQR 558–841) days follow-up: (A) The primary efficacy outcome occurred in 26 (43%) and 36 (49%) of trial and registry participants, respectively, HR 1.14, 95% CI 0.69 to 1.89. (B) The primary safety outcome occurred in 17 (28%) and 16 (22%) of trial and registry participants, respectively, HR 0.74, 95% CI 0.37 to 1.46. DISCUSSION outcome. When considered together (efficacy or safety), the rates were very similar between the groups. There was a lower proportion in the registry group experiencing both outcomes, but these events were very small in number. The main findings of our study are, compared with the trial group, in the registry group: (1) multimorbidity, func- tional limitation, frailty and impaired health status were more pronounced; (2) changes to medication were more often made because of recurrent angina but less often made for standard optimisation of secondary prevention; (3) in invasively managed patients, the extent of jeopard- ised myocardium was similarly high and the culprit lesion was identified in half, and revascularisation by PCI was performed on one third; (4) health-related quality of life was lower at baseline, 6 and 12 months and (5) there was a fourfold increased risk of cardiovascular death, although power was limited. Overall, our study provides novel, contemporary insights into an understudied sub- group of ACS patients with a substantial health burden. Kaplan-Meier survival curves (figure 2) reveal that health outcomes in the trial and registry groups were similar during follow-up. Compared with the trial group, all-cause mortality and cardiovascular death occurred more often in the registry group, but non-fatal MI and worsening renal function during the index hospitalisa- tion occurred less often (table 4). Two-thirds of patients in both groups experienced a serious adverse event (table 4). Redo CABG occurred in only one registry patient and in none of the trial participants (table 4). Health status Our registry-based trial provided a framework for information to be prospectively gathered on patients who may have been eligible for randomisation but were not, including the reasons for not being randomised. Registry participation reflected physician and/or patient preferences for one form of treatment over another.16 These beliefs substantially limited enrolment into the randomised trial. This finding has implications for the design and funding of future trials in this popula- tion. Moreover, the finding in the comparison of the Compared to the trial group, the median EQ-VAS health status in the registry group was 25 points lower (worse) at 6 months, and 12 points lower at 12 months (table 4). guest. Protected by copyright. Coronary artery disease (C) The primary efficacy and safety outcome occurred in 10 (17%) and 8 (11%) of trial and registry participants, respectively, HR 0.64, 95% CI 0.25 to 1.63. (D) The primary efficacy or safety outcome occurred in 33 (55%) and 44 (60%) of trial and registry participants, respectively, HR 1.05, 95% CI 0.67 to 1.65. Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloade Figure 2 Kaplan-Meier survival curves: during a median of 694 (IQR 558–841) days follow-up: (A) The primary efficacy outcome occurred in 26 (43%) and 36 (49%) of trial and registry participants, respectively, HR 1.14, 95% CI 0.69 to 1.89. (B) The primary safety outcome occurred in 17 (28%) and 16 (22%) of trial and registry participants, respectively, HR 0.74, 95% CI 0.37 to 1.46. (C) The primary efficacy and safety outcome occurred in 10 (17%) and 8 (11%) of trial and registry participants, respectively, HR 0.64, 95% CI 0.25 to 1.63. (D) The primary efficacy or safety outcome occurred in 33 (55%) and 44 (60%) of trial and registry participants, respectively, HR 1.05, 95% CI 0.67 to 1.65. Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 Angina The CCS angina class was similar between the groups at 6 months (median (IQR) trial 3 (1-3) vs registry 3 (2-4)) and 12 months (median (IQR) trial 3 (3-4) vs registry 3 (2-4)) (table 4). 8 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 on March 5, 2021 by guest. Protected by copyrigh http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Coronary artery disease High event rates in patients with an NSTE-ACS and prior CABG Almost half of the participants in both groups expe- rienced a primary efficacy outcome event (table 4). In contemporary trials involving NSTE-ACS patients, the 12-month major adverse cardiac event (MACE) rate is usually 8%–10%, which is very much lower than observed in this study’s patients. The proportion of affected patients increased substantially during longer- term follow-up beyond 12 months. Again, this progressive accrual of adverse cardiac events over time contrasts with other trials in NSTE-ACS patients in which cardiac events may plateau over time. The older age and universal presence of multi-morbidity probably explain the differ- ences in prognosis between NSTE-ACS patients with versus without prior CABG. Considering future trials in NSTE-ACS patients with prior CABG, there are consider- able logistical challenges to enrolment but, however, the event rate implies that the sample size may be lower than for other populations in which primary outcome event rates are expectedly lower. randomised trial groups that health outcomes were not different with invasive management versus conserva- tive management supports the notion that enrolment rates could be increased by education of physicians and patients.21 Our trial results broadly reflect equi- poise between the randomised strategies which should enhance confidence to support enrolment into a future randomised trial. Compared with trial participation, registry participa- tion was associated with a fourfold higher likelihood of cardiovascular death, with the caveat that event rates were very low for this outcome. This prognostic associ- ation may be partly explained by the greater burden of cardiovascular health problems at baseline, including HF and valve disease. Compared with trial patients, registry patients had more medication changes for recurrent angina—this may be partly explained by some registry patients having symptoms which were not stabilised and/ or refractory ischaemia (both reasons for exclusion from the randomised trial) (figure 1). Author affiliations 1 1Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK 1Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK Advances in interventional management In our trial, invasive management was selected in 36 (49%) of registry participants at baseline, but PCI was performed in only 13 (36%) of these patients. The lower PCI rate in our population may be explained by the complex nature of native vessel and graft disease, lack of a clear culprit (in almost half), and, arguably, lack of definitive evidence in support of the benefits of PCI in this population. CONCLUSION AND POTENTIAL VALUE OF RESULTS Since clinical trials usually excluded patients with prior CABG, practice guidelines are not evidence based with respect to this group. In real-world practice, clinicians lack relevant information to inform decision making. Our trial and registry may be helpful in the design of clin- ical trials in this patient group. on March 5, 2021 by guest. Protected by copyright. ttp://openheart.bmj.com/ p p PCI continues to evolve with technical advances poten- tially leading to improvements in safety and procedural success (online supplemental discussion). Limitations Our pilot trial was not powered to assess for between- group differences in the rates of the serious adverse events contributing to the prespecified efficacy and safety outcomes. The sample size was small, with resultant wide confidence intervals. Both groups included patients that were managed differently (PCI vs medical therapy), thereby confounding between-group comparisons. Angina However, up-titration of medical therapy for secondary prevention occurred less often in the registry group, implying less intensive management, less scope for therapy improvements or both. The results highlight the substantial levels of morbidity, polypharmacy and adverse health outcomes in this group. The clinical course of two participants is illus- trated (online supplemental figures 1 and 2). Feasibility of a future substantive trial in patients with an NSTE-ACS and prior CABG 2West of Scotland Heart and Lung Centre, Golden Jubilee National Hospital, Clydebank, UK 3Cardiology, Glasgow Royal Infirmary, Glasgow, UK 4Cardiology, Western Infirmary, Glasgow, UK 5Cardiology, Royal Alexandra Hospital, Paisley, UK 6Cardiology, Royal Blackburn Hospital, Blackburn, UK 7Robertson Centre for Biostatistics, University of Glasgow, Glasgow, UK 8Health Economics and Health Technology Assessment, University of Glasgow, Glasgow, UK 9Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK 2West of Scotland Heart and Lung Centre, Golden Jubilee National Hospital, Clydebank, UK 3i About one in 10–15 NSTE-ACS patients have a prior CABG (online supplemental table 1).6 This proportion is likely to remain stable in the coming years reflecting revascularisation practices in the past decade and increasing longevity. Many participants in this study were elderly, frail and multimorbid. Screening and obtaining informed consent were time-consuming for research staff. Medical decisions during urgent care may happen out-with office hours when research staff availability was limited. Medical information was commonly lacking at the time of hospitalisation, for example, graft history, limited recall by patients. These considerations present logistical barriers to enrolling patients into a randomised trial. h 5, 2021 by guest. Protected by copyright. guest. Protected by copyright. Contributors All authors meet all four ICMJE criteria for authorship. MMYL and AB coordinated study, enrolled patients, collected clinical data; MMYL also coordinated Clinical Event Committee. MCP, PR, JS and CEJ: clinical Event Committee. APR, CB, INFi and KPB: local principal investigators. MBM, AS, SHMH, EEP, CLM, AJC and NBB: enrolled patients, collected clinical data. DSC and KM: collected clinical data. PC, NPS: angiographic analyses for BCIS-1 Jeopardy Score. OW: study design and health economics. IFo, AM and SJEB: biostatistics. CB: chief investigator. 9 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 ORCID iDs Matthew M Y Lee http://orcid.org/0000-0001-9213-2067 Mark C Petrie http://orcid.org/0000-0003-1350-1544 Paul Rocchiccioli http://orcid.org/0000-0003-1350-1544 David S Corcoran http://orcid.org/0000-0001-5047-0885 Kenneth Mangion http://orcid.org/0000-0002-3505-7440 Ian Ford http://orcid.org/000-0001-5927-1823 Olivia Wu http://orcid.org/0000-0002-0570-6016 Alex McConnachie http://orcid.org/0000-0002-7262-7000 Sarah J E Barry http://orcid.org/0000-0003-3039-8729 Colin Berry http://orcid.org/0000-0002-4547-8636 14 Fanning JP, Nyong J, Scott IA, et al. Routine invasive strategies versus selective invasive strategies for unstable angina and non-ST elevation myocardial infarction in the stent era. Cochrane Database Syst Rev 2016:CD004815. y 15 Asrar Ul Haq M, Rudd N, Mian M, et al. Predictors and outcomes of early coronary angiography in patients with prior coronary artery bypass surgery presenting with non-ST elevation myocardial infarction. Open Heart 2014;1:e000059. 16 Feit F, Brooks MM, Sopko G, et al. Long-term clinical outcome in the Bypass Angioplasty Revascularization Investigation Registry: comparison with the randomized trial. BARI Investigators. Circulation 2000;101:2795–802. Data availability statement The data that support the findings of this study are available from the corresponding author on reasonable request. 11 Elbarasi E, Goodman SG, Yan RT, et al. Management patterns of non-ST segment elevation acute coronary syndromes in relation to prior coronary revascularization. Am Heart J 2010;159:40–6. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 12 Al-Aqeedi R, Asaad N, Al-Qahtani A, et al. Acute coronary syndrome in patients with prior coronary artery bypass surgery: observations from a 20-year registry in a Middle-Eastern country. PLoS One 2012;7:e40571. 13 Nikolsky E, McLaurin BT, Cox DA, et al. Outcomes of patients with prior coronary artery bypass grafting and acute coronary syndromes: analysis from the ACUITY (Acute Catheterization and Urgent Intervention Triage Strategy) trial. JACC Cardiovasc Interv 2012;5:919–26. REFERENCES 17 Sanchis J, Núñez E, Barrabés JA, et al. 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Heart 2015;101:1182–9. on March 5, 2021 by g http://openheart.bmj.com/ Open Heart: first published as 10.1136/openhrt-2020-001453 on 26 February 2021. Downloaded from Funding CB has received research support from the British Heart Foundation (PG/17/2532884, FS/17/26/32744, and RE/18/6134217) and the Medical Research Council (MR/S005714/1). KM and NS were supported by BHF Clinical Training Fellowships (FS/15/54/31639; FS/17/26/32744). This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Myocardial Revascularization of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic surgery (EACTS). Developed with the special contribution of the European Association of Percutaneous Cardiovascular Interventions (EAPCI). Eur Heart J 2014;35:2541–619. Myocardial Revascularization of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic surgery (EACTS). Developed with the special contribution of the European Association of Percutaneous Cardiovascular Interventions (EAPCI). Eur Heart J 2014;35:2541–619. 8 Hamm CW, Bassand JP, Agewall S, et al. ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: the task force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J 2011;32:2999–3054. Competing interests CB is employed by the University of Glasgow, which holds consultancy and/or research agreements with companies that have commercial interests in the diagnosis and treatment of ischaemic heart disease, including Abbott Vascular, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, HeartFlow, Menarini Farmaceutica, Opsens, Philips and Siemens Healthcare. There are no other relevant personal disclosures. 9 National Institute for Health and Care Excellence (NICE). Unstable angina and NSTEMI: early management. Clinical guideline 94, 2010. Available: https://www.nice.org.uk/guidance/cg94 [Accessed 17 Sep 2020]. Patient consent for publication Not required. Patient consent for publication Not required. Ethics approval The study was reviewed and approved by the West of Scotland National Health Service Research Ethics Service (Reference 11-WS-0116). 10 Kim MS, Wang TY, Ou FS, et al. Association of prior coronary artery bypass graft surgery with quality of care of patients with non-ST- segment elevation myocardial infarction: a report from the National Cardiovascular Data Registry Acute Coronary Treatment and Intervention Outcomes Network Registry-Get With the Guidelines. Am Heart J 2010;160:951–7. Provenance and peer review Not commissioned; internally peer reviewed. Provenance and peer review Not commissioned; internally peer reviewed. Data availability statement The data that support the findings of this study are available from the corresponding author on reasonable request. Data availability statement The data that support the findings of this study are available from the corresponding author on reasonable request. doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Setting 3 Study participants were enrolled in two large urban hospitals and two regional district general 4 hospitals in the National Health Service (NHS) in the United Kingdom (UK). The hospitals 5 differed in geography, availability of catheter laboratory facilities on-site (or not), and hospital 6 type (academic vs. regional). Royal Blackburn Hospital was the only hospital with an on-site 7 cardiac catheterisation laboratory. In the other hospitals, patients were triaged for invasive 8 management by referral and transferred to the regional cardiothoracic centre (Golden Jubilee 9 National Hospital). 10 REFERENCES 5 Bhatnagar P, Wickramasinghe K, Williams J, et al. The epidemiology of cardiovascular disease in the UK 2014. Heart 2015;101:1182–9. 6 Shoaib A, Kinnaird T, Curzen N, et al. Outcomes following percutaneous coronary intervention in Non-ST-Segment-Elevation myocardial infarction patients with coronary artery bypass grafts. Circ Cardiovasc Interv 2018;11:e006824. 22 Mehran R, Rao SV, Bhatt DL, et al. Standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the Bleeding Academic Research Consortium. Circulation 2011;123:2736–47. 7 Windecker S, Kolh P, et al, Authors/Task Force members. 2014 ESC/ EACTS guidelines on myocardial revascularization: the Task Force on 7 Windecker S, Kolh P, et al, Authors/Task Force members. 2014 ESC/ EACTS guidelines on myocardial revascularization: the Task Force on 10 Lee MMY, et al. Open Heart 2021;8:e001453. doi:10.1136/openhrt-2020-001453 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance rial placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Open Heart Supplemental material Supplemental Methods 2 SUPPLEMENTAL MATERIAL SUPPLEMENTAL MATERIAL 1 Supplemental Methods Invasive group 30 Invasive management was performed early (i.e. ≤72 hours wherever possible) after hospital 31 admission. Invasive management included native coronary and bypass graft angiography and 32 coronary and/or graft revascularisation with percutaneous coronary intervention (PCI) and/or 33 CABG, as clinically appropriate. 34 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Screening 11 The clinical research team on each site screened for patients aged ≥18 years, of either sex, 12 admitted during unscheduled emergency care with a suspected acute non-ST segment elevation 13 acute coronary syndrome and prior coronary artery bypass graft (CABG). Screening took place 14 in the acute medical and cardiology wards during the course of routine healthcare. Patients 15 eligible for either invasive (with coronary and graft angiography) or non-invasive management 16 were invited to participate. Eligible patients were given an information sheet prior to 17 participation. All randomised and registry patients provided written informed consent as soon as 18 feasible after hospital admission and prior to referral for coronary angiography. Each patient was 19 given a site and study number and entered into a screening log which only contained de- 20 identified information. Patients who did not consent were included in the „screen failure‟ log. 21 1 1 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart The community health index (CHI) or NHS number was recorded to enable electronic record 22 linkage. 23 The community health index (CHI) or NHS number was recorded to enable electronic record 22 linkage. 23 Non-invasive group 27 g p Participants who had been randomised to the non-invasive group could be referred for invasive 28 management if any pre-specified criteria were met (Supplemental Methods). 29 Participants who had been randomised to the non-invasive group could be referred for invasive 28 management if any pre-specified criteria were met (Supplemental Methods). 29 Randomisation 24 Randomisation was stratified by centre, using randomised permuted blocks of length 4 and 6, 25 with block lengths chosen at random. 26 Randomisation was stratified by centre, using randomised permuted blocks of length 4 and 6, 25 with block lengths chosen at random. 26 Optimal medical therapy 35 Optimal medical therapy was intended for all of the participants. Guidance on up-titration of 36 medical therapy was provided in an investigator guideline. Medical therapy included dual anti- 37 platelet, anti-thrombotic, and anti-ischaemic therapies as per local protocols and international 38 guidelines.[1,2] 39 Optimal medical therapy was intended for all of the participants. Guidance on up-titration of 36 medical therapy was provided in an investigator guideline. Medical therapy included dual anti- 37 platelet, anti-thrombotic, and anti-ischaemic therapies as per local protocols and international 38 guidelines.[1,2] 39 Non-invasive group 40 2 Study participants who had been randomised to the non-invasive group could be referred for 41 invasive management if one of the following pre-specified criteria are met: 1) recurrent or 42 2 Study participants who had been randomised to the non-invasive group could be referred for 41 invasive management if one of the following pre-specified criteria are met: 1) recurrent or 42 Study participants who had been randomised to the non-invasive group could be referred for 41 invasive management if one of the following pre-specified criteria are met: 1) recurrent or 42 2 2 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance lemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart refractory (class III or IV) angina with documented ischaemic electrocardiogram (ECG) changes 43 whilst on “optimal” anti-ischaemic therapy, 2) new ST-segment elevation in two contiguous 44 leads without Q waves or T wave inversion greater than 3 mm or development of haemodynamic 45 instability, or 3) a deterioration in heart failure (HF) status (consistent with Killip class 3 or 4) 46 that the attending clinician judges to be ischaemia-related based on the presence of symptoms, 47 ECG changes and cardiac biomarker elevation. 48 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Clinical Event Committee 64 The Clinical Event Committee (CEC) reviewed cases of interest to determine if they meet the 65 criteria defined in the pre-specified charter. Causality assessments were not made by the CEC. 66 The CEC was blinded to all information relating to the randomisation group. The CEC included 67 4 cardiovascular physicians who have expertise in the diagnosis and treatment of cardiovascular 68 disorders and in the medical aspects of clinical trials. The CEC had a Chairman (M.C.P.) and 69 coordinator (M.M.Y.L.) to assist with preparation of de-identified source clinical data, reports 70 and communication with the Trials Unit. The CEC followed a pre-determined adjudication 71 charter. 72 Follow-up and outcome collection 49 63 3 3 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance mental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Follow-up and outcome collection 49 Clinical research nurses and clinicians who were independent of the study teams and aware of 50 the group allocations supported enrolment and follow-up assessments on all sites. They 51 prospectively gathered information on screening, recruitment, randomisation (to medical therapy 52 or invasive management), crossover rates, and serious adverse events in patients with prior 53 coronary artery bypass graft and a recent non-ST segment elevation acute coronary syndrome. 54 Data will be held for up to 20 years to enable long-term follow-up analyses. Following 55 randomisation, clinical assessments involved gathering information from standard-of-care 56 clinical reviews (end of hospitalisation, 30-42 days and 1 year) and also from clinical contacts 57 recorded in the patients‟ medical records. In West of Scotland hospitals, a single system of 58 electronic patient records is used for all hospital attendances and correspondence with primary 59 care. 60 Clinical research nurses and clinicians who were independent of the study teams and aware of 50 the group allocations supported enrolment and follow-up assessments on all sites. They 51 prospectively gathered information on screening, recruitment, randomisation (to medical therapy 52 or invasive management), crossover rates, and serious adverse events in patients with prior 53 coronary artery bypass graft and a recent non-ST segment elevation acute coronary syndrome. 54 Data will be held for up to 20 years to enable long-term follow-up analyses. Following 55 randomisation, clinical assessments involved gathering information from standard-of-care 56 clinical reviews (end of hospitalisation, 30-42 days and 1 year) and also from clinical contacts 57 recorded in the patients‟ medical records. In West of Scotland hospitals, a single system of 58 electronic patient records is used for all hospital attendances and correspondence with primary 59 care. 60 Serious adverse events during the index admission and follow-up were evaluated from review of 61 patient records obtained during usual care, and electronic health databases, using the CHI and 62 NHS number. All outcomes were prospectively entered into an electronic Case Report Form. doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Refractory ischaemia 74 This definition 80 is in line with the Timing of Intervention in Acute Coronary Syndromes (TIMACS) trial.[3] 81 D h 82 All-cause, sudden cardiac death, death due to MI, death due to HF, death due to stroke, death due 83 to extra-axial haemorrhage, death due to cardiovascular operation, death due to other 84 All-cause, sudden cardiac death, death due to MI, death due to HF, death due to stroke, death due 83 to extra-axial haemorrhage, death due to cardiovascular operation, death due to other 84 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance plemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance pplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart cardiovascular cause (e.g. infective endocarditis), presumed cardiovascular death (undetermined 85 cause of death), non-cardiovascular death.[4] 86 Worsening renal function 95 Defined as deterioration in estimated Glomerular Filtration Rate ≥ 25% of baseline during the 96 index admission. 97 Crossover 98 A crossover between groups was defined as a change of treatment strategy from invasive to non- 99 invasive management, or vice versa. In addition, we pre-defined crossover as occurring within 30 100 days after randomisation. 101 Crossover 98 A crossover between groups was defined as a change of treatment strategy from invasive to non- 99 invasive management, or vice versa. In addition, we pre-defined crossover as occurring within 30 100 days after randomisation. 101 Refractory ischaemia 74 Recurrent ischaemic symptoms lasting more than 5 minutes, whilst on optimal medical therapy 75 (at least 2 anti-anginal treatments) with documented characteristic ECG changes indicative of 76 ischaemia and requiring an additional intervention. An additional intervention was defined as 77 reperfusion therapy for myocardial infarction (MI), cardiac catheterisation, and insertion of intra- 78 aortic balloon pump or revascularisation procedure (percutaneous coronary intervention or 79 coronary artery bypass graft surgery) within 48 hours of the onset of this episode. This definition 80 is in line with the Timing of Intervention in Acute Coronary Syndromes (TIMACS) trial.[3] 81 Recurrent ischaemic symptoms lasting more than 5 minutes, whilst on optimal medical therapy 75 (at least 2 anti-anginal treatments) with documented characteristic ECG changes indicative of 76 ischaemia and requiring an additional intervention. An additional intervention was defined as 77 reperfusion therapy for myocardial infarction (MI), cardiac catheterisation, and insertion of intra- 78 aortic balloon pump or revascularisation procedure (percutaneous coronary intervention or 79 coronary artery bypass graft surgery) within 48 hours of the onset of this episode. This definition 80 is in line with the Timing of Intervention in Acute Coronary Syndromes (TIMACS) trial.[3] 81 Death 82 All-cause, sudden cardiac death, death due to MI, death due to HF, death due to stroke, death due 83 to extra-axial haemorrhage, death due to cardiovascular operation, death due to other 84 Recurrent ischaemic symptoms lasting more than 5 minutes, whilst on optimal medical therapy 75 (at least 2 anti-anginal treatments) with documented characteristic ECG changes indicative of 76 ischaemia and requiring an additional intervention. An additional intervention was defined as 77 reperfusion therapy for myocardial infarction (MI), cardiac catheterisation, and insertion of intra- 78 aortic balloon pump or revascularisation procedure (percutaneous coronary intervention or 79 coronary artery bypass graft surgery) within 48 hours of the onset of this episode. Procedure-related MI 87 5 Procedure-related MI 87 According to the Universal Definition of MI (Type 4b).[5] A post-procedure ECG was used to 88 diagnose Q-wave vs. non-Q-wave MI. 89 Stroke 90 Defined as the presence of a new focal neurologic deficit thought to be vascular in origin, with 91 signs or symptoms lasting more than 24 hours;[3] subdural haemorrhage. 92 Major bleeding 93 Defined according to the Bleeding Academic Research Consortium criteria.[6] 94 Worsening renal function 95 Defined as deterioration in estimated Glomerular Filtration Rate ≥ 25% of baseline during the 96 index admission. 97 Crossover 98 A crossover between groups was defined as a change of treatment strategy from invasive to non- 99 invasive management, or vice versa. In addition, we pre-defined crossover as occurring within 30 100 days after randomisation. 101 Sample Size 102 Since CABG-ACS was an exploratory pilot trial, no sample size calculation was performed. The 103 sample size was n=60 based on the number of participants projected to be enrolled in 4 hospitals 104 within a 12–18 month period. We chose this number across different secondary care settings to 105 According to the Universal Definition of MI (Type 4b).[5] A post-procedure ECG was used to 88 diagnose Q-wave vs. non-Q-wave MI. 89 According to the Universal Definition of MI (Type 4b).[5] A post-procedure ECG was used to 88 diagnose Q-wave vs. non-Q-wave MI. 89 Sample Size 102 5 Sample Size 102 Since CABG-ACS was an exploratory pilot trial, no sample size calculation was performed. The 103 sample size was n=60 based on the number of participants projected to be enrolled in 4 hospitals 104 within a 12–18 month period. We chose this number across different secondary care settings to 105 5 Sample Size 102 Since CABG-ACS was an exploratory pilot trial, no sample size calculation was performed. The 103 sample size was n=60 based on the number of participants projected to be enrolled in 4 hospitals 104 within a 12–18 month period. We chose this number across different secondary care settings to 105 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart be broadly representative of the diversity in UK hospitals. The sample size was selected to 106 enable the feasibility of randomisation, and the reasons for not being randomised were 107 prospectively recorded. The trial was designed but not powered to assess for between-group 108 differences in the rates of the serious adverse events contributing to the prespecified efficacy and 109 safety outcomes. 110 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Data management and biostatistics 111 The Robertson Centre for Biostatistics acted as an independent coordinating centre for data 112 management and statistical analyses. The Centre is registered with a Clinical Trials Unit 113 (National Institute for Health Research Registration number: 16). The Chief Investigator 114 (Professor Berry) had full access to all the data in the study and takes responsibility for its 115 integrity and the data analysis. 116 Patient confidentiality 117 Patients were assigned an identification code at the time of recruitment. 118 Patients were assigned an identification code at the time of recruitment. 118 Ethics 134 Potential benefits to participants include avoidance of harmful invasive management and 135 avoidance of longer-term stent failure. No additional interventions were proposed nor were 136 procedures withdrawn that would be needed on clinical grounds. While the intention-to-treat in 137 each group was either with non-invasive or invasive management, all treatment options remained 138 available according to patient and physician preference i.e. patients initially randomised to 139 medical therapy could have undergone invasive management and vice versa. 140 Baseline data 120 Baseline characteristics were summarised using mean (standard deviation (SD)) or median 121 (interquartile range (IQR) for skewed data) for continuous variables, and count (%) for 122 categorical variables. Baseline characteristics for randomised vs. registry participants were 123 compared using t-tests, Mann-Whitney tests and chi-squared tests (or Fisher's exact tests) as 124 appropriate. 125 6 6 doi: 10.1136/openhrt-2020-00145 :e001453. 8 2021; Open Heart , et al. Lee MMY BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance emental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Efficacy and safety outcomes 126 Numbers of events and numbers (%) of patients with adverse events were summarised. The 127 proportion of patients with adverse events was compared between the registry and trial groups 128 with a chi-squared test. Kaplan-Meier curves were produced for time to occurrence of the 129 primary efficacy and safety outcomes. The hazard ratios (HR) of the primary outcomes, 130 comparing the registry to trial group, were calculated with corresponding 95% confidence 131 interval (CI) from a Cox model. Secondary outcomes were presented as descriptive statistics 132 only, since this was a pilot trial with insufficient power for statistical testing of these outcomes. 133 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Trial management 141 A Trial Management Group including the researchers and Local Principal Investigator on each of 142 the 4 sites coordinated the study‟s activities on a day-to-day basis. The NHS Sponsor monitored 143 the trial. Since the trial was a pilot, there was no Independent Data and Safety Monitoring 144 Committee. 145 7 doi: 10.1136/openhrt-2020-00145 :e001453. 8 2021; Open Heart , et al. Lee MMY BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance mental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Supplemental Discussion 166 8 8 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Registry patients who were selected for invasive management may have been identified by 167 clinicians as being potentially amenable to gaining symptomatic or prognostic benefit from 168 revascularisation. Conversely, registry patients who were selected for medical management may 169 have been judged as having little to gain and at risk of harm from invasive management and with 170 non-modifiable chronic health impairment. 171 Registry patients who were selected for invasive management may have been identified by 167 clinicians as being potentially amenable to gaining symptomatic or prognostic benefit from 168 revascularisation. Conversely, registry patients who were selected for medical management may 169 have been judged as having little to gain and at risk of harm from invasive management and with 170 non-modifiable chronic health impairment. 171 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental Discussion 146 Pivotal trials excluded patients with prior CABG, limiting the applicability of practice guidelines 147 that recommend invasive management in non-ST segment elevation acute coronary syndrome 148 (NSTE-ACS) with prior CABG.[1,2,7] Our results provide real-world insights into the baseline 149 characteristics, treatment and outcomes of patients who were ineligible for randomisation but 150 provided informed consent for registry participation. Commonly, this information is not gathered 151 in clinical trials due to resource implications and logistics. Bypass Angioplasty Revascularization 152 Investigation (BARI) was a trial of percutaneous transluminal coronary angioplasty (PTCA) 153 versus CABG.[8] BARI included a registry of eligible patients who were not randomised based 154 on physician and/or patient preference.[8] The main reason for not being randomised was 155 physician and patient preference for PTCA. In BARI, the physicians selected PTCA rather than 156 CABG for 65% of registry patients who underwent revascularisation without compromising 157 long-term survival either in the overall population or in patients with treated diabetes. This result 158 is in contrast to the randomised trial where patients with treated diabetes who underwent CABG 159 gained a survival advantage compared to those patients with treated diabetes who had PTCA. 160 We also gathered information on the selection process for trial participation, providing insights 161 into the reasons for this decision. Within the registry, invasive management was substantially the 162 preferred strategy by physicians and cardiologists. However, the proportions of patients treated 163 by PCI in the registry group and the invasive group in the randomised trial were similarly low. A 164 registry can disclose information on patient subsets in whom an intervention may have 165 differential effects (harm or benefit). 166 We also gathered information on the selection process for trial participation, providing insights 161 into the reasons for this decision. Within the registry, invasive management was substantially the 162 preferred strategy by physicians and cardiologists. However, the proportions of patients treated 163 by PCI in the registry group and the invasive group in the randomised trial were similarly low. A 164 registry can disclose information on patient subsets in whom an intervention may have 165 differential effects (harm or benefit). doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Advances in interventional management 172 In recent years, radial artery access has become the standard approach for invasive management 173 rather than femoral artery access. The left radial artery can provide arterial access in patients 174 with a left internal mammary graft. However, Complete High risk Indicated Patient (CHIP) 175 procedures may require simultaneous left and right coronary artery access which necessitates 176 vascular access via the femoral artery. Overall, our results support the safety of invasive 177 management in selected NSTE-ACS patients with prior CABG. 178 Advances in CHIP procedures lead to new possibilities for revascularisation in patients with 179 complex disease.[9] Specialist techniques have developed with „antegrade‟ and „retrograde‟ 180 approaches to recanalize chronic totally occluded (CTO) coronary arteries such that CTO PCI in 181 native vessel CTOs has become increasingly feasible.[9] However, CTO PCI procedures are 182 complex, require advanced skills, only undertaken by a minority of interventional cardiologists, 183 and are usually pre-planned on an elective basis. Equipment can be expensive. Some of these 184 techniques evolved very recently and so were not routinely implemented in the invasively 185 managed patients. Whether CHIP procedures would increase revascularisation rates, comparable 186 safety, and improvements in prognosis merit prospective evaluation in a substantive multicentre 187 trial.[9] 188 9 9 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Abbreviations: Acronyms of trials 189 189 FRISC II = FRagmin and Fast Revascularisation during InStability in Coronary artery disease 190 ICTUS = Invasive versus Conservative Treatment in Unstable Coronary Syndromes 191 ISAR-COOL = Intracoronary Stenting with Antithrombotic Regimen Cooling-Off 192 LIPSIA-NSTEMI = The Leipzig Immediate versus early and late PercutaneouS coronary 193 Intervention triAl in NSTEMI 194 MATE = Medicine versus Angiography in Thrombolytic Exclusion 195 MOSCA = coMOrbilidades en el Síndrome Coronario Agudo 196 OASIS-5 = Fifth Organization to Assess Strategies in Ischemic Syndromes 197 RINCAL = Revascularisation or Medical Therapy in Elderly Patients with Acute Anginal 198 Syndromes 199 RITA 3 = Randomized Intervention Trial of unstable Angina 200 TACTICS-TIMI 18 = Treat Angina with Aggrastat and Determine Cost of Therapy with an 201 Invasive or Conservative Strategy – Thrombolysis in Myocardial Infarction 18 202 TIMACS = Timing of Intervention in Acute Coronary Syndromes 203 TIMI IIIB = Thrombolysis in Myocardial Ischemia 204 TRUCS = Treatment of Refractory Unstable angina in geographically isolated areas without 205 Cardiac Surgery 206 VANQWISH = Veterans Affairs Non-Q-Wave Infarction Strategies in Hospital 207 VINO = Value of first day angiography/angioplasty In evolving Non-ST segment elevation 208 myocardial infarction: an Open multicenter randomized trial 209 VANQWISH = Veterans Affairs Non-Q-Wave Infarction Strategies in Hospital 207 VANQWISH = Veterans Affairs Non-Q-Wave Infarction Strategies in Hospital 207 VINO = Value of first day angiography/angioplasty In evolving Non-ST segment elevation 208 myocardial infarction: an Open multicenter randomized trial 209 VINO = Value of first day angiography/angioplasty In evolving Non-ST segment elevation 208 myocardial infarction: an Open multicenter randomized trial 209 10 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance ntal material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Supplemental Tables 210 Supplemental Tables Supplemental Table 1. Trials of patients with non-ST elevation acute coronary syndromes. doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Abbreviations: Acronyms of trials 189 1 Trials which included patients with prior coronary artery bypass grafting (CABG) Trial Year published N N (%) with prior CABG VANQWISH[10] 1998 920 156 (17.0%) (CABG >3 months before randomisation) MATE[11] 1998 201 19 (9.5%) TRUCS[12] 2000 148 18 (12.2%) TACTICS-TIMI 18[13] 2001 2220 484 (21.8%) (CABG >6 months before randomisation) ISAR-COOL[14] 2003 410 48 (11.7%) ICTUS[15] 2005 1200 105 (8.8%) OASIS-5[16] 2009 20078 1643 (8.2%) Italian Elderly ACS[17] 2012 313 29 (9.3%) LIPSIA-NSTEMI[18] 2012 600 41 (6.8%) CABG-ACS pilot[19,20] 2016 60 60 (100.0%) After Eighty study[21] 2016 457 76 (16.6%) MOSCA[22] 2016 106 14 (13.2%) Trials which excluded patients with prior CABG Trial Year published N Exclusion TIMI IIIB[23] 1994 1473 CABG at any time FRISC-II[24] 1999 2457 Previous open-heart surgery VINO[25] 2002 131 CABG less than 6 months RITA 3[26] 2002 1810 CABG at any time 2 Supplemental Table 1. Trials of patients with non-ST elevation acute coronary syndromes. 211 Supplemental Table 1. Trials of patients with non-ST elevation acute coron 211 lemental Table 1. Trials of patients with non-ST elevation acute coronary syndromes. 212 11 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance tal material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart 213 Supplemental Figures doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental Figure 1: 217 Clinical case example: This 72-year-old male in the registry had an inpatient coronary angiogram 218 at baseline which showed a (A) blocked right coronary artery (red arrow) and a (B) tight stenosis 219 in his saphenous vein graft supplying the obtuse marginal artery (orange arrow). He underwent 220 (C) successful percutaneous coronary intervention to this saphenous vein graft (yellow arrow), 221 thereby restoring flow to his (D) right coronary artery (green arrow) which was collateralised by 222 his circumflex artery. This case highlights a diseased culprit vessel supplying collaterals to 223 another territory that is not supplied by another patent saphenous vein graft or native artery. This 224 participant experienced serious adverse events during follow-up including bleeding (Bleeding 225 Academic Research Consortium type 2) and heart failure hospitalisation. 226 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental Figure 1. 214 Supplemental Figure 1. 214 A B C D B A B D C D C 12 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Open Heart Supplemental material Supplemental Figure 2. 215 Supplemental Figure 2. 215 A B C D B A B D C D C 13 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Supplemental Figure Legends 216 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental Figure 2: 228 Clinical case example: This 63-year-old male with a history of previous coronary artery bypass 229 grafting and multiple coronary stents for intractable angina, was admitted with a non-ST 230 elevation acute coronary syndrome and was recruited to the CABG-ACS registry due to 231 physician preference for invasive management. Urgent inpatient coronary angiography revealed 232 a (A) occluded native right coronary artery (red arrow) and (B) diseased saphenous vein graft- 233 right coronary artery (orange arrow). (C and D) Percutaneous coronary intervention to his 234 saphenous vein graft-right posterior descending artery (yellow) was unsuccessful (plain old 235 balloon angioplasty only, no stents). Following multi-disciplinary team discussion, he 236 subsequently underwent redo coronary artery bypass grafting (long saphenous vein to posterior 237 14 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance mental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance upplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart descending artery) but unfortunately after a protracted post-operative recovery period, he did not 238 survive. 239 15 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance mental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance pplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart 240 doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental References 1 Windecker S, Kolh P, Alfonso F, et al. 2014 ESC/EACTS Guidelines on myocardial 241 revascularization: The Task Force on Myocardial Revascularization of the European 242 Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery 243 (EACTS). Eur Heart J 2014;35:2541–619. doi:10.1093/eurheartj/ehu278 244 2 Hamm CW, Bassand JP, Agewall S, et al. ESC Guidelines for the management of acute 245 coronary syndromes in patients presenting without persistent ST-segment elevation. Eur 246 Heart J 2011;32:2999–3054. doi:10.1093/eurheartj/ehr236 247 3 Mehta SR, Granger CB, Boden WE, et al. Early versus delayed invasive intervention in 248 acute coronary syndromes. N Engl J Med 2009;360:2165–75. 249 doi:10.1056/NEJMoa0807986 250 4 Cutlip DE, Windecker S, Mehran R, et al. Clinical end points in coronary stent trials: a 251 case for standardized definitions. 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Long-term clinical outcome in the Bypass 262 1 Windecker S, Kolh P, Alfonso F, et al. 2014 ESC/EACTS Guidelines on myocardial 241 revascularization: The Task Force on Myocardial Revascularization of the European 242 Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery 243 (EACTS). Eur Heart J 2014;35:2541–619. doi:10.1093/eurheartj/ehu278 244 1 Windecker S, Kolh P, Alfonso F, et al. 2014 ESC/EACTS Guidelines on myocardial 241 revascularization: The Task Force on Myocardial Revascularization of the European 242 Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery 243 (EACTS). Eur Heart J 2014;35:2541–619. doi:10.1093/eurheartj/ehu278 244 8 Feit F, Brooks MM, Sopko G, et al. Supplemental References Long-term clinical outcome in the Bypass 262 16 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart Angioplasty Revascularization Investigation Registry: comparison with the randomized 263 trial. 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Eur Heart J 2000;21:1954–9. doi:10.1053/euhj.2000.2397 280 13 Cannon CP, Weintraub WS, Demopoulos LA, et al. Comparison of Early Invasive and 281 Conservative Strategies in Patients with Unstable Coronary Syndromes Treated with the 282 Glycoprotein IIb/IIIa Inhibitor Tirofiban. N Engl J Med 2001;344:1879–87. 283 doi:10.1056/NEJM200106213442501 284 14 Neumann FJ, Kastrati A, Pogatsa-Murray G, et al. 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Early Invasive versus Selectively Invasive 289 Management for Acute Coronary Syndromes. N Engl J Med 2005;353:1095–104. 290 doi:10.1056/nejmoa044259 291 16 Jolly SS, Faxon DP, Fox KAA, et al. Efficacy and safety of fondaparinux versus 292 enoxaparin in patients with acute coronary syndromes treated with glycoprotein IIb/IIIa 293 inhibitors or thienopyridines: results from the OASIS 5 (Fifth Organization to Assess 294 Strategies in Ischemic Syndromes) trial. J Am Coll Cardiol 2009;54:468–76. 295 doi:10.1016/j.jacc.2009.03.062 296 17 Savonitto S, Cavallini C, Petronio AS, et al. Early Aggressive Versus Initially 297 Conservative Treatment in Elderly Patients With Non–ST-Segment Elevation Acute 298 Coronary Syndrome: A Randomized Controlled Trial. JACC Cardiovasc Interv 299 2012;5:906–16. doi:10.1016/j.jcin.2012.06.008 300 18 Thiele H, Rach J, Klein N, et al. doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. 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Early Aggressive Versus Initially 297 Conservative Treatment in Elderly Patients With Non–ST-Segment Elevation Acute 298 Coronary Syndrome: A Randomized Controlled Trial. JACC Cardiovasc Interv 299 2012;5:906–16. doi:10.1016/j.jcin.2012.06.008 300 18 Thiele H, Rach J, Klein N, et al. Optimal timing of invasive angiography in stable non- 301 ST-elevation myocardial infarction: The Leipzig Immediate versus early and late 302 PercutaneouS coronary Intervention triAl in NSTEMI (LIPSIA-NSTEMI Trial). Eur 303 Heart J 2012;33:2035–43. doi:10.1093/eurheartj/ehr418 304 18 Thiele H, Rach J, Klein N, et al. Optimal timing of invasive angiography in stable non- 301 ST-elevation myocardial infarction: The Leipzig Immediate versus early and late 302 PercutaneouS coronary Intervention triAl in NSTEMI (LIPSIA-NSTEMI Trial). Eur 303 Heart J 2012;33:2035–43. doi:10.1093/eurheartj/ehr418 304 19 Lee MMY, Petrie MC, Rocchiccioli P, et al. Non-invasive versus invasive management in 305 patients with prior coronary artery bypass surgery with a non-ST segment elevation acute 306 coronary syndrome: study design of the pilot randomised controlled trial and registry 307 (CABG-ACS). Open Heart 2016;3:e000371. doi:10.1136/openhrt-2015-000371 308 19 Lee MMY, Petrie MC, Rocchiccioli P, et al. Non-invasive versus invasive management in 305 patients with prior coronary artery bypass surgery with a non-ST segment elevation acute 306 coronary syndrome: study design of the pilot randomised controlled trial and registry 307 (CABG-ACS). Open Heart 2016;3:e000371. doi:10.1136/openhrt-2015-000371 308 20 Lee MMY, Petrie MC, Rocchiccioli P, et al. Invasive Versus Medical Management in 309 Patients With Prior Coronary Artery Bypass Surgery With a Non-ST Segment Elevation 310 20 Lee MMY, Petrie MC, Rocchiccioli P, et al. Supplemental References Invasive Versus Medical Management in 309 Patients With Prior Coronary Artery Bypass Surgery With a Non-ST Segment Elevation 310 18 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart 19 Acute Coronary Syndrome: A Pilot Randomized Controlled Trial. Circ Cardiovasc Interv 311 2019;12:e007830. doi:10.1161/CIRCINTERVENTIONS.119.007830 312 21 Tegn N, Abdelnoor M, Aaberge L, et al. Invasive versus conservative strategy in patients 313 aged 80 years or older with non-ST-elevation myocardial infarction or unstable angina 314 pectoris (After Eighty study): an open-label randomised controlled trial. Lancet 315 2016;387:1057–65. doi:10.1016/S0140-6736(15)01166-6 316 22 Sanchis J, Núñez E, Barrabés JA, et al. Randomized comparison between the invasive and 317 conservative strategies in comorbid elderly patients with non-ST elevation myocardial 318 infarction. Eur J Intern Med 2016;35:89–94. doi:10.1016/j.ejim.2016.07.003 319 23 The TIMI IIIB Investigators. Effects of tissue plasminogen activator and a comparison of 320 early invasive and conservative strategies in unstable angina and non-Q-wave myocardial 321 infarction. Results of the TIMI IIIB Trial. Thrombolysis in Myocardial Ischemia. 322 Circulation 1994;89:1545–56. doi:10.1016/S0196-0644(94)70199-7 323 24 FRagmin and Fast Revascularisation during InStability in Coronary artery disease (FRISC 324 II) Investigators. Invasive compared with non-invasive treatment in unstable coronary- 325 artery disease: FRISC II prospective randomised multicentre study. Lancet 1999;354:708– 326 15. 327 25 Spacek R, Widimský P, Straka Z, et al. Value of first day angiography/angioplasty In 328 evolving Non-ST segment elevation myocardial infarction: an Open multicenter 329 randomized trial: The VINO study. Eur Heart J 2002;23:230–8. 330 doi:10.1053/euhj.2001.2735 331 26 Fox KAA, Poole-Wilson PA, Henderson RA, et al. Interventional versus conservative 332 treatment for patients with unstable angina or non-ST-elevation myocardial infarction: the 333 British Heart Foundation RITA 3 randomised trial. Randomized Intervention Trial of 334 Acute Coronary Syndrome: A Pilot Randomized Controlled Trial. Circ Cardiovasc Interv 311 2019;12:e007830. doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY Supplemental References doi:10.1161/CIRCINTERVENTIONS.119.007830 312 21 Tegn N, Abdelnoor M, Aaberge L, et al. Invasive versus conservative strategy in patients 313 aged 80 years or older with non-ST-elevation myocardial infarction or unstable angina 314 pectoris (After Eighty study): an open-label randomised controlled trial. Lancet 315 2016;387:1057–65. doi:10.1016/S0140-6736(15)01166-6 316 22 Sanchis J, Núñez E, Barrabés JA, et al. Randomized comparison between the invasive and 317 conservative strategies in comorbid elderly patients with non-ST elevation myocardial 318 infarction. Eur J Intern Med 2016;35:89–94. doi:10.1016/j.ejim.2016.07.003 319 23 The TIMI IIIB Investigators. Effects of tissue plasminogen activator and a comparison of 320 early invasive and conservative strategies in unstable angina and non-Q-wave myocardial 321 infarction. Results of the TIMI IIIB Trial. Thrombolysis in Myocardial Ischemia. 322 Circulation 1994;89:1545–56. doi:10.1016/S0196-0644(94)70199-7 323 19 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Open Heart BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Open Heart Supplemental material unstable Angina. Lancet 2002;360:743–51. doi:10.1016/S0140-6736(02)09894-X 335 336 20 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Open Heart doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material Supplemental material Open Heart doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY doi: 10.1136/openhrt-2020-001453 :e001453. 8 2021; Open Heart , et al. Lee MMY
https://openalex.org/W2194616187	https://europepmc.org/articles/PMC4889471?pdf=render	English	null	Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia	Leukemia	2,015	cc-by	5,225	Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia binding of both proteins is highly enriched within ± 3 kb from transcriptional start sites (Figure 1b). Leukemia (2016) 30, 1436–1440; doi:10.1038/leu.2015.331 We analyzed the effect of Ikaros and HDAC1 DNA binding on the surrounding chromatin. First, the genome-wide distribution of histone H3 trimethylation at lysine 4 (H3K4me3), lysine 27 (H3K27me3), lysine 36 (H3K36me3), or lysine 9 (H3K9me3), or acetylated at lysine 9 (H3K9ac) was determined by ChIP-Seq experiments in Nalm6 cells. ChIP-Seq data for histone modi- ﬁcations were validated by qChIP analysis of the high- and low- rank ChIP-Seq peak values (Supplementary Figures S3–S7). Next, we analyzed the distribution of chromatin modiﬁcations relative to (1) Ikaros peaks; (2) Ikaros–HDAC1 overlapped peaks; and (3) HDAC1 peaks. Most of the Ikaros and HDAC1 binding occurs within the promoters of target genes (Figure 1b). Thus, we compared the epigenetic changes that we observed in chroma- tin surrounding Ikaros, Ikaros–HDAC1 and HDAC1 peaks (Figures 1c–e), which are located within the promoter region, to epigenetic markers present in chromatin surrounding promoters across the genome, regardless of Ikaros and/or HDAC1 occupancy (Figure 1f). IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a master regulatory of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL).1–4 The deletion and/or mutation of Ikaros is associated with the development of B-cell acute lymphoblastic leukemia (B-ALL) with poor outcome.5–11 Ikaros directly associates with components of the histone deacetylase complex (NuRD), HDAC1, HDAC2 and Mi-2.12–14 Although Ikaros is hypothesized to regulate the transcription of target genes by recruiting the NuRD complex, the mechanism of Ikaros-mediated transcriptional regulation in leukemia is still unknown. Here we use a systems biology approach to determine the mechanism through which Ikaros and HDAC1 regulate gene expression in human B-ALL. To study the role of Ikaros and Ikaros–HDAC1 complexes in ALL, we determined the genome-wide occupancy of Ikaros and HDAC1 using chromatin immunoprecipitation followed by deep sequen- cing (ChIP-Seq) in human B-ALL cells (Nalm6 cell line). We identiﬁed 12 464 distinct binding sites for Ikaros and 9971 for HDAC1, and these were associated with 6722 and 6182 target genes, respectively (Figure 1a). Of these, 12% of the Ikaros-binding sites overlapped by at least 1 bp with 14.6% of the HDAC1-binding sites. The overlapping binding sites correlated with 934 gene targets (Figure 1a). REFERENCES 8 Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Luthar N et al. AXL mediates resistance to cetuximab therapy. Cancer Res 2014; 74: 5152–5164. 1 Moroni E, Dell'Era P, Rusnati M, Presta M. Fibroblast growth factors and their receptors in hematopoiesis and hematological tumors. J Hematother Stem Cell Res 2002; 11: 19–32. 9 Sinha S, Boysen J, Nelson M, Secreto C, Warner SL, Bearss DJ et al. Targeted Axl inhibition primes chronic lymphocytic leukemia B cells to apoptosis and shows synergistic/additive effects in combination with BTK inhibitors. Clin Cancer Res 2015; 21: 2115–2126. 2 Ghosh AK, Kay NE. Critical signal transduction pathways in CLL. Adv Exp Med Biol 2013; 792: 215–239. 10 Chase A, Grand FH, Cross NC. Activity of TKI258 against primary cells and cell lines with FGFR1 fusion genes associated with the 8p11 myeloproliferative syndrome. Blood 2007; 110: 3729–3734. 3 Turner N, Grose R. Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 2010; 10: 116–129. 4 Johnston CL, Cox HC, Gomm JJ, Coombes RC. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem 1995; 270: 30643–30650. 11 Korah RM, Sysounthone V, Golowa Y, Wieder R. Basic ﬁbroblast growth factor confers a less malignant phenotype in MDA-MB-231 human breast cancer cells. Cancer Res 2000; 60: 733–740. 5 Mohammadi M, Dikic I, Sorokin A, Burgess WH, Jaye M, Schlessinger J. Identiﬁcation of six novel autophosphorylation sites on ﬁbroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol 1996; 16: 977–989. 12 Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W et al. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res 2010; 70: 1544–1554. 13 L'Hote CG, Knowles MA. Cell responses to FGFR3 signalling: growth, differentia- tion and apoptosis. Exp Cell Res 2005; 304: 417–431. 6 Ghosh AK, Secreto C, Boysen J, Sassoon T, Shanafelt TD, Mukhopadhyay D et al. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy. Blood 2011; 117: 1928–1937. 14 Azab AK, Azab F, Quang P, Maiso P, Sacco A, Ngo HT et al. Accepted article preview online 7 December 2015; advance online publication, 22 January 2016 Letters to the Editor 1436 1436 REFERENCES FGFR3 is overexpressed waldenstrom macroglobulinemia and its inhibition by Dovitinib induces apoptosis and overcomes stroma-induced proliferation. Clin Cancer Res 2011; 17: 4389–4399. 7 Boysen J, Sinha S, Price-Troska T, Warner SL, Bearss DJ, Viswanatha D et al. The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients. Leukemia 2014; 28: 451–455. 7 Boysen J, Sinha S, Price-Troska T, Warner SL, Bearss DJ, Viswanatha D et al. The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients. Leukemia 2014; 28: 451–455. 15 Hynes NE, Ingham PW, Lim WA, Marshall CJ, Massague J, Pawson T. Signalling change: signal transduction through the decades. Nat Rev Mol Cell Biol 2013; 14: 393–398. 15 Hynes NE, Ingham PW, Lim WA, Marshall CJ, Massague J, Pawson T. Signalling change: signal transduction through the decades. Nat Rev Mol Cell Biol 2013; 14: 393–398. entary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) 15 Hynes NE, Ingham PW, Lim WA, Marshall CJ, Massague J, Pawson T. Signalling change: signal transduction through the decades. Nat Rev Mol Cell Biol 2013; 14: 393–398. pplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia ChIP-Seq data for Ikaros and HDAC1 were validated by quantitative chromatin immunoprecipitation (qChIP) analysis of the high- and low-rank ChIP-Seq peak values (Supplementary Figures S1 and S2). The peak distributions of Ikaros and of HDAC1 relative to target genes revealed that the p y g We found that unique epigenetic changes are associated with Ikaros, Ikaros–HDAC1 and HDAC1 peaks. Ikaros peaks are associated with the presence of H3K4me3, H3K9me3 and H3K9ac histone modiﬁcations (Figure 1c). Ikaros–HDAC1 over- lapped peaks correlated with a different chromatin environment that is characterized by the very strong presence of H3K4me3 and H3K27me3, moderate H3K9me3 and virtually absent H3K9ac (Figure 1d). HDAC1 peaks were also associated with the very strong presence of H3K27me3 and H3K4me3, and virtually absent H3K9ac. However, H3K9me3 was reduced as compared with Ikaros or Ikaros–HDAC1 peaks (Figure 1e). © 2016 Macmillan Publishers Limited Leukemia (2016) 1399 – 1448 Letters to the Editor 1437 Figure 1. Genome-wide mapping of Ikaros and HDAC1 binding B-ALL cells. (a) Ikaros and HDAC1 target genes identiﬁed by ChI Seq analysis of Nalm6 B-ALL cells. The overlapping gene targe have Ikaros and HDAC1 peaks overlapped by at least 1 bp. (b) Th distribution of Ikaros and HDAC1 peaks around the transcription start sites (TSS). Peak numbers were normalized by treating th These results indicate that the binding of Ikaros, Ikaros–HDAC1 or HDAC1 is each associated with a distinct characteristic chromatin change that likely affects the expression of target genes. The speciﬁc distribution of histone modiﬁcations around Ikaros, Ikaros–HDAC1 or HDAC1 peaks were similar, regardless of whether these peaks were localized within promoter regions or other regions across the genome (Supplementary Figures S8–S10). Most of the speciﬁc epigenetic changes occur within 1 kb of the center of the Ikaros, Ikaros–HDAC1 or HDAC1 peaks. This suggests that binding of these proteins has a direct effect on chromatin remodeling and the observed epigenetic changes. g Our analysis demonstrates a strong association between HDAC1 occupancy and H3K27me3 (Supplementary Table S1). This is particularly pronounced at promoter regions—85% of all promo- ters with H3K27me3 showed HDAC1 binding (Figure 1g). This suggests that HDAC1 occupancy is the major determinant of the H3K27me3 marker. Further analysis demonstrates Ikaros–HDAC1 occupancy at 21% of all promoters with H3K27me3 in leukemia cells (Figure 1g). This suggests that Ikaros binding to promoters of its target genes can result in H3K27me3 via recruitment of HDAC1. Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia These results show the importance of Ikaros’ recruitment of HDAC1 in determining the global epigenetic signature in leukemia. We tested whether histone deacetylase activity is required for the formation of H3K27me3 in Nalm6 cells. Treatment of Nalm6 cells with the histone deacetylase inhibitor trichostatin resulted in strong reduction of H3K27me3 by western blot (Figure 1h), suggesting that histone deacetylase activity is essential for the presence of H3K27me3. These results demon- strate an essential role for histone deacetylase in the formation of H3K27me3 in B-ALL. Figure 1. Genome-wide mapping of Ikaros and HDAC1 binding in B-ALL cells. (a) Ikaros and HDAC1 target genes identiﬁed by ChIP- Seq analysis of Nalm6 B-ALL cells. The overlapping gene targets have Ikaros and HDAC1 peaks overlapped by at least 1 bp. (b) The distribution of Ikaros and HDAC1 peaks around the transcriptional start sites (TSS). Peak numbers were normalized by treating the maximum possible peak number at a location as 100. (c–f) Speciﬁc epigenetic changes associated with Ikaros and HDAC1 occupancy. The distribution of histone modiﬁcations relative to the center of (c) the Ikaros peak; (d) the Ikaros peak in Ikaros (IK)-HDAC1 overlapped peaks; (e) the HDAC1 peak; (f) all TSS, genome wide. Graphed is the frequency of each particular epigenetic modiﬁca- tions per 100 Ikaros, or 100 Ikaros–HDAC1 peaks, over a 1 kb span. (g) Association of H3K27me3 with IK and HDAC1 occupancy, genome wide (left), or within the promoter region (right). Graphed are the number of H3K27me3 peaks located within 1 kb of IK, HDAC1, or IK–HDAC1 peaks, or outside of these regions (non-IK and non-HDAC1). (h) Effect of pan-HDAC inhibitor (TSA) on H3K27me3 level. Western blot of H3K27me3 in untreated Nalm6 cells and following TSA treatment at speciﬁc days are shown. The total level of histone H3 was used for normalization (bottom). WT, wild type. ChIP-Seq analysis of the epigenetic signature around Ikaros occupancy led to the hypothesis that DNA binding of Ikaros or Ikaros–HDAC1 complexes alters the transcription of their respective target genes by induction of distinct epigenetic changes. To test this hypothesis, we analyzed the effect of increased Ikaros expression on chromatin remodeling at promoters of genes that are regulated by Ikaros-only or by Ikaros–HDAC1 complexes. © 2016 Macmillan Publishers Limited Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia Recently, we reported that Ikaros represses the transcription of a large number of genes that promote cell cycle progression in leukemia.15 The epigenetic signatures at promoters of the cell cycle-promoting genes CDC7 and ANAPC7 (Ikaros-only targets), and CDC2 and ANAPC1 (Ikaros–HDAC1 targets) were compared in Nalm6 cells trans- duced with Ikaros or empty vector (control) using serial qChIP assays. Results showed that increased Ikaros expression is associated with unchanged H3K27me3, increased H3K9me3 and decreased H3K9ac in regulatory elements of the Ikaros- only targets, CDC7 and ANAPC7 (Figure 2a, Supplementary Figure S11a, red vs black lines). In contrast, in regulatory elements of the Ikaros–HDAC1 target genes CDC2 and ANAPC1, increased Ikaros expression is associated with increased H3K27me3, unchanged H3K9me3 and decreased H3K9ac (Figure 2b, Supplementary Figure S11b, red vs black lines). These data identify speciﬁc epigenetic signatures induced by binding of Ikaros-only and Ikaros–HDAC1 complexes to promoters of Ikaros target genes in B-ALL. Figure 1. Genome-wide mapping of Ikaros and HDAC1 binding in B-ALL cells. (a) Ikaros and HDAC1 target genes identiﬁed by ChIP- Seq analysis of Nalm6 B-ALL cells. The overlapping gene targets have Ikaros and HDAC1 peaks overlapped by at least 1 bp. (b) The distribution of Ikaros and HDAC1 peaks around the transcriptional start sites (TSS). Peak numbers were normalized by treating the maximum possible peak number at a location as 100. (c–f) Speciﬁc epigenetic changes associated with Ikaros and HDAC1 occupancy. The distribution of histone modiﬁcations relative to the center of (c) the Ikaros peak; (d) the Ikaros peak in Ikaros (IK)-HDAC1 overlapped peaks; (e) the HDAC1 peak; (f) all TSS, genome wide. Graphed is the frequency of each particular epigenetic modiﬁca- tions per 100 Ikaros, or 100 Ikaros–HDAC1 peaks, over a 1 kb span. (g) Association of H3K27me3 with IK and HDAC1 occupancy, genome wide (left), or within the promoter region (right). Graphed are the number of H3K27me3 peaks located within 1 kb of IK, HDAC1, or IK–HDAC1 peaks, or outside of these regions (non-IK and non-HDAC1). (h) Effect of pan-HDAC inhibitor (TSA) on H3K27me3 level. Western blot of H3K27me3 in untreated Nalm6 cells and following TSA treatment at speciﬁc days are shown. The total level of histone H3 was used for normalization (bottom). WT, wild type. Figure 1. Genome-wide mapping of Ikaros and HDAC1 binding in B-ALL cells. (a) Ikaros and HDAC1 target genes identiﬁed by ChIP- Seq analysis of Nalm6 B-ALL cells. Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia The overlapping gene targets have Ikaros and HDAC1 peaks overlapped by at least 1 bp. (b) The distribution of Ikaros and HDAC1 peaks around the transcriptional start sites (TSS). Peak numbers were normalized by treating the maximum possible peak number at a location as 100. (c–f) Speciﬁc epigenetic changes associated with Ikaros and HDAC1 occupancy. The distribution of histone modiﬁcations relative to the center of (c) the Ikaros peak; (d) the Ikaros peak in Ikaros (IK)-HDAC1 overlapped peaks; (e) the HDAC1 peak; (f) all TSS, genome wide. Graphed is the frequency of each particular epigenetic modiﬁca- tions per 100 Ikaros, or 100 Ikaros–HDAC1 peaks, over a 1 kb span. (g) Association of H3K27me3 with IK and HDAC1 occupancy, genome wide (left), or within the promoter region (right). Graphed are the number of H3K27me3 peaks located within 1 kb of IK, HDAC1, or IK–HDAC1 peaks, or outside of these regions (non-IK and non-HDAC1). (h) Effect of pan-HDAC inhibitor (TSA) on H3K27me3 level. Western blot of H3K27me3 in untreated Nalm6 cells and following TSA treatment at speciﬁc days are shown. The total level of histone H3 was used for normalization (bottom). WT, wild type. Next, we studied how Ikaros loss-of-function or gain-of- function affects the transcriptional regulation and epigenetic signature of Ikaros target genes in primary high-risk B-ALL cells. In high-risk B-ALL, Ikaros function as a transcriptional regulator is severely impaired due to the deletion of one Ikaros allele and/or functional inactivation of Ikaros protein by Casein Kinase II (CK2) phosphorylation.15 Inhibition of CK2 has been shown to restore Ikaros activity as transcriptional regulator, resulting in transcriptional repression of Ikaros target genes that promote cell cycle progression.15 We analyzed the epigenetic signature at promoters of Ikaros and Ikaros–HDAC1 Leukemia (2016) 1399 – 1448 © 2016 Macmillan Publishers Limited Letters to the Editor 1438 1438 Figure 2. Ikaros-mediated chromatin changes in promoter regions of Ikaros target genes. (a, b) Epigenetic signature at promoters of Ikaros target genes following overexpression of Ikaros in Nalm6 cells (red line) or in control Nalm6 cells (black line). The binding of Ikaros and HDAC1, and the histone modiﬁcation markers, H3K27me3, H3K9me3 and H3K9ac were detected by serial qChIP assays in a representative (a) Ikaros-only target gene (CDC7) and (b) IK–HDAC1 target gene (CDC2) in Nalm6 B-ALL cells. AUTHOR CONTRIBUTIONS C Song1,8, X Pan1,8, Z Ge1,2, C Gowda1, Y Ding1, H Li1, Z Li1,3, G Yochum4, M Muschen5, Q Li6, KJ Payne7 and S Dovat1 1Department of Pediatrics, Pennsylvania State University Medical College, Hershey, PA, USA; 2 C Song1,8, X Pan1,8, Z Ge1,2, C Gowda1, Y Ding1, H Li1, Z Li1,3, G Yochum4, M Muschen5, Q Li6, KJ Payne7 and S Dovat1 1Department of Pediatrics, Pennsylvania State University Medical College, Hershey, PA, USA; 2 2Department of Hematology, The First Afﬁliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China; 3Jilin Province Animal Embryo Engineering Key Laboratory, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China; 4Department of Biochemistry and Molecular Biology, Pennsylvania State University Medical College, Hershey, PA, USA; 5 The distinct epigenetic changes that occur following the restoration of Ikaros binding to promoters of Ikaros-only and Ikaros–HDAC1 target genes were reproduced in cells derived from three different primary high-risk B-ALL following treatment with CK2 inhibitor CX-4945 (Figures 2c and d, Supplementary Figure S12). These results were also reproduced following treatment of high-risk primary B-ALL cells with a different CK2 inhibitor, TBB, (Supplementary Figures S13a and b compared with Figures 2a–d and Supplementary Figures S12 and S13c and d compared with Supplementary Figure 11). 5University of California San Francisco, San Francisco, CA, USA; 6Department of Statistics, Pennsylvania State University, University Park, State College, PA, USA and g 7Department of Pathology and Human Anatomy and Center for Health Disparities and Molecular Medicine, Loma Linda University, Loma Linda, CA, USA g 7Department of Pathology and Human Anatomy and Center for Health Disparities and Molecular Medicine, Loma Linda University, Loma Linda, CA, USA p 8These authors contributed equally to this work In summary, our data reveal the mechanism by which chromatin remodeling and target gene expression are regulated by Ikaros alone and in complex with HDAC1 in B-ALL (Figure 2e). These data suggest that Ikaros can repress transcription of its target genes by inducing the formation of repressive chromatin via two distinct mechanisms: (1) direct Ikaros binding resulting in the formation of heterochromatin due to increased H3K9me3 and reduced H3K9ac; or (2) Ikaros recruitment of HDAC1, where the most prominent change is a strong increase in H3K27me3 along with reduced H3K9ac. In high-risk B-ALL, Ikaros ability to regulate chromatin remodeling of its target genes is impaired. AUTHOR CONTRIBUTIONS target genes in primary high-risk B-ALL (with loss of Ikaros function), and in primary high-risk B-ALL cells following treat- ment with CK2 inhibitors (with restored Ikaros function). In high-risk B-ALL, Ikaros DNA binding to the promoters of its target genes is impaired (Figures 2c and d, Supplementary Figures S12 and S13 black lines). Inhibition of CK2 with a speciﬁc CK2 inhibitor, CX-4945, restored Ikaros DNA binding to promoters and induced an epigenetic signature with high-level H3K9me3, reduced H3K9ac and the absence of H3K27me3 at the Ikaros-only target gene, CDC7 (Figure 2c, Supplementary Figure S12a, red vs black lines). However, for the Ikaros–HDAC1 target, CDC2, restoration of Ikaros binding following CK2 inhibition results in a high level of H3K27me3, the loss of H3K9ac and largely unchanged H3K9me3 (Figure 2d, Supplementary Figure S12b, red vs black lines). Results obtained following the restoration of Ikaros function demonstrate that treatment of high-risk B-ALL cells with the CK2 inhibitor CX-4945 results in epigenetic changes that are remarkably similar to those found with increased Ikaros expression in Nalm6 (Figures 2c and d and Supplementary Figures S12 and S13c and d as compared with Figures 2a and b). CS performed the majority of the biological experiments, analyzed the data and participated in the manuscript preparation. XP provided bioinformatics and biostatistical analysis of the data and participated in the manuscript preparation. CG, YD, HL and ZG performed experiments. GY assisted in the data interpretation, participated in the experimental design and provided conceptual advice. MM provided vital reagents and conceptual advice. QL participated in the interpretation of biostatistical data. KJP provided vital reagents and conceptual advice and participated in the experimental design and in writing the manuscript. SD designed the experiments, interpreted the data and wrote the manuscript. CONFLICT OF INTEREST 9 Martinelli G, Iacobucci I, Storlazzi CT, Vignetti M, Paoloni F, Cilloni D et al. IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are asso- ciated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol 2009; 27: 5202–5207. The authors declare no conﬂict of interest. relapse: a GIMEMA AL WP report. J Clin Oncol 2009; 27: 5202–520 AUTHOR CONTRIBUTIONS In high-risk B-ALL with deletion of one Ikaros allele, inhibition of CK2 restores Ikaros-mediated epigenetic repression of the cell cycle-promoting genes. These data suggest that the ability to regulate chromatin remodeling is an essential part of Ikaros tumor-suppressor function. These studies provide new insight into the epigenetic regulation of gene expression in B-ALL and a rationale for the use of CK2 inhibitors as a novel treatment. Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia (c, d) Epigenetic signature at promoters of Ikaros target genes in primary high-risk B-ALL cells that carry deletion of one Ikaros allele (patient 1; black line) and following treatment with the CK2 inhibitor, CX-4945 (red line). The binding of Ikaros, HDAC1 and histone modiﬁcation markers were detected by serial qChIP assays in the representative (c) Ikaros-only target gene (CDC7) and (d) IK–HDAC1 target gene (CDC2) in primary cells from patient 1. Patient characteristics are shown in Supplementary Table S2. Graphed data are means ± s.d. of data obtained using ﬁve primer pairs that span the transcription start site (TSS) of indicated genes. In addition to the presented data, the serial qChIP assays for H3K4me3 did not show any changes following treatment with CX-4945 (data not shown). (e) Model of proposed epigenetic mechanisms for Ikaros- and IK–HDAC1-mediated regulation of gene expression. Figure 2. Ikaros-mediated chromatin changes in promoter regions of Ikaros target genes. (a, b) Epigenetic signature at promoters of Ikaros target genes following overexpression of Ikaros in Nalm6 cells (red line) or in control Nalm6 cells (black line). The binding of Ikaros and HDAC1, and the histone modiﬁcation markers, H3K27me3, H3K9me3 and H3K9ac were detected by serial qChIP assays in a representative (a) Ikaros-only target gene (CDC7) and (b) IK–HDAC1 target gene (CDC2) in Nalm6 B-ALL cells. (c, d) Epigenetic signature at promoters of Ikaros target genes in primary high-risk B-ALL cells that carry deletion of one Ikaros allele (patient 1; black line) and following treatment with the CK2 inhibitor, CX-4945 (red line). The binding of Ikaros, HDAC1 and histone modiﬁcation markers were detected by serial qChIP assays in the representative (c) Ikaros-only target gene (CDC7) and (d) IK–HDAC1 target gene (CDC2) in primary cells from patient 1. Patient characteristics are shown in Supplementary Table S2. Graphed data are means ± s.d. of data obtained using ﬁve primer pairs that span the transcription start site (TSS) of indicated genes. In addition to the presented data, the serial qChIP assays for H3K4me3 did not show any changes following treatment with CX-4945 (data not shown). (e) Model of proposed epigenetic mechanisms for Ikaros- and IK–HDAC1-mediated regulation of gene expression. © 2016 Macmillan Publishers Limited Leukemia (2016) 1399 – 1448 Letters to the Editor 1439 REFERENCES 1 Lo K, Landau NR, Smale ST. LyF-1, a transcriptional regulator that interactswith a novel class of promoters for lymphocyte-speciﬁc genes. Mol CellBiol 1991; 11: 5229–5243. 2 Georgopoulos K, Moore DD, Derﬂer B. Ikaros, an early lymphoid-speciﬁc tran- scription factor and a putative mediator for T cell commitment. Science 1992; 258: 808–812. 3 Georgopoulos K, Bigby M, Wang JH, Molnar A, Wu P, Winandy S et al. The Ikaros gene is required for the development of all lymphoid lineages. Cell 1994; 79: 143–156. 4 Winandy S, Wu P, Georgopoulos K. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 1995; 83: 289–299. pid development of leukemia and lymphoma. Cell 1995; 83: 289–29 5 Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 2007; 446: 758–764. 6 Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008; 453: 110–114. 7 Mullighan CG, Su X, Zhang J, Radtke I, Phillips LA, Miller CB et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med 2009; 360: 470–480. 8 Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012; 481: 157–163. © 2016 Macmillan Publishers Limited A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients Leukemia (2016) 30, 1440–1443; doi:10.1038/leu.2015.333 Factors independently associated with TFS were included in the PRS. To account for differences in the magnitude of the association between individual independent factors and TFS, we assigned a weighted-risk score to each factor based on ranges of their corresponding hazard ratios (HR) (that is, 1 point for HR 1.1–1.9; 2 points for HR 2.0–2.9 and so on).9 The total risk score was then calculated by summing the ratings of each individual factor. Risk groups were identiﬁed combining risk categories with a non-statistically different TFS (see Supplementary Methods). Several phenotypic, molecular and chromosomal markers of chronic lymphocytic leukemia (CLL) cells have been identiﬁed that are signiﬁcantly associated with patient prognosis.1–6 However, these markers used singularly are inaccurate predictors of outcome for individual patients. Recent efforts have focused on combining markers to predict either treatment-free survival (TFS)4,7,8 or overall survival (OS),9–11 however, further effort is worthwhile to determine how to combine prognostic parameters, optimize risk stratiﬁcation, simplify calculations and/or identify new prognostic variables. Baseline patient features of the training cohort are listed in Supplementary Table S1. Patients with Rai stage I and II were grouped for analysis according to convention.12 Given the limited number of patients with del(11q23) and del(17p13), cytogenetic abnormalities identiﬁed by FISH were clustered in three risk groups (that is, low risk (del(13q14) and normal), intermediate risk (trisomy 12) and high risk (del(11q23) and del(17p13)). After a median 42 months follow-up (range, 6–82 months), 84/337 (24.9%) cases required treatment. Herein analyzing data from a cohort of Binet A patients, enrolled in a prospective multicenter observational study, we developed a weighted, multivariate score (progression-risk score (PRS)) integrating clinical, laboratory and biological parameters independently associated with TFS. The PRS was subsequently validated using an external cohort of CLL patients from the Mayo Clinic, Minnesota, USA. q In multivariate analysis, Rai stage I–II, absolute lymphocyte count ⩾10 × 109/l, elevated β2-microglobulin levels, and IGHV-UM remained associated with shorter TFS (Table 1). The multivariate model was conﬁrmed by bootstrap resampling (data not shown). Considering the HR of the independent factors, a risk score was assigned to each marker (Table 1); the total risk score was deﬁned as the sum of the risk scores of the four individual parameters (range, 0–7). Letters to the Editor 1440 1440 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 inthecreditline; ifthe materialisnotincludedundertheCreativeCommonslicense, 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/ 14 O'Neill DW, Schoetz SS, Lopez RA, Castle M, Rabinowitz L, Shor E et al. An ikaros- containing chromatin-remodeling complex in adult-type erythroid cells. Mol Cell Biol 2000; 20: 7572–7582. 14 O'Neill DW, Schoetz SS, Lopez RA, Castle M, Rabinowitz L, Shor E et al. An ikaros- containing chromatin-remodeling complex in adult-type erythroid cells. Mol Cell Biol 2000; 20: 7572–7582. 15 Song C, Gowda C, Pan X, Ding Y, Tong Y, Tan BH et al. Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efﬁcacy in high-risk leukemia. Blood 2015; 126: 1813–1822. 15 Song C, Gowda C, Pan X, Ding Y, Tong Y, Tan BH et al. Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efﬁcacy in high-risk leukemia. Blood 2015; 126: 1813–1822. pplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) A ACKNOWLEDGEMENTS 10 Kuiper RP, Waanders E, van der Velden VH, van Reijmersdal SV, Venkatachalam R, Scheijen B et al. IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL. Leukemia 2010; 24: 1258–1264. We would like to thank S Smale for critical reading of the manuscript. This work has been supported by National Institutes of Health (NIH) R01 HL095120, a St Baldrick’s Foundation Career Development Award, a Hyundai Hope on Wheels Scholar Grant Award, the Four Diamonds Fund of the Pennsylvania State University, College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (to SD) a St Baldrick’s Foundation Fellows Award and Hyundai Hope on Wheels Fellowship Grant Award (to CG). Additional funding for this work is from NSFC 81270613 (ZG), NIH R01GM109453 and 1UL1RR033184 (to QL). This work was also supported by NIH R21CA162259, P20MD006988 a St Baldrick's Research Grant and a Hyundai Hope on Wheels Award (KJP). 11 Iacobucci I, Iraci N, Messina M, Lonetti A, Chiaretti S, Valli E et al. IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia. PLoS One 2012; 7: e40934. 12 Kim J, Sif S, Jones B, Jackson A, Koipally J, Heller E et al. Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity 1999; 10: 345–355. 13 Zhang J, Jackson AF, Naito T, Dose M, Seavitt J, Liu F et al. Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leukemogenesis. Nat Immunol 2012; 13: 86–94. Leukemia (2016) 1399 – 1448 Leukemia (2016) 1399 – 1448 Leukemia (2016) 1399 – 1448 © 2016 Macmillan Publishers Limited cepted article preview online 9 December 2015; advance online publication, 19 January 2016 14 O'Neill DW, Schoetz SS, Lopez RA, Castle M, Rabinowitz L, Shor E et al. An ikaros- containing chromatin-remodeling complex in adult-type erythroid cells. Mol Cell Biol 2000; 20: 7572–7582. A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients According to the predeﬁned criteria (Supplementary Table S2), three different risk categories for TFS were determined: low (score 0–2), intermediate (score 3–5) and high risk (score 6–7; Supplementary Table S3). We analyzed data from 480 newly diagnosed CLL patients enrolled in the O-CLL1-GISL protocol (clinicaltrial.gov identiﬁer: NCT00917540). Of these, 337 cases with available biological (CD38, ZAP-70, IGHV mutational status and ﬂuoresent in situ hybridization (FISH)) and clinical/laboratory parameters (sex, age, absolute lymphocyte count, Rai-modiﬁed stage, lactate dehydro- genase (normal range, 313–618 IU/l) and β2-microglobulin level (normal range, 0.6–2.0 mg/l),11 were included in this analysis (see Supplementary Methods). Table 1. Univariate and multivariate Cox proportional Hazards Models for TFS Variable Univariate analysis Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value Score Age (years) o60/ ⩾60 1.12 (0.73–1.74) 0.59 − − − Sex male/female 0.93 (0.6–1.44) 0.93 − − − Rai stage 0/I–II 2.30 (1.47–3.50) o0.0001 1.76 (1.11–2.78) 0.015 0/1 ALC (109/l) o10/ ⩾10 3.43 (1.99–5.92) o0.0001 2.70 (1.54–4.72) 0.001 0/2 β2-microglobulin normal/elevated 3.04 (1.96–4.70) o0.0001 2.65 (1.66–4.21) o0.0001 0/2 LDH normal/elevated 1.25 (0.57–2.71) 0.57 − − − CD38 negative/positive 3.22 (2.06–5.02) o0.0001 1.40 (0.80–2.42) 0.24 − ZAP-70 negative/positive 2.34 (1.51–3.61) o0.0001 1.0 (0.98–1.01) 0.72 − IGHV mutated/unmutated 3.57 (2.32–5.50) o0.0001 2.39 (1.27–4.50) 0.007 0/2 FISH risk low+int/high 2.93 (1.46–5.90) 0.002 1.80 (0.84–3.88) 0.13 − Abbreviations: ALC, absolute lymphocyte count; CI, conﬁdence interval; HR, hazard ratio; LDH, lactate dehydrogenase. Table 1. Univariate and multivariate Cox proportional Hazards Models for TFS Accepted article preview online 9 December 2015; advance online publication, 19 January 2016 © 2016 Macmillan Publishers Limited
https://openalex.org/W2219113137	https://europepmc.org/articles/pmc2968496?pdf=render	English	null	Bis[4-(2-benzylidenepropylideneamino)phenyl] ether	Acta crystallographica. Section E	2,009	cc-by	3,598	organic compounds Experimental Crystal data C32H28N2O Mr = 456.6 Monoclinic, P21=n a = 7.4737 (3) A˚ b = 55.929 (3) A˚ c = 6.0275 (3) A˚ = 90.022 (4) V = 2519.5 (2) A˚ 3 Z = 4 Cu K radiation = 0.56 mm1 T = 295 K 0.51 0.38 0.02 mm Data collection Oxford Diffraction Gemini diffractometer with Atlas CCD detector Absorption correction: multi-scan (CrysAlis RED; Oxford Diffraction, 2008) Tmin = 0.682, Tmax = 0.99 15440 measured reﬂections 3838 independent reﬂections 3428 reﬂections with I > 3(I) Rint = 0.016 max = 61.4 Reﬁnement R[F 2 > 2(F 2)] = 0.036 wR(F 2) = 0.120 S = 2.32 3838 reﬂections 317 parameters H-atom parameters constrained max = 0.19 e A˚ 3 min = 0.18 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Related literature Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: BT2870). For the synthesis of the title compound, see: Khalaji & Ng (2008). For related structures, see: Hu et al. (2008); Xu et al. (2008). For background to transition metal complexes, see: Laye (2007); Huang et al. (2005); Chu & Huang (2007). Mansoor Movahedi,a Hassan Hadadzadeh,b Karla Fejfarova,c Michal Dusekc* and Aliakbar Dehno Khalajid aDepartment of Science, Golestan University, Gorgan, Iran, bDepartment of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran, cInstitute of Physics of the ASCR, Na Slovance 2, 182 21 Prague 8, Czech Republic, and dDepartment of Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan 49189-43464, Iran Correspondence e-mail: dusek@fzu.cz Reﬁnement R[F 2 > 2(F 2)] = 0.036 wR(F 2) = 0.120 S = 2.32 3838 reﬂections 317 parameters H-atom parameters constrained max = 0.19 e A˚ 3 min = 0.18 e A˚ 3 317 parameters H-atom parameters constrained max = 0.19 e A˚ 3 min = 0.18 e A˚ 3 Received 10 February 2009; accepted 11 February 2009 Key indicators: single-crystal X-ray study; T = 295 K; mean (C–C) = 0.003 A˚; R factor = 0.036; wR factor = 0.120; data-to-parameter ratio = 12.1. Data collection: CrysAlis CCD (Oxford Diffraction, 2008); cell reﬁnement: CrysAlis RED (Oxford Diffraction, 2008); data reduc- tion: CrysAlis RED; program(s) used to solve structure: SIR2002 (Burla et al., 2003); program(s) used to reﬁne structure: JANA2006 (Petrˇı´cˇek et al., 2008); molecular graphics: DIAMOND (Brandenburg & Putz, 2005); software used to prepare material for publication: JANA2006. The title compound, C32H28N2O, is a ﬂexible Schiff base displaying a trans conﬁguration across the C N double bond. It was prepared in high yield by condensation of -methyl- cinnamaldehyde and bis(4-aminophenyl) ether in methanol at room temperature. The sample, with pseudo-orthorhombic cell parameters, exhibited merohedral twinning by rotation 180 around a, with a reﬁned twin domain fraction of 0.722 (1). The elongated shape of the elementary cell corresponds to the shape and direction of the molecules. The dihedral angle between the O-linked aromatic rings is 57.86 (8). We thank Gorgan University of Agricultural Sciences and Natural Resources (GUASNR) and the Grant Agency of the Czech Republic (grant No. 202/07/J007) for supporting this study. ADK thanks Dr Jan Fabry (Institute of Physics of ASCR) for his collaboration. Bis[4-(2-benzylidenepropylideneamino)- phenyl] ether Mansoor Movahedi,a Hassan Hadadzadeh,b Karla Fejfarova,c Michal Dusekc and Aliakbar Dehno Khalajid aDepartment of Science, Golestan University, Gorgan, Iran, bDepartment of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran, cInstitute of Physics of the ASCR, Na Slovance 2, 182 21 Prague 8, Czech Republic, and dDepartment of Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan 49189-43464, Iran Correspondence e-mail: dusek@fzu.cz o538 Movahedi et al. References Brandenburg, K. & Putz, H. (2005). DIAMOND. Crystal Impact GbR, Bonn, Germany. y Burla, M. C., Camalli, M., Carrozzini, B., Cascarano, G., Giacovazzo, C. Polidori, G. & Spagna, R. (2003). J. Appl. Cryst. 36, 1103. Chu, Z. & Huang, W. (2007). J. Mol. Struct. 837, 15-22. Hu, S.-L., Li, Y.-T. & Cao, L.-P. (2008). Acta Cryst. E64, o115. Huang, Z., Tian, J.-L. & Bu, X. H. (2005). Inorg. Chem. Commun. 8, 194-198 Khalaji, A. D. & Ng, S. W. (2008). Acta Cryst. E64, o1771. Laye, R. H. (2007). Inorg. Chim. Acta, 360, 439-447. Burla, M. C., Camalli, M., Carrozzini, B., Cascarano, G., Giacovazzo, C., Polidori, G. & Spagna, R. (2003). J. Appl. Cryst. 36, 1103. p g ( ) pp y Chu, Z. & Huang, W. (2007). J. Mol. Struct. 837, 15-22. g ( ) Hu, S.-L., Li, Y.-T. & Cao, L.-P. (2008). Acta Cryst. E64, o115. Huang, Z., Tian, J.-L. & Bu, X. H. (2005). Inorg. Chem. Commun. 8, 194-198. Khalaji, A. D. & Ng, S. W. (2008). Acta Cryst. E64, o1771. j g ( ) Laye, R. H. (2007). Inorg. Chim. Acta, 360, 439-447. Oxford Diffraction (2008). CrysAlis CCD and CrysAlis RED. Oxford Diffraction Ltd, Abingdon, England. Petrˇı´cˇek, V., Dusˇek, M. & Palatinus, L. (2008). JANA2006. Institute of Physics, Praha, Czech Republic. Xu, H.-W., Li, J.-X. & Li, Y.-H. (2008). Acta Cryst. E64, o1145. o538 Movahedi et al. Acta Cryst. (2009). E65, o538 doi:10.1107/S1600536809005005 Comment Flexible Schiff-base ligands have received a lot of attention in the field of supramolecular coordination chemistry (Laye, 2007; Huang et al., 2005; Chu & Huang, 2007). Because of easy syntheses of these compounds by condensation between aldehydes (or ketones) and amines, many of them were designed and sued to prepare transition metal complexes in recent years. Here, we report the synthesis and crystal structure of a new flexible Schiff-base compound (I). The molecule of (I) is shown in Fig. 1. Bond lengths and angles are comparable with those observed in similar compounds (Hu et al., 2008; Xu et al., 2008). The C(7)=N(1) and C(23)=N(2) bond lengths of 1.266 (3) and 1.270 (3) Å, respectively, conform to the usual value for a C=N double bond. Each half of the molecule displays a trans configuration across the C=N double bond. The prolongated shape of the molecule is reflected by very long b axis about 55 Å. The molecules are isolated (Fig. 2), with no intermolecular contacts. From crystallographic point of view merohedric twinning by rotation 180° around a* and pseudo-orthorhombic cell parameters should be noted. The twinning has occurred regularly in all tested samples. Experimental The title compound was synthesized using a method analogous to the literature procedure of Khalaji and Ng (2008). Crystals appropriate for data collection were obtained by slow evaporation from a methanol-chloroform mixture (1:5 v/v) at room temperature (yield; 88%). supplementary materials Acta Cryst. (2009). E65, o538 [ doi:10.1107/S1600536809005005 ] Bis[4-(2-benzylidenepropylideneamino)phenyl] ether M. Movahedi, H. Hadadzadeh, K. Fejfarova, M. Dusek and A. Dehno Khalaji supplementary materials supplementary materials Refinement All H atoms were found in difference Fourier maps, but they were constrained to ideal positions. The isotropic atomic displacement parameters of hydrogen atoms were set to 1.2Ueq of the parent atom. The sample had a pseudo-orthorhombic cell parameters and exhibited a merohedric twinning by rotation 180° around a* (with twinning matrix 1 0 0 / 0 - 1 0 / 0 0 - 1 applied to indices expressed like row vectors). The volume fraction of the second domain was refined to 0.278 (1). sup-1 supplementary materials Figures Fig. 1. The molecule of (I) viewed along a with atom-labeling scheme. Displacement ellips- oids are drawn at the 50% probability level. Figures Fig. 1. The molecul oids are drawn at th Fig. 1. The molecule of (I) viewed along a with atom-labeling scheme. Displacement ellips- oids are drawn at the 50% probability level. Fig. 1. The molecule of (I) viewed along a with atom-labeling scheme. Displacement ellips- oids are drawn at the 50% probability level. sup-2 supplementary materia Fig. 2. The packing of (I) viewed along c. Bis[4-(2-benzylidenepropylideneamino)phenyl] ether Crystal data C32H28N2O1 F000 = 968 Mr = 456.6 Dx = 1.203 Mg m−3 Monoclinic, P21/n Cu Kα radiation λ = 1.54184 Å Hall symbol: -P 2yn Cell parameters from 9034 reflections a = 7.4737 (3) Å θ = 3.2–61.2º b = 55.929 (3) Å µ = 0.56 mm−1 c = 6.0275 (3) Å T = 295 K β = 90.022 (4)º Plate, yellow V 2519 5 (2) Å3 0 51 × 0 38 × 0 02 mm supplementary materials supplementary Fig. 2. The packing of (I) viewed along c. Bis[4-(2-benzylidenepropylideneamino)phenyl] ether Fig. 2. The packing of (I) viewed along c. Fig. 2. The packing of (I) viewed along c. Bis[4-(2-benzylidenepropylideneamino)phenyl] ether Bis[4-(2-benzylidenepropylideneamino)phenyl] ether Crystal data C32H28N2O1 F000 = 968 Mr = 456.6 Dx = 1.203 Mg m−3 Monoclinic, P21/n Cu Kα radiation λ = 1.54184 Å Hall symbol: -P 2yn Cell parameters from 9034 reflections a = 7.4737 (3) Å θ = 3.2–61.2º b = 55.929 (3) Å µ = 0.56 mm−1 c = 6.0275 (3) Å T = 295 K β = 90.022 (4)º Plate, yellow V = 2519.5 (2) Å3 0.51 × 0.38 × 0.02 mm Z = 4 Data collection Oxford Diffraction Gemini 3838 independent reflections 3838 independent reflections sup-3 supplementary materials diffractometer with Atlas CCD detector Radiation source: X-ray tube 3428 reflections with I > 3σ(I) Monochromator: mirrors Rint = 0.016 Detector resolution: 20.7567 pixels mm-1 θmax = 61.4º T = 295 K θmin = 3.2º Rotation method data acquisition using ω scans h = −8→8 Absorption correction: multi-scan (CrysAlis RED; Oxford Diffraction, 2008) k = −62→62 Tmin = 0.682, Tmax = 0.99 l = −6→6 15440 measured reflections diffractometer with Atlas CCD detector Radiation source: X-ray tube 3428 reflections with I > 3σ(I) Monochromator: mirrors Rint = 0.016 Detector resolution: 20.7567 pixels mm-1 θmax = 61.4º T = 295 K θmin = 3.2º Rotation method data acquisition using ω scans h = −8→8 Absorption correction: multi-scan (CrysAlis RED; Oxford Diffraction, 2008) k = −62→62 Tmin = 0.682, Tmax = 0.99 l = −6→6 15440 measured reflections Refinement Refinement on F2 Secondary atom site location: difference Fourier map Least-squares matrix: full Hydrogen site location: difference Fourier map R[F2 > 2σ(F2)] = 0.036 H-atom parameters constrained wR(F2) = 0.120 Weighting scheme based on measured s.u.'s w = 1/ [σ2(I) + 0.0016I2] S = 2.32 (Δ/σ)max = 0.001 3838 reflections Δρmax = 0.19 e Å−3 317 parameters Δρmin = −0.18 e Å−3 112 constraints Extinction correction: none Primary atom site location: structure-invariant direct methods Refinement Refinement on F2 Secondary atom site location: difference Fourier map Least-squares matrix: full Hydrogen site location: difference Fourier map R[F2 > 2σ(F2)] = 0.036 H-atom parameters constrained wR(F2) = 0.120 Weighting scheme based on measured s.u.'s w = 1/ [σ2(I) + 0.0016I2] S = 2.32 (Δ/σ)max = 0.001 3838 reflections Δρmax = 0.19 e Å−3 317 parameters Δρmin = −0.18 e Å−3 112 constraints Extinction correction: none Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: difference Fourier map H-atom parameters constrained Weighting scheme based on measured s.u.'s w = 1/ [σ2(I) + 0.0016I2] (Δ/σ)max = 0.001 Δρmax = 0.19 e Å−3 Δρmin = −0.18 e Å−3 Extinction correction: none Extinction correction: none Special details Refinement. The refinement was carried out against all reflections. The conventional R-factor is always based on F. The goodness of fit as well as the weighted R-factor are based on F and F2 for refinement carried out on F and F2, respectively. The threshold expres- sion is used only for calculating R-factors etc. and it is not relevant to the choice of reflections for refinement. The program used for refinement, Jana2006, uses the weighting scheme based on the experimental expectations, see _refine_ls_weighting_details, that does not force S to be one. Therefore the values of S are usually larger than the ones from the SHELX program. supplementary materials pp y sup-6 H16c 0.549168 0.380782 0.445248 0.0936* H32a 0.493257 0.132162 0.434681 0.0927* H32b 0.486302 0.104148 0.435939 0.0927* H32c 0.308313 0.118963 0.442292 0.0927* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0715 (8) 0.0641 (7) 0.0559 (7) −0.0001 (5) 0.0032 (7) 0.0004 (6) N1 0.0581 (8) 0.0672 (9) 0.0598 (9) −0.0006 (6) 0.0011 (7) 0.0000 (7) N2 0.0611 (8) 0.0675 (9) 0.0585 (9) −0.0007 (7) 0.0045 (7) 0.0004 (7) C1 0.0445 (8) 0.0641 (10) 0.0559 (9) 0.0018 (7) 0.0061 (7) −0.0017 (8) C2 0.0571 (9) 0.0713 (11) 0.0554 (9) 0.0006 (8) −0.0064 (8) −0.0025 (9) C3 0.0600 (10) 0.0670 (11) 0.0634 (11) −0.0034 (8) −0.0054 (8) −0.0080 (9) C4 0.0503 (8) 0.0659 (10) 0.0543 (9) 0.0003 (7) 0.0039 (8) −0.0023 (8) C5 0.0541 (9) 0.0719 (11) 0.0543 (9) 0.0012 (7) −0.0042 (8) −0.0032 (8) C6 0.0505 (8) 0.0688 (11) 0.0655 (11) −0.0027 (7) −0.0066 (8) −0.0065 (9) C7 0.0698 (11) 0.0701 (11) 0.0508 (9) 0.0010 (8) 0.0035 (9) 0.0014 (9) C8 0.0553 (9) 0.0703 (11) 0.0543 (10) 0.0018 (8) 0.0024 (8) 0.0043 (8) C9 0.0703 (11) 0.0720 (11) 0.0495 (9) 0.0028 (8) 0.0028 (8) 0.0042 (9) C10 0.0555 (9) 0.0704 (11) 0.0547 (10) 0.0015 (8) −0.0040 (8) 0.0020 (8) C11 0.0742 (11) 0.0766 (13) 0.0662 (11) 0.0011 (9) 0.0119 (10) 0.0048 (10) C12 0.0964 (15) 0.0807 (14) 0.0807 (15) −0.0061 (11) 0.0132 (12) 0.0151 (11) C13 0.0925 (15) 0.0701 (13) 0.1000 (17) −0.0013 (11) −0.0004 (14) 0.0106 (12) C14 0.0870 (14) 0.0745 (13) 0.0912 (16) 0.0068 (10) 0.0064 (13) −0.0108 (12) C15 0.0702 (12) 0.0789 (13) 0.0645 (11) −0.0009 (9) 0.0054 (9) −0.0040 (9) C16 0.0973 (16) 0.0812 (13) 0.0555 (11) −0.0032 (11) 0.0008 (11) 0.0019 (9) C17 0.0456 (8) 0.0639 (10) 0.0572 (9) 0.0009 (7) −0.0010 (7) 0.0014 (8) C18 0.0559 (9) 0.0714 (11) 0.0568 (10) 0.0012 (8) 0.0096 (8) 0.0040 (9) C19 0.0580 (9) 0.0670 (11) 0.0634 (11) −0.0028 (8) 0.0117 (8) 0.0062 (9) C20 0.0493 (8) 0.0668 (10) 0.0562 (9) 0.0013 (7) 0.0027 (8) 0.0026 (8) C21 0.0560 (9) 0.0718 (11) 0.0541 (9) 0.0001 (7) 0.0104 (8) 0.0029 (8) C22 0.0539 (9) 0.0674 (11) 0.0623 (10) −0.0032 (7) 0.0109 (8) 0.0061 (8) C23 0.0715 (11) 0.0708 (11) 0.0522 (9) 0.0025 (8) 0.0015 (9) −0.0015 (9) C24 0.0593 (10) 0.0709 (11) 0.0524 (10) 0.0004 (8) 0.0033 (8) −0.0021 (8) C25 0.0706 (11) 0.0733 (11) 0.0493 (9) −0.0008 (8) 0.0002 (8) −0.0044 (9) C26 0.0570 (9) 0.0723 (11) 0.0543 (10) −0.0016 (8) 0.0022 (8) −0.0002 (8) C27 0.0707 (11) 0.0774 (12) 0.0667 (12) −0.0016 (9) −0.0099 (10) −0.0022 (10) C28 0.0925 (15) 0.0827 (14) 0.0776 (14) −0.0149 (11) −0.0013 (12) −0.0131 (12) C29 0.0934 (15) 0.0725 (13) 0.0901 (16) −0.0043 (11) 0.0137 (13) −0.0041 (12) C30 0.0947 (15) 0.0747 (13) 0.0892 (16) 0.0011 (11) −0.0021 (13) 0.0167 (12) C31 0.0793 (13) 0.0799 (14) 0.0630 (11) −0.0062 (10) −0.0057 (10) 0.0078 (10) C32 0.0954 (15) 0.0798 (13) 0.0567 (11) −0.0036 (11) 0.0032 (11) −0.0016 (9) Geometric parameters (Å, °) O1—C1 1.386 (2) C16—H16a 0.96 O1—C17 1.385 (2) C16—H16b 0.96 N1—C4 1.412 (2) C16—H16c 0.96 Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-4 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å ) x y z Uiso/Ueq O1 0.41159 (19) 0.25020 (2) 1.1527 (2) 0.0638 (4) N1 0.4123 (2) 0.33936 (3) 0.7401 (3) 0.0617 (5) N2 0.4264 (2) 0.16126 (3) 0.7377 (3) 0.0623 (5) C1 0.4131 (2) 0.27183 (3) 1.0403 (3) 0.0548 (5) C2 0.4984 (2) 0.29047 (3) 1.1464 (3) 0.0613 (6) C3 0.4970 (2) 0.31299 (3) 1.0534 (3) 0.0635 (6) C4 0.4118 (2) 0.31709 (3) 0.8503 (3) 0.0568 (5) C5 0.3295 (2) 0.29795 (3) 0.7467 (3) 0.0601 (6) C6 0.3268 (2) 0.27553 (3) 0.8402 (3) 0.0616 (6) C7 0.4096 (2) 0.35875 (3) 0.8483 (3) 0.0636 (6) sup-4 supplementary materials C8 0.4215 (2) 0.38208 (3) 0.7420 (3) 0.0600 (6) C9 0.4227 (2) 0.40156 (3) 0.8727 (3) 0.0639 (6) C10 0.4388 (2) 0.42689 (3) 0.8176 (3) 0.0602 (6) C11 0.5287 (3) 0.43555 (4) 0.6318 (4) 0.0723 (7) C12 0.5396 (3) 0.45964 (4) 0.5896 (4) 0.0859 (9) C13 0.4641 (3) 0.47591 (4) 0.7318 (4) 0.0875 (9) C14 0.3778 (3) 0.46796 (4) 0.9185 (4) 0.0842 (8) C15 0.3661 (3) 0.44386 (4) 0.9620 (4) 0.0712 (7) C16 0.4273 (3) 0.38214 (4) 0.4939 (3) 0.0780 (8) C17 0.4135 (2) 0.22865 (3) 1.0395 (3) 0.0555 (5) C18 0.3308 (2) 0.20971 (3) 1.1439 (3) 0.0613 (6) C19 0.3356 (2) 0.18727 (3) 1.0517 (3) 0.0628 (6) C20 0.4223 (2) 0.18353 (3) 0.8484 (3) 0.0574 (5) C21 0.5012 (2) 0.20285 (3) 0.7441 (3) 0.0606 (6) C22 0.5005 (2) 0.22525 (3) 0.8389 (3) 0.0612 (6) C23 0.4375 (2) 0.14192 (4) 0.8456 (3) 0.0648 (6) C24 0.4369 (2) 0.11855 (3) 0.7384 (3) 0.0609 (6) C25 0.4481 (2) 0.09907 (3) 0.8688 (3) 0.0644 (6) C26 0.4490 (2) 0.07366 (3) 0.8136 (3) 0.0612 (6) C27 0.3678 (3) 0.06393 (4) 0.6255 (4) 0.0716 (7) C28 0.3726 (3) 0.03972 (4) 0.5842 (4) 0.0843 (8) C29 0.4589 (3) 0.02459 (4) 0.7274 (4) 0.0854 (8) C30 0.5381 (3) 0.03360 (4) 0.9145 (4) 0.0862 (9) C31 0.5324 (3) 0.05778 (4) 0.9586 (4) 0.0741 (7) C32 0.4306 (3) 0.11845 (4) 0.4907 (3) 0.0773 (8) H2 0.558692 0.287765 1.284787 0.0735 H3 0.555012 0.325979 1.128755 0.0762* H5 0.272697 0.300391 0.605623 0.0721* H6 0.265779 0.262608 0.76752 0.0739* H11 0.584073 0.424477 0.531363 0.0868* H12 0.600738 0.46515 0.459163 0.1031* H13 0.471712 0.492723 0.700898 0.105* H14 0.325198 0.479257 1.01914 0.1011* H15 0.306821 0.438587 1.094548 0.0855* H18 0.26953 0.212226 1.281899 0.0736* H19 0.279415 0.174115 1.126614 0.0753* H21 0.557768 0.200601 0.602612 0.0728* H22 0.559553 0.238363 0.76685 0.0734* H27 0.307393 0.074305 0.523036 0.0859* H28 0.315201 0.033414 0.454239 0.1011* H29 0.463802 0.00776 0.696717 0.1024* H30 0.597998 0.023017 1.01551 0.1035* H31 0.586783 0.063783 1.091686 0.0889* H7 0.399203 0.358045 1.006914 0.0763* H9 0.410947 0.398259 1.028338 0.0767* H23 0.446597 0.142663 1.004279 0.0778* H25 0.457048 0.102463 1.024492 0.0773* H16a 0.359347 0.36888 0.438036 0.0936* H16b 0.37714 0.396808 0.439336 0.0936* sup-5 supplementary materials Atomic displacement parameters (Å2) sup-6 supplementary materials 1.266 (2) C17—C18 1.414 (2) C17—C22 1.264 (2) C18—C19 1.379 (2) C18—H18 1.384 (3) C19—C20 1.379 (3) C19—H19 0.96 C20—C21 1.398 (3) C21—C22 0.96 C21—H21 1.384 (2) C22—H22 1.375 (3) C23—C24 0.96 C23—H23 0.96 C24—C25 1.456 (3) C24—C32 0.96 C25—C26 1.345 (3) C25—H25 1.496 (3) C26—C27 1.460 (3) C26—C31 0.96 C27—C28 1.393 (3) C27—H27 1.398 (3) C28—C29 1.374 (3) C28—H28 0.96 C29—C30 1.372 (3) C29—H29 0.96 C30—C31 1.372 (4) C30—H30 0.96 C31—H31 1.376 (3) C32—H32a 0.96 C32—H32b 0.96 C32—H32c 121.25 (14) H16b—C16—H16c 120.89 (16) O1—C17—C18 120.83 (16) O1—C17—C22 115.92 (15) C18—C17—C22 123.52 (15) C17—C18—C19 120.45 (16) C17—C18—H18 119.92 (17) C19—C18—H18 120.0388 C18—C19—C20 120.0374 C18—C19—H19 120.58 (17) C20—C19—H19 119.7093 N2—C20—C19 119.7097 N2—C20—C21 123.73 (15) C19—C20—C21 118.11 (16) C20—C21—C22 118.09 (16) C20—C21—H21 121.81 (17) C22—C21—H21 119.0935 C17—C22—C21 119.0919 C17—C22—H22 119.12 (16) C20—C22—H22 1.266 (2) C17—C18 1.378 (2) 1.414 (2) C17—C22 1.386 (3) 1.264 (2) C18—C19 1.373 (3) 1.379 (2) C18—H18 0.96 1.384 (3) C19—C20 1.402 (3) 1.379 (3) C19—H19 0.96 0.96 C20—C21 1.383 (3) 1.398 (3) C21—C22 1.377 (3) 0.96 C21—H21 0.96 1.384 (2) C22—H22 0.96 1.375 (3) C23—C24 1.458 (3) 0.96 C23—H23 0.96 0.96 C24—C25 1.346 (3) 1.456 (3) C24—C32 1.494 (3) 0.96 C25—C26 1.459 (3) 1.345 (3) C25—H25 0.96 1.496 (3) C26—C27 1.396 (3) 1.460 (3) C26—C31 1.393 (3) 0.96 C27—C28 1.377 (3) 1.393 (3) C27—H27 0.96 1.398 (3) C28—C29 1.370 (3) 1.374 (3) C28—H28 0.96 0.96 C29—C30 1.370 (4) 1.372 (3) C29—H29 0.96 0.96 C30—C31 1.379 (3) 1.372 (4) C30—H30 0.96 0.96 C31—H31 0.96 1.376 (3) C32—H32a 0.96 0.96 C32—H32b 0.96 0.96 C32—H32c 0.96 121.25 (14) H16b—C16—H16c 109.4707 120.89 (16) O1—C17—C18 116.07 (16) 120.83 (16) O1—C17—C22 123.64 (15) 115.92 (15) C18—C17—C22 120.20 (16) 123.52 (15) C17—C18—C19 120.40 (17) 120.45 (16) C17—C18—H18 119.7986 119.92 (17) C19—C18—H18 119.8003 120.0388 C18—C19—C20 120.14 (16) 120.0374 C18—C19—H19 119.9296 120.58 (17) C20—C19—H19 119.9325 119.7093 N2—C20—C19 123.66 (15) 119.7097 N2—C20—C21 117.67 (16) 123.73 (15) C19—C20—C21 118.60 (16) 118.11 (16) C20—C21—C22 121.35 (17) 118.09 (16) C20—C21—H21 119.326 121.81 (17) C22—C21—H21 119.3271 119.0935 C17—C22—C21 119.28 (16) 119.0919 C17—C22—H22 120.3631 119.12 (16) C20—C22—H22 149.7198 1.266 (2) C17—C18 1.414 (2) C17—C22 1.264 (2) C18—C19 1.379 (2) C18—H18 1.384 (3) C19—C20 1.379 (3) C19—H19 0.96 C20—C21 1.398 (3) C21—C22 0.96 C21—H21 1.384 (2) C22—H22 1.375 (3) C23—C24 0.96 C23—H23 0.96 C24—C25 1.456 (3) C24—C32 0.96 C25—C26 1.345 (3) C25—H25 1.496 (3) C26—C27 1.460 (3) C26—C31 0.96 C27—C28 1.393 (3) C27—H27 1.398 (3) C28—C29 1.374 (3) C28—H28 0.96 C29—C30 1.372 (3) C29—H29 0.96 C30—C31 1.372 (4) C30—H30 0.96 C31—H31 1.376 (3) C32—H32a 0.96 C32—H32b 0.96 C32—H32c 17 121.25 (14) H16b—C16—H16c 7 120.89 (16) O1—C17—C18 C23 120.83 (16) O1—C17—C22 2 115.92 (15) C18—C17—C22 6 123.52 (15) C17—C18—C19 6 120.45 (16) C17—C18—H18 3 119.92 (17) C19—C18—H18 2 120.0388 C18—C19—C20 2 120.0374 C18—C19—H19 4 120.58 (17) C20—C19—H19 3 119.7093 N2—C20—C19 3 119.7097 N2—C20—C21 3 123.73 (15) C19—C20—C21 5 118.11 (16) C20—C21—C22 5 118.09 (16) C20—C21—H21 6 121.81 (17) C22—C21—H21 5 119.0935 C17—C22—C21 5 119.0919 C17—C22—H22 5 119.12 (16) C20—C22—H22 sup-7 supplementary materials supplementary materials C1—C6—H6 120.4391 N2—C23—C24 122.61 (18) C5—C6—H6 120.4404 N2—C23—H23 118.6934 N1—C7—C8 122.68 (17) C24—C23—H23 118.6931 N1—C7—H7 118.6563 C23—C24—C25 117.85 (17) C8—C7—H7 118.6616 C23—C24—C32 116.50 (16) C7—C8—C9 117.94 (17) C25—C24—C32 125.61 (17) C7—C8—C16 116.35 (16) C24—C25—C26 130.95 (17) C9—C8—C16 125.70 (17) C24—C25—H25 114.5252 C8—C9—C10 130.80 (17) C26—C25—H25 114.5237 C8—C9—H9 114.598 C25—C26—C27 124.25 (17) C10—C9—H9 114.6038 C25—C26—C31 118.67 (17) C9—C10—C11 124.07 (17) C27—C26—C31 117.06 (18) C9—C10—C15 119.02 (17) C26—C27—C28 121.3 (2) C11—C10—C15 116.87 (18) C26—C27—H27 119.375 C10—C11—C12 121.3 (2) C28—C27—H27 119.3729 C10—C11—H11 119.3764 C27—C28—C29 120.4 (2) C12—C11—H11 119.3736 C27—C28—H28 119.8062 C11—C12—C13 120.7 (2) C29—C28—H28 119.8072 C11—C12—H12 119.6501 C28—C29—C30 119.7 (2) C13—C12—H12 119.6503 C28—C29—H29 120.1738 C12—C13—C14 119.4 (2) C30—C29—H29 120.1722 C12—C13—H13 120.2841 C29—C30—C31 120.4 (2) C14—C13—H13 120.2831 C29—C30—H30 119.7998 C13—C14—C15 120.3 (2) C31—C30—H30 119.7949 C13—C14—H14 119.8703 C26—C31—C30 121.2 (2) C15—C14—H14 119.8702 C26—C31—H31 119.3916 C10—C15—C14 121.5 (2) C30—C31—H31 119.3905 C10—C15—H15 119.2735 C24—C32—H32a 109.4703 C14—C15—H15 119.2764 C24—C32—H32b 109.4721 C8—C16—H16a 109.4718 C24—C32—H32c 109.4709 C8—C16—H16b 109.4708 H32a—C32—H32b 109.4716 C8—C16—H16c 109.4712 H32a—C32—H32c 109.4711 H16a—C16—H16b 109.4705 H32b—C32—H32c 109.4713 H16a—C16—H16c 109.4723 ?—?—?—? ? Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A ?—?···? ? ? ? ? Hydrogen-bond geometry (Å, °) sup-8 supplementary materials Fig. 1 sup-9 supplementary materials supplementary materials Fig. 2 sup-10
https://openalex.org/W2136215480	https://europepmc.org/articles/pmc2810790?pdf=render	English	null	Silicone migration to the contralateral axillary lymph nodes and breast after highly cohesive silicone gel implant failure: a case report	Cases journal	2,009	cc-by	1,421	Abstract Highlycohesivesiliconegelimplantsareadvertisedforaestheticandsafetyadvantages.Ourcaseisthefourth reportdescribingearlyimplantruptureandcontralateralmigrationofsiliconoma.Despitethegreaterdegree of gel cohesiveness, a continued vigilance for signs and symptoms of migration is highly recommended. reconstruction, the patient was treated for multifocal invasive ductal carcinoma with adjuvant chemotherapy and radiation to the chest. During reconstruction, symme- trization of the right side was achieved by performing a superior pedicle mammoplasty and insertion of a Poly Implant Prosthesis (PIP) gel implant. After 2 years of routine follow-up, the patient experienced rapid enlarge- ment of her reconstructed left breast (Figure 1). Findings were suspicious for implant rupture and seroma forma- tion, however; a palpable mass of the augmented right breast was also noted on examination as well as right axillary lymphadenopathy. Biopsy was performed on both the right breast mass palpable axillary node to rule out malignancy. The biopsy demonstrated findings consistent with siliconoma. Axillary dissection revealed 3 large rubbery nodes, the greatest measuring approximately 2cm in diameter. A capsular mass was identified on the right side and the implant shell appeared to be intact. Examination of the left breast demonstrated seroma and Introduction The introduction of highly cohesive silicone gel implants (HCGI) advertised favorable aesthetic and safety advan- tages over standard cohesive gel implants. These included greater durability of overall shape particularly with regards to the upper-pole volume and a reduction in incidence of outer shell folding. The safety profile also improved with the greater degree of gel viscosity by limiting migration and loco-regional spread of silicone gel after compromise of the implant shell. Since the introduction of HCGI in 1993 there have only been 3 published case reports of regional spread and axillary lymph node involvement after capsular rupture of an HCGI [1, 2, 3]. Case report Address: Department of breast cancer and reconstructive surgery, Institut du Sein, Paris Breast Center, 7 Avenue Bugeaud, 75116 Paris, France Email: GK - Kaufman_gabriel@msn.com; RS* - ritasakr@yahoo.com; CI - inguenaultcyrille@yahoo.fr; IS - isa.sarfati@wanadoo.fr; CN - claude. nos@hop.egp.ap-hop-paris.fr; KC - Krishna.clough@orange.fr * Corresponding author †Equal contribution Received: 15 December 2008 Accepted: 09 February 2009 Cases Journal 2009, 2:6420 doi: 10.1186/1757-1626-2-6420 This article is available from: http://casesjournal.com/casesjournal/article/view/2/3/6420 © 2009 A. Sakr et al; licensee Cases Network Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 3 (page number not for citation purposes) Abbreviations HCGI, Highly cohesive silicone gel implants; PIP, Poly Implant Prosthesis; MRI, Magnetic resonance imaging. Discussion Silicone gel entering the lymphatics, either through overt implant rupture or slow leakage across the intact outer shell, can result in regional migration to the draining lymph node basins [1, 2]. Axillary lymphadenopathy in any patient with a history of breast cancer should raise concern for recurrence and prompt aggressive evaluation to avoid delays in diagnosis. Migration of silicone is not always limited to the corresponding axillary lymph nodes and spread to the internal mammary and inguinal nodes as well as the abdominal wall and lower back have all been reported in the literature [1, 2, 3, 4]. Our report is the first to describe a silicone granuloma within the capsule of the contralateral breast and axillary lymph nodes. Figure 2 Implant rupture with extrusion of the high cohesive gel into the upper pole. Figure 2 Implant rupture with extrusion of the high cohesive gel into the upper pole. The causes of implant rupture are varied, but those commonly reported are compression from closed capsu- lotomy, mammography or trauma with the actual cause often unknown [4]. The causes of implant rupture are varied, but those commonly reported are compression from closed capsu- lotomy, mammography or trauma with the actual cause often unknown [4]. With the introduction of highly cohesive silicone gel matrix implants in the 1990’s the risk of local-regional spread after rupture was thought to have been amelio- rated. The early experience with highly cohesive implants resulted in low complication rates without evidence for silicone migration [1]. MRI has proven to be sensitive in the detection of implant rupture. Comparison studies have demonstrated higher rates of sensitivity using MRI compared to mammography or ultrasonography when the appropriate breast coil is utilized [5]. The role of fine needle aspiration for palpable lesions in the axilla and breast after breast augmentation is a useful tool in differentiating between cancer recurrence and silicone granulomas [6]. The 3 year results of the highly cohesive silicone breast implant core study reported a less than 1% device rupture rate [2]. Magnetic resonance imaging (MRI) was used to evaluate patients for evidence of rupture in this study. An additional European series from Sweden found a 0.3% incidence of rupture based on MRI evaluation, thus confirming low rupture rates in this type of implant [3]. Case presentation An European Caucasian 59-year-old patient had delayed reconstruction with a latissimus dorsi flap and McGhan 410 highly cohesive silicone implant after a modified radical mastectomy of the left breast. Prior to Page 1 of 3 (page number not for citation purposes) Cases Journal 2009, 2:6420 http://casesjournal.com/casesjournal/article/view/2/3/6420 Figure 2 Implant rupture with extrusion of the high cohesive gel into the upper pole. implant rupture with extrusion of the high cohesive gel into the upper pole (Figure 2). The left and right implants were removed and both replaced with PIP standard profile silicone implants. Final pathology was consistent with siliconoma for both the enlarged lymph nodes and right breast mass. Conclusion Early implant failure of HCGI is rare, but despite the increased gel viscosity the potential for regional migration remains. This is the fourth case report describing regional migration. Our case report adds to a growing awareness of this phenomenon and emphasizes the need for continued vigilance for signs and symptoms of migration despite the greater degree of gel cohesiveness. Figure 1 Early enlargement of the patient’s left reconstructed breast. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Figure 1 Early enlargement of the patient’s left reconstructed breast. Figure 1 Early enlargement of the patient’s left reconstructed breast. Page 2 of 3 (page number not for citation purposes) http://casesjournal.com/casesjournal/article/view/2/3/6420 http://casesjournal.com/casesjournal/article/view/2/3/6420 http://casesjournal.com/casesjournal/article/view/2/3/6420 Cases Journal 2009, 2:6420 Authors’ contribution GK and RS performed the writing of the manuscript. CI, IS and CN contributed to analysis. KC contributed to revision, supervision and approval of the work. GK and RS performed the writing of the manuscript. CI, IS and CN contributed to analysis. KC contributed to revision, supervision and approval of the work. References 1. Brown MH, Shenker R, Silver SA: Cohesive silicone gel breast implants in aesthetic and reconstructive breast surgery. Plast Reconstr Surg 2005, 16(3):768-79. g ( ) 2. Bengtson BP, Van Natta BW, Murphy DK, et al.: Style 410 highly cohesive silicone breast implant core study results at 3 years. Plast Reconstr Surg 2007, 120(Suppl 1):40S-48S. g ( pp ) 3. Heden P, Bone B, Murphy DK, et al.: Style 410 cohesive silicone breast implants: safety and effectiveness at 5 to 9 years after implantation. Plast Reconstr Surg 2006, 118(6):1281-1287. p g , ( ) 4. Brown SL, Silverman BG, Berg WA: Rupture of silicone-gel breast implants: causes, sequelae, and diagnosis. Lancet 1997, 350 (9090):1531-1537. ( ) 5. Berg WA, Caskey CI, Hamper UM, et al.: Diagnosing breast implant rupture with MR imaging, US and mammography. Radiographics 1993, 13(6):1323-1336. ( ) 5. Berg WA, Caskey CI, Hamper UM, et al.: Diagnosing breast implant rupture with MR imaging, US and mammography. Radiographics 1993, 13(6):1323-1336. g p ( ) 6. Kulber DA, Mackenzie D, Steiner JH, et al: Monitoring the axilla in patients with silicone gel implants. Ann Plast Surg 1995, 35 (6):580-584. g p ( ) 6. Kulber DA, Mackenzie D, Steiner JH, et al: Monitoring the axilla in patients with silicone gel implants. Ann Plast Surg 1995, 35 (6):580-584. Competing interests p g The authors declare that they have no competing interests. Page 3 of 3 (page number not for citation purposes) Do you have a case to share? Submit your case report today • Rapid peer review • Fast publication • PubMed indexing • Inclusion in Cases Database Any patient, any case, can teach us something www.casesnetwork.com Page 3 of 3 (page number not for citation purposes) Do you have a case to share? Submit your case report today • Rapid peer review • Fast publication • PubMed indexing • Inclusion in Cases Database Any patient, any case, can teach us something www.casesnetwork.com
https://openalex.org/W2553569692	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-016-3848-5	English	null	User satisfaction with the structure and content of the NEXit intervention, a text messaging-based smoking cessation programme	BMC public health	2,016	cc-by	7,245	* Correspondence: ulrika.mussener@liu.se 1Department of Medical and Health Sciences, Linköping University, Linköping 581 83, Sweden Full list of author information is available at the end of the article © The Author(s). 2016 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. User satisfaction with the structure and content of the NEXit intervention, a text messaging-based smoking cessation programme Ulrika Müssener1* , Marcus Bendtsen2, Jim McCambridge1,3 and Preben Bendtsen4 Müssener et al. BMC Public Health (2016) 16:1179 DOI 10.1186/s12889-016-3848-5 Müssener et al. BMC Public Health (2016) 16:1179 DOI 10.1186/s12889-016-3848-5 Short description of the NEXit intervention p The NEXit intervention was developed in several steps based on existing recommended smoking cessation manuals used in Sweden [4]. Using the BCT taxonomy suggested by Mitchie et al. [10], which includes 43 BCTs previously found to be reliable in coding the context of smoking cessation interventions, the intervention used 32 different BCTs, for example: “provide information on consequences of smoking and smoking cessation”, “prompt review of goals”, and “boost motivation and self-efficacy”. Slightly more than half of the messages clearly combined more than one BCT [5] (see Table 1). As part of the formative development, 10 smoking cessa- tion experts and 10 smokers evaluated and provided comments and suggestions on each message [4]. The content of the intervention was then revised. Thereafter, a shortened version (8 weeks) was evaluated in a pilot study to test and improve the technical platform devel- oped by the research group. There is a limited amount of evidence for effective smok- ing cessation interventions among young people and a pre- vailing reluctance to seek help in stopping smoking. To address this, a text messaging-based smoking intervention, the NEXit intervention, was developed, aimed specifically at this group [4, 5]. A text messaging-based intervention was chosen because mobile phones are increasingly being used as a medium for reminders about medication and self-management of chronic diseases, such as diabetes, as well as behavioural change interventions on problematic drinking, obesity, tobacco cessation and sedentary lifestyle [6–9]. Young people use mobile phones extensively, there- fore this mode of delivery was deemed especially suitable for overcoming barriers to treatment by offering easy ac- cess at all times. The intervention included the following elements: making a public declaration about quitting (i.e. telling friends about the quit attempt), encouraging asking for support from family and friends, distraction techniques, and the possibility of requesting more text messages when the participant experienced strong cravings or a temporary relapse. The intervention began with a motiv- ational phase with 2 SMS (short message services) per day. The messages in the motivational phase contained relevant information in advance of quitting i.e. symp- toms to expect on quitting, tips to avoid weight gain, tips to cope with cravings and to avoid smoking triggers, motivational support, and how to distract one’s mind from smoking. The motivational phase lasted for a mini- mum of 1 week and a maximum of 4 weeks pending on the participant’s preference. Short description of the NEXit intervention During this motivational phase, participants were free to decide when to set a quit date [4, 5]. If no quit date had been set after 4 weeks, participants were given a quit date 3 days later. The core programme started on the quit date and, lasted for 12 weeks. The formative development and content of the NEXit 12-week intervention has been reported previously [4], following the steps recommended by Abroms et al. [9]. As well as developing the content based on existing evidence for effective smoking cessation messages, we also analysed the behaviour change techniques (BCTs) included in the intervention using the BCT taxonomy [4, 9], as suggested by Mitchie et al. [10]. The next step was to test the effect- iveness of the intervention in a randomized controlled trial (RCT), which included 1590 participants recruited from all colleges and universities in Sweden. The short-term effect, measured immediately after the 12-week intervention, revealed that 25.9% of smokers in the intervention group had prolonged abstinence (not having smoked in the last 8 weeks) compared with 14.6% in the control group [5]. This does not mean that the intervention was flawless and there is no room for improvement from the individual user’s perspective, so the present study was performed to get information for subsequent revisions, thus adding user knowledge to optimize the text-based intervention. The 12-week core programme consisted of 157 mes- sages. At participants’ discretion, extra messages were sent regarding cravings, temporary relapses or concerns about weight gain. During the last 3 days of the motivation phase, participants were sent 5 messages per day in order to prepare the participants for the quit day with tips on getting rid of cigarettes and ashtrays, writing a list of rea- sons for quitting and renewed information on how the body might react to abstinence. The core programme Abstract Background: Smoking is still the leading cause of preventable ill health and death. There is a limited amount of evidence for effective smoking cessation interventions among young people. To address this, a text messaging-based smoking cessation programme, the NEXit intervention, was developed. Short-term effectiveness, measured immediately after the 12-week intervention revealed that 26% of smokers in the intervention group had prolonged abstinence compared with 15% in the control group. The present study was performed to explore the users’ experiences of the structure and content of the intervention in order to further develop the intervention. Methods: Students participating in the main NEXit randomized controlled trial were invited to grade their experiences of the structure and content of the intervention after having completed follow-up. The participants received an e-mail with an electronic link to a short questionnaire. Descriptive analysis of the distribution of the responses to the questionnaire was performed. Free-text comments to 14 questions were analysed. Results: The response rate for the user feedback questionnaire was 35% (n = 289/827) and 428 free-text comments were collected. The first motivational phase of the intervention was appreciated by 55% (158/289) of the participants. Most participants wanted to quit smoking immediately and only 124/289 (43%) agreed to have to decide a quit-date in the future. Most participants 199/289 (69%) found the content of the messages in the core programme to be very good or good, and the variability between content types was appreciated by 78% (224/289). Only 34% (97/289) of the participants thought that all or nearly all messages were valuable, and some mentioned that it was not really the content that mattered, but that the messages served as a reminder about the decision to quit smoking. Conclusions: The programme was largely perceived satisfactory in most aspects concerning structure and content by young people and most participants stated that they would recommend it to a friend who wants to quit smoking. The motivational phase might be worth shortening and the number of messages around the quit date itself reduced. Shorter messages seemed to be more acceptable. Trial registration: ISRCTN75766527. Keywords: Tobacco, Smoking cessation, Students, Text messages, Mobile phones, SMS Müssener et al. BMC Public Health (2016) 16:1179 Page 2 of 8 Background smoking cessation intervention. Specifically, we report to what extent the users read the text messages, and explore their views on the content to get user input for further development of the intervention. Although the number of smokers is decreasing in most populations in high income countries around the world, smoking is still the leading cause of preventable ill health and death. The ratio between death and ill health due to smoking is 1:20; thus smoking may lead to premature death, and it is also a major contributing factor of ill health [1]. In Sweden, smoking is responsible for approximately 9.6% of the total disease burden, which means that that around 6000 people die every year due to smoking [2]. The number of young people aged 16–29 years who smoke has been stable for the last 5 years; approximately 11–13% of young women and 7–10% of young men are daily smokers and twice as many are occasional smokers [3]. Questionnaire h The participants from the NEXit study intervention group were asked 14 questions, with 5–7 fixed response options (Additional file 1). The first 2 questions were about change in smoking habits and possible reasons for having stopped smoking or smoking less if applicable. A free-text option gave the participants an opportunity to describe other factors of importance not covered by the fixed response options (response options: participation in the study/people around me urged me to stop/negative consequences of smoking/too expensive to smoke/ don´t know why I managed to quit). Methods The aim of the present study was to explore the users’ experiences of the structure and content of the NEXit Müssener et al. BMC Public Health (2016) 16:1179 Page 3 of 8 Table 1 Baseline characteristics of the responders and non-responders in the follow-up Variables Non-responders (n = 538) Responders (n = 289) Significance Gender, n (% female) 367 (68.2) 206 (71.3) NS Age, n (%) NS < 21 years 46 (8.6) 24 (8.3) 21–25 years 245 (45.5) 127 (8.3) 26–30 years 118 (21.9) 63 (21.8) ≥31 years 129 (24.0) 75 (26.0) Marital status, n (% single) 288 (53.5) 138 (47.8) NS Duration of smoking in years, median, (IQR) 8 (7) 8 (8) NS Number of cigarettes per weeka median (IQR) 70 (70.0) 56 (57.8) P = 0.016b Using snuff, n (% yes) 127 (23.6) 67 (23.2) NS Importance of quitting smoking, median (IQR) (Scale: 1–10 where 1 = not important at all, 10 = very important) 9 (3) 9 (2) NS a24 participants did not state how many cigarettes they smoked. Percentages for this variable is based on those that did state the number of cigarettes smoked. Percentages for all other variables are based on the full sample bMann–Whitney U test indicated that the distributions in the two groups differed significantly (Mann–Whitney U = 81292, P = 0.016) ble 1 Baseline characteristics of the responders and non-responders in the follow-up by SMS, and 10 attempts were made to phone those who had still not responded. started with 4 messages per day. In weeks 2–4, the partici- pants received 2 messages per day, which was reduced to 2 messages every second day during weeks 5–7. The num- ber of messages per day was reduced to 1 in the final weeks [4, 5]. After this intensive follow-up procedure, we then offered the intervention group the opportunity to give their opinion on the structure and content of the inter- vention. The intervention group received an e-mail with an electronic link to a short questionnaire with 2 reminders sent 1 week apart to non-responders. The messages were sent from a GSM modem and administered from a web-based technical platform that has been developed by one of the authors (MB) and jointly owned by 2 of the authors (PB and MB). Using this platform, sending of all SMS was fully automated, including the extra SMS requested by the participants. Study population and procedure Our findings are presented under the headings pre- sented in the Methods section. We report the responses to the relevant questions and include citations from the free-text comments for each heading. to receive extra messages (response options: positive and something I used/positive but I chose not to use it/nega- tive, I used the function but did not benefit from it/nega- tive, but I did not use it/don´t know). (6) how the participants experienced the total duration of the inter- vention (response options: far too long /somewhat too long/just right/somewhat too short/too short/don´t know). Just under half (45%) of the participants provided 428 comments to the 14 questions; the other 55% did not offer any additional comments. Most comments were on the question about what kind of other help the participants had sought during the intervention (n = 87). The lowest number of comments were provided for the questions about the number of messages close to the quit date (n = 16). On average, around 30 comments were received for each question. g j g Experiences of the content of the intervention was explored by 5 questions: (1) the perceived usefulness of the content of the messages in the 4-week motivation phase (response options: very good/good/not particular god/bad/don´t know); (2) the perceived usefulness of the content of the 12 week core-program (response options: very good/good/not particular good/bad/don´t know); (3) the proportion of the total numbers of messages that the participants perceived to be useful (response options: all/nearly all/about half/some/nearly none/none/don´t know). Two questions were asked as a proxy for satisfac- tion with the content; (4) what proportion of all mes- sages were read (response options: all/nearly all/about half/some/nearly none/none/don´t know); (5) would the participants recommend the intervention to a friend who ought to quit smoking (response options: yes/no, definitely not/don´t know). Our findings are presented under the headings pre- sented in the Methods section. We report the responses to the relevant questions and include citations from the free-text comments for each heading. Data analysis Descriptive analysis of the distribution of the responses to the 14 questions was performed. In a first step of the analysis all free-text comments to each question were read through by the first and last author (UM and PB). In the second analysis step, the free-text comments were discussed between of the authors and the comments that captured the main content of the specific question with regard to the aim of the study were chosen. The free-text com- ments are used to underline and illustrate the pattern of response to the fixed response options. The figure after each comment represents the code that were assigned to each of the respondents. Most of the participants who stopped smoking during the intervention (n = 84 of 289) gave negative consequences as the main reason for quitting smok- ing (54%); 31% attributed participation in the study and receipt of the intervention as their main reason. Among the negative consequences mentioned was being a poor role model for children, frequent colds and coughs, bad breath/smell and environmental pollution. Some also suggested that trying to smoke on fewer days worked as positive reinforcement because they felt better on days when they did not smoke. Changes in smoking habits and reasons for smoking cessation A total of 73/289 participants (25%) smoked at the same level as previously, 116 (40%) stated that they were smoking less, 84 (29%) stated that they had quit smoking (compared with 26% in the intervention group as a whole), 9 (3%) participants stated that they now smoked more than before and 7 (2%) answered that they did not know. A number of participants expressed gratitude in the free-text comment for help they received in quitting smoking during the study. The last question explored whether the participants had used any additional support during the intervention (response options: no, I did not need additional support/ yes, I needed and used additional support (type of sup- port was to be specified)). When I received the first message from the programme, I said to myself, “let’s do it.” From that day until now, over a year later, I have smoked 3 cigarettes. No nicotine gum since the New Year, and I don’t smoke any longer. (275) Study population and procedure For this study, college and university students participat- ing in the main NEXit RCT were invited to give feed- back on the intervention after completing the 12-week intervention, and after participating in the formal follow-up of the RCT [4, 5]. The participants were recruited from all colleges and universities in Sweden except 1 university that participated in a pilot study. The participants came from all levels and disciplines [4, 5]. All participants gave informed consent to participate by clicking on a link in an e-mail invitation and the study was approved by Regional Ethical Committee in Linköp- ing, Sweden (Dnr 2014/217-31). A total of 827 partici- pants were allocated to the intervention group and 763 to the control group (a waiting list group that got access to the intervention after the follow-up). In the main study, we managed to get information on the primary outcome from approximately 95% of the participants in the intervention group and 94% of the controls [5]. This was achieved by sending a high number of reminders over a 4-week period. Non-responders to the follow-up were sent up to 6 reminders by e-mail and 3 reminders Experiences of the structure of the intervention was explored by 6 questions: (1) Perception of the usefulness of the 1–4 week motivation phase (response options: large support/some support/weak support/no support/ don´t know 2) Having to set a stop date within the next 4–6 days (Response options: very simple/simple/some- what difficult/difficult /don´t know) (3) perceptions of the large numbers of messages 3 days before the quit date (response options: far too many/somewhat too many/just right/somewhat too few/too few/don´t know). (4) the mix of motivating, supporting and factual mes- sages (response options: very good/good/not particular god/bad/don´t know). (5) the helpfulness of the option Müssener et al. BMC Public Health (2016) 16:1179 Müssener et al. BMC Public Health (2016) 16:1179 Page 4 of 8 Page 4 of 8 Just under half (45%) of the participants provided 428 comments to the 14 questions; the other 55% did not offer any additional comments. Most comments were on the question about what kind of other help the participants had sought during the intervention (n = 87). The lowest number of comments were provided for the questions about the number of messages close to the quit date (n = 16). On average, around 30 comments were received for each question. Perception of the experiences of the structure of the intervention Very good variety. However, I understand that you don’t want to “disturb” too much. But imagine a smoker’s weekday, for example, the need to receive a text message early in the morning, at 12:30 when lunch is served, at the “two forty-five coffee” for the worker, after supper, and the “late noon cigarette”. Maybe the programme should be more adjustable in relation to weekdays and weekends also. If you could you solve that, I think you would really create something unique. (70) The intervention started with a motivational phase of up to 4 weeks before setting a quit date, depending on the individual’s preference. Just over half (158/289, 55%) of the participants agreed that having the motivational phase before the quit date provided good or very good support before setting a quit date. On the other hand, 40% (n = 115/289) did not agree; some thought the mes- sages were repetitive and some said they could be unhelpful reminders about cigarettes. Among those who did managed to quit smoking, 84/289 (67%) stated that the motivational phase gave good or very good support. The variation in the content of the messages between facts and motivational and practical advice was appreci- ated by most (224/289) of the participants (78%) and particularly among those who quit smoking, the great majority agreed that the variation was good or very good. The participants underlined the need for different content and support over time. That I myself could decide was good because I was in control of when I would stop and be able to plan a little in advance. (52) I experienced it only as sick tedious. It was just the same things that others say to you when they want you to stop smoking, and that has never motivated anyone to quit.(19) I thought it was very good, I never knew what was coming and the variation contributed so that I didn’t become tired of the messages. (28) At any point during the motivational phase, partici- pants had the option to decide a quit date within 4–6 days. Just less than half, 124/289 of the participants (43%), found this structure with 3 days preparation before the quit date as constraining and did not see why they could not should stop smoking immediately. Perception of the experiences of the structure of the intervention Some days you need more motivation, and other days it was very nice and good that you got a message that was supportive! So I definitely think it was great that the purposes of the messages that I received were different. (46) At first, I thought it was tough, and I felt very pressured when I got the first message. But on the other hand, to set a stop date so close in time was good I think. (46) A total of 122/289 (42%) of the participants requested, at least once, extra messages concerning relapse, craving or fear of gaining weight. Mean numbers of request for extra messages was 2,4 times (range 1–13) during the entire intervention time. The option to get extra mes- sages was reported as a resource, but a technical delay in message delivery was experienced as disappointing. Because it is not possible to brush it aside so to speak, like I have done other times when I tried to quit smoking. (30) The duration of the 12 weeks’ intervention was perceived to be adequate for about half of the partici- pants. Thirty-eight participants (13%) stated that it was too short and 69/289 participants (24%) stated that it was too long. It was nice to be able to decide, to plan what day I would stop so I did not have any important commitment the first days after the stop date. (52) I do not think it was specifically this process of setting a stop date that made it difficult, but rather the fact that you actually decide that after this date, I’ll never smoke again. It’s like breaking up with your best friend on that particular date. (164) I felt a bit frustrated. I wanted to set a stop date the d Wh it? (70) Results Of course, the driving force to quit smoking was mainly the negative consequences of my role as a parent and the health, environment, economy, but it was really thanks to the messages that I The response rate was 35% (n = 289/827). The baseline characteristics of the participants was similar to non- participants concerning sex, age, marital status, duration of smoking in years, proportion using snuff and perceived importance to quit smoking (Table 1). How- ever, the responders smoked significant fewer cigarettes per week (56 cigarettes (IQR 57.8)) compared to non- responders (70 cigarettes (IQR 70.0)). Especially, I have noticed the positive effects of not smoking. On days when I do not smoke everything is really lovely! To participate in the study was an incredible incentive! (84) Müssener et al. BMC Public Health (2016) 16:1179 Page 5 of 8 Page 5 of 8 I had a constant cold which was motivation enough to stop smoking. And the fact that my condition changed for the worse and I got smoker’s cough. (214) I had a constant cold which was motivation enough to stop smoking. And the fact that my condition changed for the worse and I got smoker’s cough. (214) about right. Among those, 20 participants suggested better timing of the messages so they would receive them in relation to meals and social situations when smoking might be most likely. Perception of the experiences of the content of the intervention Concerning the content of the motivational messages, 65% (n = 158/289) of all participants found the content very helpful/helpful. A number of participants empha- sized that the messages increased their motivation and helped them make a decision. Some thought that the messages were helpful initially but then became less I felt a bit frustrated. I wanted to set a stop date the same day. Why wait? (70) Nearly half, 47% (n = 137), thought there were too many messages during the 3 days before the quit date. However, a similar proportion found the number to be Müssener et al. BMC Public Health (2016) 16:1179 Page 6 of 8 If a person is really motivated, yes. I was not as motivated, which is why the messages did not give as good results, as I believe they would have if I were more motivated. (190) Yes! Great programme! Made me quit smoking after 10 years! (51) useful, and suggested reducing the number of messages. In general, short messages were perceived as most help- ful and the longer messages tended to be seen as repeti- tive. Some participants who were still smoking perceived the messages as impersonal and more formulated to frighten people. If a person is really motivated, yes. I was not as motivated, which is why the messages did not give as good results, as I believe they would have if I were more motivated. (190) Yes! Great programme! Made me quit smoking after 10 years! (51) The messages were good with support and advice as well as motivational information such as what happens when one starts and stops smoking. The messages helped me to stand by my decision to quit smoking. (257) Discussion The aim of this study was to explore the users experi- ences of the structure and content of the SMS-based NEXit smoking cessation intervention aimed at young people [4]. The effectiveness of the intervention has been reported previously [5]. The user evaluation is seen as the last step in a formative development process as suggested by Abroms et al. [9] when designing a text message intervention. This evaluation offers additional knowledge about the barriers and facilitating factors with regard to how users perceive and react to the struc- ture and content of the intervention that can serve as a basis for further revisions of the intervention before large-scale implementation. Despite the effectiveness of the programme, some participants were not helped by the intervention, some disengaged from the intervention and others found it counterproductive. Most of the messages were very long, and very numerous also. It got a little too much and too repetitive. (20) I thought the short, more supportive messages were very good. (138) I thought the short, more supportive messages were very good. (138) The majority, 199/289 participants (69%), found the content of the 12 week core programme good or very good. Among those who liked the content, some empha- sized that the messages changed their thinking about smoking and they were reminded about why they wanted to quit smoking. Among those 25% who did not value the content, some were concerned that the mes- sages were not tailored to the individual person and cer- tain messages were too basic and just common sense. Despite the response rate of 35%, participants in this user evaluation study provided valuable information that can be used in further development of the intervention. The proportion who quit smoking in this study and in the main study was similar to most baseline characteris- tics with the only significant difference concerning num- bers of cigarettes smoked per week where participants smoked somewhat less (56 versus 70 cigarettes per week). Still, the participants had smoked for the same number of years (8 years) as non-participants. Thus, the participants in this study were regarded as broadly representative of the participants in the intervention group in the RCT [5]. Good! The messages helped me to remember why am I doing this, and why I feel irritated, and that cravings don’t last forever! (227) Good! The messages helped me to remember why am I doing this, and why I feel irritated, and that cravings don’t last forever! (227) The mech- anisms of the effect of this intervention remain to be iden- tified; is it the actual content of the messages or is it the frequent reminders and reinforcement of having commit- ted oneself to stop smoking, as was pointed out by some participants. This is important to study further. The theoretical perspectives underpinning interven- tions such as this emphasize the need for preparation before quitting [12, 13]. Alternatively, if a person feels ready to stop immediately, then it may be more appro- priate to encourage them to do so, as about half of quit attempts are made in the moment the smoker has decided to quit [14, 15]. If medication or some other kind of cessation support is going to be used, one needs to plan in advance. For smokers who prefer to quit abruptly, rather than planning ahead, smoking cessation interventions may need to place greater emphasis on the dynamic nature of motivation, allowing for an immedi- ate start to a quit attempt rather than postponing it for motivating preparatory messages [15, 16]. We designed the structure of NEXit so that the frequency of messages per day/week decreased over time because previous research has shown this to be more effective than a con- stant number of messages [17]. However, as seen in pre- vious studies, the number of messages at certain periods, especially around the quit date, was perceived by many to be too high and might act as a trigger to keep on smoking [18]. On the other hand, other studies show the opposite; that few participants perceived the number of messages to be too few [11]. Allowing users to set their own preferences for message frequency and duration may be worth exploring. As seen in previous studies, a majority of the partici- pants read all/nearly all messages [9, 18] emphasizing the advantage of automatic delivery of a message-based intervention that does not require the user to engage in the intervention by opening an app or logging on to a web site. The study is not without limitations due to the low response rate and relative short questionnaire used to explore the views of the participants. The strength is that the participants were recruited from all colleges and universities in Sweden and also the amount of free-text comments to all questions. However, the optimal duration of the intervention has still to be established. Good! The messages helped me to remember why am I doing this, and why I feel irritated, and that cravings don’t last forever! (227) The messages did not seem appealing to me. I would prefer messages that were more suited to me and my habits, personally. (88) However, not all messages were perceived as helpful. Only 97/289 participants (34%) thought that all/nearly all messages were valuable. Ten participants suggested that it was not really the content that mattered but the fact that the messages served as a reminder of their deci- sion to quit smoking. The content of the intervention was based on existing evidence-based face-to-face practices including compo- nents derived from expert guidance and official smoking cessation manuals recommended in Sweden [4]. Thus, the overall structure and content of the message-based intervention was well received by most of the partici- pants, regardless of whether they quit or not. Unsurpris- ingly, participants who were still smoking were less satisfied with the intervention. One possible conclusion also noted in previous research may be that text-based messaging smoking cessation interventions are more suitable for smokers who are motivated to use these types of programmes, and those who are not fully moti- vated or not determined to stop smoking may find other types of support more suitable [11]. In particular, partici- pants who were still smoking disliked the motivational phase, experienced by many as unnecessary and Perhaps 70% of the ones I read were useful; but for me it was not the content itself, but the fact that the messages arrived and reminded me that “yeah, I’m not a smoker”. (134) The majority of participants (n = 196/289, 68%) stated that they had read all/nearly all messages. Only 20/289 participants (7%) said that they only read a few of the messages. Some saved the messages and read them the next day. Most of the participants (n = 244/289, 84%) indicated that they would recommend the intervention to a friend and 12% (n = 34/289) would not. Müssener et al. BMC Public Health (2016) 16:1179 Page 7 of 8 Page 7 of 8 unhelpful. Greater frequency of messages immediately before the quit date was perceived as problematic because many wanted to quit immediately after having decided to stop. These observations reflect the limita- tions imposed by untailored interventions. whereas others show benefits of tailoring [22]. Conclusions h The NEXit smoking cessation intervention seems to have supported an encouragingly high proportion of smokers to quit smoking but might have missed the opportunity to help others. On the basis of this study, we are inclined to reduce the duration of the motiv- ational phase and offer fewer messages on the days immediately before the quit date. In general, shorter messages were more acceptable and longer messages will be avoided. Most participants found the intervention satisfactory and would recommend it to a friend who is ready to set a quit date. The intervention was fully automated and did not require the user to provide any information. The variation in content between facts and motivational and practical advice was appreciated by most participants, and the tim- ing of specific messages was perceived as satisfactory. However, the content was not tailored to the individuals’ perceived need for support other than participants being able to ask for additional supportive messages when experiencing cravings, fear of weight gain and after a relapse. As seen in other studies, this function with extra messages was used by a minority [11, 19, 20] but this pos- sibility may still be perceived as important, perhaps creat- ing some sense of tailoring and personal control of engagement. Other participants pointed out the lack of tailoring of the messages. Tailoring messages would increase the technical complexity of implementing the intervention and it remains to be seen if this would increase the effectiveness and cost effectiveness of a smok- ing cessation intervention. The evidence on whether tailoring messages is useful is complex. In some studies, tailoring internet-based interventions for smoking cessa- tion does not increase quit rates in the long term, and seems not to mediate the effect of the intervention [21] Acknowledgements Availability of data and materials: data can be received by contacting the main author Ulrika Müssener email: ulrika.mussener@liu.se Abbreviations BCT: Behaviour Change Technique; RCT: Randomized Clinical Trial; SMS: Short Message Service Good! The messages helped me to remember why am I doing this, and why I feel irritated, and that cravings don’t last forever! (227) In our study, the duration of the intervention was experi- enced as adequate for only about half of the participants. Most previous message-based quit smoking interven- tions have been between 8 and 12 weeks long although some have been somewhat shorter. Additional file 1: NEXit evaluation questionaire. (DOCX 20 kb) Additional file 1: NEXit evaluation questionaire. (DOCX 20 kb) Received: 4 June 2016 Accepted: 15 November 2016 Received: 4 June 2016 Accepted: 15 November 2016 Funding The study was funded by the Swedish Research Council, grant number 521–2012–2865. The funding source had no role in the study design, data collection, analysis, interpretation of data, writing the report, or the decision to submit the paper for publication. UM had full access to all data in the study and had final responsibility for the decision to submit for publication. Page 8 of 8 Page 8 of 8 Page 8 of 8 Müssener et al. BMC Public Health (2016) 16:1179 14. West R. The smokefree formula. Orion House: A revolutionary way to stop smoking now; 2013. 15. Larabie LC. To what extent do smokers plan quit attempts? Tob Control. 2005;14(6):425–8. 16. Free C, Knight R, Robertson S, Whittaker R, Edwards P, Zhou W, Rodgers A, Cairns J, Kenward MG, Roberts I. Smoking cessation support delivered via mobile phone text messaging (txt2stop): a single-blind, randomised trial. Lancet. 2011;2:49–55. 17. Head KJ, Noar SM, Iannarino NT, Grant HN. Efficacy of text messaging-based interventions for health promotion: a meta-analysis. Soc Sci Med. 2013;97:41–8. 18. Abroms LC, Ahuja M, Kodl Y, Thaweethai L, Sims J, Winickoff JP. Windsor RA Text2Quit: results from a pilot test of a personalized, interactive mobile health smoking cessation program. J Health Commun. 2012;17 Suppl 1:44–53. 19. Whittaker R, Maddison R, McRobbie H, Bullen C, Denny S, Dorey E, Ellis- Pegler M, van Rooyen J, Rodgers A. A multimedia mobile phone-based youth smoking cessation intervention: findings from content development and piloting studies. J Med Internet Res. 2008;10(5):e49. 20. Bock B, Heron K, Jennings E, Morrow K, Cobb V, Magee J, Fava J, Deutsch C, Foster R. A text message delivered smoking cessation intervention: the initial trial of TXT-2-Quit: randomized controlled trial. JMIR Mhealth Uhealth. 2013;1:e17. 21. Wangberg S, Nilsen O, Antypas K, Gram I. Effect of tailoring in an internet- based intervention for smoking cessation: randomized controlled trial. J Med Internet Res. 2011;13(4):e121. 22. Skov-Ettrup LS, Ringgaard LW, Dalum P, Flensborg-Madsen T, Thygesen LC, Tolstrup JS. Comparing tailored and untailored text messages for smoking cessation: a randomized controlled trial among adolescent and young adult smokers. Health Educ Res. 2014;29(2):195–205. Author details 1 f 1Department of Medical and Health Sciences, Linköping University, Linköping 581 83, Sweden. 2Department of Computer and Information Science, Linköping University, Linköping 581 83, Sweden. 3Department of Health Sciences, University of York, Heslington, York YO10 5DD, UK. 4Department of Medical Specialist and Department of Medical and Health Sciences, Motala, Linköping University, Linköping 581 83, Sweden. 22. Skov-Ettrup LS, Ringgaard LW, Dalum P, Flensborg-Madsen T, Thygesen LC, Tolstrup JS. Comparing tailored and untailored text messages for smoking cessation: a randomized controlled trial among adolescent and young adult smokers. Health Educ Res. 2014;29(2):195–205. 22. Skov-Ettrup LS, Ringgaard LW, Dalum P, Flensborg-Madsen T, Thygesen LC, Tolstrup JS. Comparing tailored and untailored text messages for smoking cessation: a randomized controlled trial among adolescent and young adult smokers. Health Educ Res. 2014;29(2):195–205. Authors’ contributions 14. West R. The smokefree formula. Orion House: A revolutionary way to stop smoking now; 2013. PB, JMc and MB designed the study. UM took a leading role in developing the intervention and recruiting the participants. MB did all the programming and supervised the data collection and all technical aspects of the delivery of the intervention. UM and PB wrote the first draft of the manuscript and revised it with input from all authors. All authors read and approved the final manuscript. 15. Larabie LC. To what extent do smokers plan quit attempts? Tob Control. 2005;14(6):425–8. 16. Free C, Knight R, Robertson S, Whittaker R, Edwards P, Zhou W, Rodgers A, Cairns J, Kenward MG, Roberts I. Smoking cessation support delivered via mobile phone text messaging (txt2stop): a single-blind, randomised trial. Lancet. 2011;2:49–55. 17. Head KJ, Noar SM, Iannarino NT, Grant HN. Efficacy of text messaging-based interventions for health promotion: a meta-analysis. Soc Sci Med. 2013;97:41–8. Ethics approval and consent to participate The study was approved by the Regional Ethical Committee in Linköping, Sweden (Dnr 2014/217–31). The study was approved by the Regional Ethical Committee in Linköping, Sweden (Dnr 2014/217–31). 21. Wangberg S, Nilsen O, Antypas K, Gram I. Effect of tailoring in an internet- based intervention for smoking cessation: randomized controlled trial. J Med Internet Res. 2011;13(4):e121. Consent to publish Not applicable. 20. Bock B, Heron K, Jennings E, Morrow K, Cobb V, Magee J, Fava J, Deutsch C, Foster R. A text message delivered smoking cessation intervention: the initial trial of TXT-2-Quit: randomized controlled trial. JMIR Mhealth Uhealth. 2013;1:e17. Competing interests b d d Preben Bendtsen and Marcus Bendtsen own a private company that develops and distributes evidence-based life style interventions to be used in health care settings. All other authors do not declare any competing interest. 18. Abroms LC, Ahuja M, Kodl Y, Thaweethai L, Sims J, Winickoff JP. Windsor RA Text2Quit: results from a pilot test of a personalized, interactive mobile health smoking cessation program. J Health Commun. 2012;17 Suppl 1:44–53. 19. Whittaker R, Maddison R, McRobbie H, Bullen C, Denny S, Dorey E, Ellis- Pegler M, van Rooyen J, Rodgers A. A multimedia mobile phone-based youth smoking cessation intervention: findings from content development and piloting studies. J Med Internet Res. 2008;10(5):e49. References 1. Gilliam H. Quitting smoking brings quick health benefits. Lakartidningen. 2012;109:554–7 [in Swedish]. 1. Gilliam H. Quitting smoking brings quick health benefits. Lakartidningen. 2012;109:554–7 [in Swedish]. 2. The Public Health Agency of Sweden. Tobaksvanor. [Tobacco habits. The proportion of the population using some form of tobacco. https://www. folkhalsomyndigheten.se/folkhalsorapportering-statistik/statistikdatabaser- och-visualisering/nationella-folkhalsoenkaten/levnadsvanor/tobaksvanor/. Accessed 17 Nov 2016. 3. Agardh E, Moradi T, Allebeck P. The contribution of risk factors to the burden of disease in Sweden. A comparison between Swedish and WHO data. Lakartidningen. 2008;105:816–21 [in Swedish]. 4. Müssener U, Bendtsen M, Karlsson N, White IR, McCambridge J, Bendtsen P. SMS-based smoking cessation intervention among university students: study protocol for randomised controlled trial (NEXit trial). Trials. 2015;16:140. 5. Müssener U, Bendtsen M, Karlsson N, White IR, McCambridge J, Bendtsen P. Effectiveness of short message service text-based smoking cessation intervention among university student. JAMA Intern Med. 2016;176(3):321–8. 6. Free C, Phillips G, Watson L, Galli L, Felix L, Edwards P, Patel V, Haines A. The effectiveness of mobile-health technologies to improve health care services processes: a systematic review and meta-analysis. PLoS Med. 2013;10:e1001363. Fj ld B M h ll A Mill Y B h i h i i d li d b 7. Fjeldsoe B, Marshall A, Miller Y. Behaviour change interventions delivered by moblie telehone short-message service. Am J Prev. 2009;36:165–73. 7. Fjeldsoe B, Marshall A, Miller Y. Behaviour change interventions delivered by moblie telehone short-message service. Am J Prev. 2009;36:165–73. 8. Cole-Lewis H, Kershaw T. Text messaging as a tool for behavior change in disease prevention and management. Epidemiol Rev. 2010;32(1):56–69. 8. Cole-Lewis H, Kershaw T. Text messaging as a tool for behavior change in disease prevention and management. Epidemiol Rev. 2010;32(1):56–69. 9. Abroms LC, Whittaker R, Free C, Mendel Van Alstyne J, Schindler-Ruwisch JM. Developing and pretesting a text messaging program for health behavior change: recommended steps. JMIR Mhealth Uhealth. 2015;3(4):e107. 9. Abroms LC, Whittaker R, Free C, Mendel Van Alstyne J, Schindler-Ruwisch JM. Developing and pretesting a text messaging program for health behavior change: recommended steps. JMIR Mhealth Uhealth. 2015;3(4):e107. 22. Skov-Ettrup LS, Ringgaard LW, Dalum P, Flensborg-Madsen T, Thygesen LC, Tolstrup JS. Comparing tailored and untailored text messages for smoking cessation: a randomized controlled trial among adolescent and young adult smokers. Health Educ Res. 2014;29(2):195–205. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 10. Michie S, Hyder N, Walia A, West R. Development of a taxonomy of behaviour change techniques used in individual behavioural support for smoking cessation. Addict Behav. 2011;36:315–19. 11. Ybarra ML, Holtrop JS, Prescott TL, Rahbar MH, Strong D. Pilot RCT results of stop my smoking USA: a text messaging-based smoking cessation program for young adults. Nicotine Tob Res. 2013;15:1388–99. 13. Holm-Ivarsson B, Hjalmarson A, Pantzar M. Stödja patienter att sluta röka och snusa: rådgivning om tobak och avvänjning [Support patients to stop smoking and snuffing: counseling on tobacco cessation]. 2012. Studentlitteratur, [in Swedish].
https://openalex.org/W2593983370	https://europepmc.org/articles/pmc5344962?pdf=render	English	null	An eigenvalue localization set for tensors and its applications	Journal of inequalities and applications	2,017	cc-by	3,602	R ES EA RCH Open Access Abstract A new eigenvalue localization set for tensors is given and proved to be tighter than those presented by Li et al. (Linear Algebra Appl. 481:36-53, 2015) and Huang et al. (J. Inequal. Appl. 2016:254, 2016). As an application of this set, new bounds for the minimum eigenvalue of M-tensors are established and proved to be sharper than some known results. Compared with the results obtained by Huang et al., the advantage of our results is that, without considering the selection of nonempty proper subsets S of N = {1,2,...,n}, we can obtain a tighter eigenvalue localization set for tensors and sharper bounds for the minimum eigenvalue of M-tensors. Finally, numerical examples are given to verify the theoretical results. MSC: 15A18; 15A69; 65F10; 65F15 Keywords: M-tensors; nonnegative tensors; minimum eigenvalue; localization set Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 DOI 10.1186/s13660-017-1331-1 An eigenvalue localization set for tensors and its applications Jianxing Zhao* and Caili Sang Correspondence: zjx810204@163.com College of Data Science and Information Engineering, Guizhou Minzu University, Guiyang, Guizhou 550025, P.R. China Correspondence: zjx810204@163.com College of Data Science and Information Engineering, Guizhou Minzu University, Guiyang, Guizhou 550025, P.R. China 1 Introduction For a positive integer n, n ≥, N denotes the set {,,...,n}. C (respectively, R) denotes the set of all complex (respectively, real) numbers. We call A = (ai···im) a complex (real) tensor of order m dimension n, denoted by C[m,n](R[m,n]), if ai···im ∈C(R), where ij ∈N for j = ,,...,m. A is called reducible if there exists a nonempty proper index subset J ⊂N such that aii···im = , ∀i∈J,∀i,...,im /∈J. If A is not reducible, then we call A irreducible []. A is not reducible, then we call A irreducible []. Given a tensor A = (ai···im) ∈C[m,n], if there are λ ∈C and x = (x,x,...,xn)T ∈C\{} such that Axm–= λx[m–], © The Author(s) 2017. 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. Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 2 of 9 Page 2 of 9 then λ is called an eigenvalue of A and x an eigenvector of A associated with λ, where Axm–is an n dimension vector whose ith component is Axm– i = i,...,im∈N aii···imxi···xim and x[m–] = xm–  ,xm–  ,...,xm– n T. x[m–] = xm–  ,xm–  ,...,xm– n T. If λ and x are all real, then λ is called an H-eigenvalue of A and x an H-eigenvector of A associated with λ; see [, ]. Moreover, the spectral radius ρ(A) of A is deﬁned as If λ and x are all real, then λ is called an H-eigenvalue of A and x an H-eigenvector of A associated with λ; see [, ]. Moreover, the spectral radius ρ(A) of A is deﬁned as ρ(A) = max \|λ\| : λ ∈σ(A) , where σ(A) is the spectrum of A, that is, σ(A) = {λ : λ is an eigenvalue of A}; see [, ]. where σ(A) is the spectrum of A, that is, σ(A) = {λ : λ is an eigenvalue of A}; see [, ]. A real tensor A is called an M-tensor if there exist a nonnegative tensor B and a positive number α > ρ(B) such that A = αI – B, where I is called the unit tensor with its entries δi···im = ⎧ ⎨ ⎩  if i= ··· = im,  otherwise. δi···im = ⎨ ⎩ otherwise. Denote by τ(A) the minimal value of the real part of all eigenvalues of an M-tensor A. Then τ(A) > is an eigenvalue of A with a nonnegative eigenvector. If A is irreducible, then τ(A) is the unique eigenvalue with a positive eigenvector [–]. Denote by τ(A) the minimal value of the real part of all eigenvalues of an M-tensor A. Then τ(A) > is an eigenvalue of A with a nonnegative eigenvector. If A is irreducible, then τ(A) is the unique eigenvalue with a positive eigenvector [–]. Recently, many people have focused on locating eigenvalues of tensors and using ob- tained eigenvalue inclusion theorems to determine the positive deﬁniteness of an even- order real symmetric tensor or to give the lower and upper bounds for the spectral radius of nonnegative tensors and the minimum eigenvalue of M-tensors. For details, see [, , –]. In , Li et al. [] proposed the following Brauer-type eigenvalue localization set for tensors. Theorem ([], Theorem ) Let A = (ai···im) ∈C[m,n]. Then Theorem ([], Theorem ) Let A = (ai···im) ∈C[m,n]. Then σ(A) ⊆(A) = i,j∈N,j̸=i j i(A), where j i(A) = z ∈C : (z – ai···i)(z – aj···j) – aij···jaji···i ≤\|z – aj···j\|rj i(A) + \|aij···j\|ri j(A) , ri(A) = δii···im= \|aii···im\|, rj i(A) = δii···im=, δji···im= \|aii···im\| = ri(A) – \|aij···j\|. To reduce computations, Huang et al. [] presented an S-type eigenvalue localization set by breaking N into disjoint subsets S and ¯S, where ¯S is the complement of S in N. Page 3 of 9 Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 3 of 9 Theorem ([], Theorem .) Let A = (ai···im) ∈C[m,n], S be a nonempty proper subset of N, ¯S be the complement of S in N. Then σ(A) ⊆S(A) = i∈S,j∈¯S j i(A) ∪ i∈¯S,j∈S j i(A) . Based on Theorem , Huang et al. [] obtained the following lower and upper bounds for the minimum eigenvalue of M-tensors. Theorem ([], Theorem .) Let A = (ai···im) ∈R[m,n] be an M-tensor, S be a nonempty proper subset of N, ¯S be the complement of S in N. Then Theorem ([], Theorem .) Let A = (ai···im) ∈R[m,n] be an M-tensor, S be a nonempty proper subset of N, ¯S be the complement of S in N. Then min min i∈S max j∈¯S Lij(A),min i∈¯S max j∈S Lij(A) ≤τ(A) ≤max max i∈S min j∈¯S Lij(A),max i∈¯S min j∈S Lij(A) , where Lij(A) =   ai···i + aj···j – rj i(A) – ai···i – aj···j – rj i(A) – aij···jrj(A)   . The main aim of this paper is to give a new eigenvalue inclusion set for tensors and prove that this set is tighter than those in Theorems and without considering the selection of S. And then we use this set to obtain new lower and upper bounds for the minimum eigenvalue of M-tensors and prove that new bounds are sharper than those in Theorem . 2 Main results Now, we give a new eigenvalue inclusion set for tensors and establish the comparison between this set with those in Theorems and . Theorem Let A = (ai···im) ∈C[m,n]. Then Theorem Let A = (ai···im) ∈C[m,n]. Then Theorem Let A = (ai···im) ∈C[m,n]. Then σ(A) ⊆∩(A) = i∈N j∈N,j̸=i j i(A). Proof For any λ ∈σ(A), let x = (x,...,xn)T ∈Cn\{} be an associated eigenvector, i.e., Proof For any λ ∈σ(A), let x = (x,...,xn)T ∈Cn\{} be an associated eigenvector, i.e., Axm–= λx[m–]. () Axm–= λx[m–]. () \|xp\| = max{\|xi\| : i ∈N}. Then \|xp\| > . For any j ∈N,j ̸= p, then from () we have Let \|xp\| = max{\|xi\| : i ∈N}. Then \|xp\| > . For any j ∈N,j ̸= p, then from () we have λxm– p = δpi···im=, δji···im= api···imxi···xim + ap···pxm– p + apj···jxm– j and λxm– j = δji···im=, δpi···im= aji···imxi···xim + aj···jxm– j + ajp···pxm– p , λxm– p = δpi···im=, δji···im= api···imxi···xim + ap···pxm– p + apj···jxm– j λxm– p = δpi···im=, δji···im= api···imxi···xim + ap···pxm– p + apj···jxm– j and λxm– j = δji···im=, δpi···im= aji···imxi···xim + aj···jxm– j + ajp···pxm– p , and Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 4 of 9 equivalently, equivalently, (λ – ap···p)xm– p – apj···jxm– j = δpi···im=, δji···im= api···imxi···xim () and (λ – aj···j)xm– j – ajp···pxm– p = δji···im=, δpi···im= aji···imxi···xim. () (λ – ap···p)xm– p – apj···jxm– j = δpi···im=, δji···im= api···imxi···xim () and and (λ – aj···j)xm– j – ajp···pxm– p = δji···im=, δpi···im= aji···imxi···xim. () (λ – aj···j)xm– j – ajp···pxm– p = δji···im=, δpi···im= aji···imxi···xim. () (λ – aj···j)xm– j – ajp···pxm– p = δji···im=, δpi···im= aji···imxi···xim. () Solving xm– p from () and (), we get Solving xm– p from () and (), we get (λ – ap···p)(λ – aj···j) – apj···jajp···p xm– p = (λ – aj···j) δpi···im=, δji···im= api···imxi···xim + apj···j δji···im=, δpi···im= aji···imxi···xim. Taking absolute values and using the triangle inequality yields Taking absolute values and using the triangle inequality yields (λ – ap···p)(λ – aj···j) – apj···jajp···p \|xp\|m– ≤\|λ – aj···j\|rj p(A)\|xp\|m–+ \|apj···j\|rp j (A)\|xp\|m–. 2 Main results Furthermore, by \|xp\| > , we have (λ – ap···p)(λ – aj···j) – apj···jajp···p ≤\|λ – aj···j\|rj p(A) + \|apj···j\|rp j (A), which implies that λ ∈j p(A). From the arbitrariness of j, we have λ ∈ j∈N,j̸=p j p(A). Furthermore, we have λ ∈ i∈N j∈N,j̸=i j i(A). The conclusion follows. □ Next, a comparison theorem is given for Theorems , and . Theorem Let A = (ai···im) ∈C[m,n], S be a nonempty proper subset of N. Then Theorem Let A = (ai···im) ∈C[m,n], S be a nonempty proper subset of N. Then Theorem Let A = (ai···im) ∈C[m,n], S be a nonempty proper subset of N. Then ∩(A) ⊆S(A) ⊆(A). Proof By Theorem .in [], S(A) ⊆(A). Here, only ∩(A) ⊆S(A) is proved. Let z ∈∩(A), then there exists some i∈N such that z ∈j i(A),∀j ∈N,j ̸= i. Let ¯S be the complement of S in N. If i∈S, then taking j ∈¯S, obviously, z ∈ i∈S,j∈¯S j i(A) ⊆ S(A). If i∈¯S, then taking j ∈S, obviously, z ∈ i∈¯S,j∈S j i(A) ⊆S(A). The conclusion follows. □ Remark Theorem shows that the set ∩(A) in Theorem is tighter than those in Theorems and , that is, ∩(A) can capture all eigenvalues of A more precisely than (A) and S(A). Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 5 of 9 Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 In the following, we give new lower and upper bounds for the minimum eigenvalue of M-tensors. Theorem Let A = (ai···im) ∈R[m,n] be an irreducible M-tensor. Then eorem Let A = (ai···im) ∈R[m,n] be an irreducible M-tensor. Then min i∈N max j̸=i Lij(A) ≤τ(A) ≤max i∈N min j̸=i Lij(A). min i∈N max j̸=i Lij(A) ≤τ(A) ≤max i∈N min j̸=i Lij(A). Proof Let x = (x,x,...,xn)T be an associated positive eigenvector of A corresponding to τ(A), i.e., Proof Let x = (x,x,...,xn)T be an associated positive eigenvector of A corresponding to τ(A), i.e., Axm–= τ(A)x[m–]. () (I) Let xq = min{xi : i ∈N}. For any j ∈N,j ̸= q, we have by () that (I) Let xq = min{xi : i ∈N}. 2 Main results For any j ∈N,j ̸= q, we have by () that τ(A)xm– q = δqi···im=, δji···im= aqi···imxi···xim + aq···qxm– q + aqj···jxm– j and τ(A)xm– j = δji···im=, δqi···im= aji···imxi···xim + aj···jxm– j + ajq···qxm– q , equivalently, equivalently, () and τ(A) – aj···j xm– j – ajq···qxm– q = δji···im=, δqi···im= aji···imxi···xim. () () Solving xm– q by () and (), we get Solving xm– q by () and (), we get τ(A) – aq···q τ(A) – aj···j – aqj···jajq···q xm– q = τ(A) – aj···j δqi···im=, δji···im= aqi···imxi···xim + aqj···j δji···im=, δqi···im= aji···imxi···xim. From Theorem .in [], we have τ(A) ≤mini∈N ai···i and aq···q – τ(A) aj···j – τ(A) – aqj···jajq···q xm– q = aj···j – τ(A) δqi···im=, δji···im= \|aqi···im\|xi···xim + \|aqj···j\| δji···im=, δqi···im= \|aji···im\|xi···xim. Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 6 of 9 Page 6 of 9 Hence, aq···q – τ(A) aj···j – τ(A) – \|aqj···j\|\|ajq···q\| xm– q ≥ aj···j – τ(A) δqi···im=, δji···im= \|aqi···im\|xm– q + \|aqj···j\| δji···im=, δqi···im= \|aji···im\|xm– q . From xq > , we have From xq > , we have aq···q – τ(A) aj···j – τ(A) – \|aqj···j\|\|ajq···q\| ≥ aj···j – τ(A) δqi···im=, δji···im= \|aqi···im\| + \|aqj···j\| δji···im=, δqi···im= \|aji···im\| = aj···j – τ(A) rj q(A) + \|aqj···j\|rq j (A), equivalently, equivalently, aq···q – τ(A) aj···j – τ(A) – aj···j – τ(A) rj q(A) – \|aqj···j\|rj(A) ≥, that is, that is, τ(A)– aq···q + aj···j – rj q(A) τ(A) + aq···qaj···j – aj···jrj q(A) + aqj···jrj(A) ≥. Solving for τ(A) gives Solving for τ(A) gives τ(A) ≤  aq···q + aj···j – rj q(A) – aq···q – aj···j – rj q(A) – aqj···jrj(A)   = Lqj(A). For the arbitrariness of j, we have τ(A) ≤minj̸=q Lqj(A). Furthermore, we have For the arbitrariness of j, we have τ(A) ≤minj̸=q Lqj(A). Furthermore, we have τ(A) ≤max i∈N min j̸=i Lij(A). τ(A) ≤max i∈N min j̸=i Lij(A). (II) Let xp = max{xi : i ∈N}. Similar to (I), we have (II) Let xp = max{xi : i ∈N}. Similar to (I), we have τ(A) ≥min i∈N max j̸=i Lij(A). 2 Main results (II) Let xp = max{xi : i ∈N}. Similar to (I), we have τ(A) ≥min i∈N max j̸=i Lij(A). τ(A) ≥min i∈N max j̸=i Lij(A). The conclusion follows from (I) and (II). □ The conclusion follows from (I) and (II). □ □ The conclusion follows from (I) and (II). Similar to the proof of Theorem .in [], we can extend the results of Theorem to a more general case. Theorem Let A = (ai···im) ∈R[m,n] be an M-tensor. Then Theorem Let A = (ai···im) ∈R[m,n] be an M-tensor. Then min i∈N max j̸=i Lij(A) ≤τ(A) ≤max i∈N min j̸=i Lij(A). min i∈N max j̸=i Lij(A) ≤τ(A) ≤max i∈N min j̸=i Lij(A). min i∈N max j̸=i Lij(A) ≤τ(A) ≤max i∈N min j̸=i Lij(A). By Theorems , and in [], the following comparison theorem is obtained easily. By Theorems , and in [], the following comparison theorem is obtained easily. Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 Page 7 of 9 Theorem Let A = (ai···im) ∈R[m,n] be an M-tensor, S be a nonempty proper subset of N, ¯S be the complement of S in N. Then min i∈N Ri(A) ≤min j̸=i Lij(A) ≤min min i∈S max j∈¯S Lij(A),min i∈¯S max j∈S Lij(A) ≤min i∈N max j̸=i Lij(A) ≤max i∈N min j̸=i Lij(A) ≤max max i∈S min j∈¯S Lij(A),max i∈¯S min j∈S Lij(A) , min i∈N Ri(A) ≤min j̸=i Lij(A) ≤min min i∈S max j∈¯S Lij(A),min i∈¯S max j∈S Lij(A) ≤min i∈N max j̸=i Lij(A) A) ≤min j̸=i Lij(A) ≤min min i∈S max j∈¯S Lij(A),min i∈¯S max j∈S Lij(A) ≤min i∈N max j̸=i Lij(A) ≤max i∈N min j̸=i Lij(A) ≤max max i∈S min j∈¯S Lij(A),max i∈¯S min j∈S Lij(A) , where Ri(A) = i,...,im∈N aii···im. Remark Theorem shows that the bounds in Theorem are shaper than those in The- orem , Theorem .of [] and Theorem of [] without considering the selection of S, which is also the advantage of our results. 3 Numerical examples In this section, two numerical examples are given to verify the theoretical results. Example Let A = (aijk) ∈R[,] be an irreducible M-tensor with elements deﬁned as follows: A(:,:,) = ⎛ ⎜⎜⎜⎝  – – – – – – – – – – – – – – – ⎞ ⎟⎟⎟⎠, A(:,:,) = ⎛ ⎜⎜⎜⎝  – – – –  – – – – – – – – – – ⎞ ⎟⎟⎟⎠, A(:,:,) = ⎛ ⎜⎜⎜⎝ – – – – – – – – – –  – – – – – ⎞ ⎟⎟⎟⎠, A(:,:,) = ⎛ ⎜⎜⎜⎝ – – – – – – – – – – – – – – –  ⎞ ⎟⎟⎟⎠. By Theorem .in [], we have By Theorem .in [], we have By Theorem .in [], we have = min i∈N Ri(A) ≤τ(A) ≤min max i∈N Ri(A),min i∈N ai···i = . = min i∈N Ri(A) ≤τ(A) ≤min max i∈N Ri(A),min i∈N ai···i = . By Theorem in [], we have τ(A) ≥min j̸=i Lij(A) = .. By Theorem , we have By Theorem , we have if S = {}, ¯S = {,,}, .≤τ(A) ≤.; if S = {}, ¯S = {,,}, .≤τ(A) ≤.; if S = {}, ¯S = {,,}, .≤τ(A) ≤.; if S = {}, ¯S = {,,}, .≤τ(A) ≤.; if S = {,}, ¯S = {,}, .≤τ(A) ≤.; if S = {,}, ¯S = {,}, .≤τ(A) ≤.; Page 8 of 9 Page 8 of 9 Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 if S = {,}, ¯S = {,}, .≤τ(A) ≤.. By Theorem , we have .≤τ(A) ≤.. In fact, τ(A) = .. Hence, this example veriﬁes Theorem and Remark , that is, the bounds in Theorem are sharper than those in Theorem , Theorem .of [] and Theorem of [] without considering the selection of S. Example Let A = (aijkl) ∈R[,] be an M-tensor with elements deﬁned as follows: a= , a= –, a= –, a= , other aijkl = . By Theorem , we have ≤τ(A) ≤. In fact, τ(A) = . In fact, τ(A) = . Authors’ contributions ll h b d Authors’ contributions All authors contributed equally to this work. All authors read and approved the ﬁnal manuscript. Competing interests The authors declare that they have no competing interests. Authors’ contributions All authors contributed equally to this work. All authors read and approved the ﬁnal manuscript. 4 Conclusions In this paper, we give a new eigenvalue inclusion set for tensors and prove that this set is tighter than those in [, ]. As an application, we obtain new lower and upper bounds for the minimum eigenvalue of M-tensors and prove that the new bounds are sharper than those in [, , ]. Compared with the results in [], the advantage of our results is that, without considering the selection of S, we can obtain a tighter eigenvalue localization set for tensors and sharper bounds for the minimum eigenvalue of M-tensors. Competing interests Competing interests The authors declare that they have no competing interests. Acknowledgements Acknowledgements This work is supported by the National Natural Science Foundation of China (Nos. 11361074, 11501141), the Foundation of Guizhou Science and Technology Department (Grant No. [2015]2073) and the Natural Science Programs of Education Department of Guizhou Province (Grant No. [2016]066). Received: 15 January 2017 Accepted: 27 February 2017 Received: 15 January 2017 Accepted: 27 February 2017 Zhao and Sang Journal of Inequalities and Applications ( 2017) 2017:59 10. Li, CQ, Li, YT, Kong, X: New eigenvalue inclusion sets for tensors. Numer. Linear Algebra Appl. 21, 39-50 (2014) 11. Li, CQ, Li, YT: An eigenvalue localization set for tensor with applications to determine the positive (semi-)deﬁniteness of tensors. Linear Multilinear Algebra 64(4), 587-601 (2016) 12. Li, CQ, Jiao, AQ, Li, YT: An S-type eigenvalue location set for tensors. Linear Algebra Appl. 493, 469-483 (2016) 13. Zhao, JX, Sang, CL: Two new lower bounds for the minimum eigenvalue of M-tensors. J. Inequal. Appl. 2016, 268 (2016) 14. He, J: Bounds for the largest eigenvalue of nonnegative tensors. J. Comput. Anal. Appl. 20(7), 1290-1301 (2016) ( ) 14. He, J: Bounds for the largest eigenvalue of nonnegative tensors. J. Comput. Anal. Appl. 20(7), 1290-1301 (2016) References References 1. Li, CQ, Chen, Z, Li, YT: A new eigenvalue inclusion set for tensors and its applications. Linear Algebra Appl. 481, 36-53 (2015) e e e ces 1. Li, CQ, Chen, Z, Li, YT: A new eigenvalue inclusion set for tensors and its applications. Linear Algebra Appl. 481, 36-53 (2015) (2015) 2. Huang, ZG, Wang, LG, Xu, Z, Cui, JJ: A new S-type eigenvalue inclusion set for tensors and its applications. J. Inequal. Appl. 2016, 254 (2016) ( ) 2. Huang, ZG, Wang, LG, Xu, Z, Cui, JJ: A new S-type eigenvalue inclusion set for tensors and its applications. J. Inequal. Appl. 2016, 254 (2016) 3. Chang, KQ, Zhang, T, Pearson, K: Perron-Frobenius theorem for nonnegative tensors. Commun. Math. Sci. 6, 507-520 (2008) 4. Qi, LQ: Eigenvalues of a real supersymmetric tensor. J. Symb. Comput. 40, 1302-1324 (2005) 5. Lim, LH: Singular values and eigenvalues of tensors: a variational approach. In: Proceedings of the IEEE Internationa Workshop on Computational Advances in Multi-Sensor Adaptive Processing. CAMSAP, vol. 05, pp. 129-132 (2005) 5. Lim, LH: Singular values and eigenvalues of tensors: a variational approach. In: Proceedings of the IEEE International Workshop on Computational Advances in Multi-Sensor Adaptive Processing. CAMSAP, vol. 05, pp. 129-132 (2005) 6. Yang, YN, Yang, QZ: Further results for Perron-Frobenius theorem for nonnegative tensors. SIAM J. Matrix Anal. Appl. 31, 2517-2530 (2010) 7. Ding, WY, Qi, LQ, Wei, YM: M-tensors and nonsingular M-tensors. Linear Algebra Appl. 439, 3264-3278 (2013) 8. Zhang, LP, Qi, LQ, Zhou, GL: M-tensors and some applications. SIAM J. 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https://openalex.org/W2157391236	https://europepmc.org/articles/pmc4125195?pdf=render	English	null	Association of UGT1A1 Variants and Hyperbilirubinemia in Breast-Fed Full-Term Chinese Infants	PloS one	2,014	cc-by	6,742	Abstract A retrospective case control study of breast-fed full-term infants was carried out to determine whether variants in Uridine Diphosphate Glucuronosyl Transferase 1A1 (UGT1A1) and Heme Oxygenase-1 (HMOX1) were associated with neonatal hyperbilirubinemia. Eight genetic variants of UGT1A1 and 3 genetic variants of HMOX1 were genotyped in 170 hyperbilirubinemic newborns and 779 controls. Five significant associations with breast-fed hyperbilirubinemia were detected after adjusting for gender, birth season, birth weight, delivery mode, gestational age and False Discovery Rate (FDR) correction: the dominant effect of rs887829 (c-364t) (Odds Ratio (OR): 0.55; 95% Confidence Interval (CI): 0.34–0.89; p = 0.014), the additive effect of (TA)n repeat (OR: 0.59; 95%CI: 0.38–0.91; p = 0.017), the dominant effect of rs4148323 (Gly71Arg, G211A) (OR: 2.02; 95%CI: 1.44–2.85; p = 5.061025), the recessive effect of rs6717546 (g+914a) (OR: 0.30; 95%CI: 0.11–0.83; p = 0.021) and rs6719561 (t+2558c) (OR: 0.38; 95%CI: 0.20–0.75; p = 0.005). Neonates carrying the minor allele of rs887829 (TA)n repeat had significantly lower peak bilirubin than wild types, while the minor allele carriers of rs4148323 had significantly higher peak bilirubin than wild types. No association was found in HMOX1. Our findings added to the understanding of the significance of UGT1A1 in association with neonatal hyperbilirubinemia in East Asian population. Additional studies were required to investigate the mechanisms of the protective effects. Citation: Zhou Y, Wang S-n, Li H, Zha W, Wang X, et al. (2014) Association of UGT1A1 Variants and Hyperbilirubinemia in Breast-Fed Full-Term Chinese Infants. PLoS ONE 9(8): e104251. doi:10.1371/journal.pone.0104251 Editor: Bing-Hua Jiang, Thomas Jefferson University, United States of America Editor: Bing-Hua Jiang, Thomas Jefferson University, United States of America Received April 6, 2014; Accepted June 20, 2014; Published August 7, 2014 Received April 6, 2014; Accepted June 20, 2014; Published August 7, 2014 Copyright: 2014 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The authors confirm that all data underlying the findings are fully available without restriction. The data is available from the following: h .edu.cn/link/P1TDzrqp2az34Qd6RdlozQowiQEKtjM5ytAeerbLypbY8WEe0DiXm. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The data is available sharelink.cloud.fudan.edu.cn/link/P1TDzrqp2az34Qd6RdlozQowiQEKtjM5ytAeerbLypbY8WEe0DiXm. Abstract Funding: LJ was supported by grants from the Ministry of Science and Technology (2011BAI09B00), National Basic Research Program (2012CB944600), and Ministry of Health (201002007); YC was partially supported by the ‘‘Twelfth Five’’ Key Medical Talent’s Project in Science and Education of Jiangsu Province (RC2011036), Suzhou Science and Technology Development Project (SYSD2011101). 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. * Email: lijin@fudan.edu.cn increased [6]. However, in East Asian populations, such as Japanese and Chinese, the minor allele of rs4148323 (Gly71Arg, G211A) in exon 1, but not (TA)n repeat, is found to be associated with neonatal hyperbilirubinemia [7,8,9]. Youyou Zhou1, San-nan Wang2, Hong Li3, Weifeng Zha2, Xuli Wang2, Yuanyuan Liu2, Jian Sun3, Qianqian Peng1, Shilin Li1, Ying Chen3, Li Jin1* 1 Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China, 2 Department of Neonatology, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, China, 3 Center for Reproduction and Genetics and Suzhou Maternal-Child Medical Center, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, China Association of UGT1A1 Variants and Hyperbilirubinemia in Breast-Fed Full-Term Chinese Infants Youyou Zhou1, San-nan Wang2, Hong Li3, Weifeng Zha2, Xuli Wang2, Yuanyuan Liu2, Jian Sun3, Qianqian Peng1, Shilin Li1, Ying Chen3, Li Jin1* August 2014 \| Volume 9 \| Issue 8 \| e104251 Materials and Methods This study retrospectively enrolled infants born between February and October in 2008 from The Municipal Hospital in Suzhou, China. Eligible infants were exclusively breast-fed, had an estimated gestational age of 37 weeks or above, had a birth weight of 2500 grams or above, and had no major abnormalities. Hardy-Weinberg equilibrium (HWE) for the (GT)n repeat of HMOX1 was performed using an exact test [18]. HWE for other variants, including bi-allelic-grouped (TA)n repeat of UGT1A1 were examined by Fisher’s exact test. Every morning, transcutaneous bilirubin (TcB) was measured on the forehead of each neonate before discharge. Five transcu- taneous bilirubinometers, including 3 KONICA MINOLTA JM- 103 and 2 local branded were randomly used by the neonatol- ogists. The hospital has adopted a discharge policy of 72 hours or above for newborns delivered vaginally and 120 hours or above for newborns delivered by cesarean section. Once the infant was discharged, no TcB was measured any more. Peak TcB of each neonate before discharge was reviewed. Haplotypes and their frequencies were inferred using PHASE version 2.1.1 software (www.stat.washington.edu/stephens/phase/ download.html). The Haploview program (www.broad.mit.edu/ haploview/haploview) was adopted to identify block structures, and to calculate \|D9\| and r2 to measure linkage disequilibrium (LD). Continuous variables and categorical variables were compared by t test and Pearson’s x2 test, respectively. Univariate logistic regression analysis was performed to compute unadjusted and adjusted ORs with 95% CIs. Peak TcB comparison according to genotypes were examined by t test and adjusted by covariance (ANOCVA). All analyses were performed with SPSS version 16.0 software (SPSS Inc, Chicago, IL, USA). A two-sided probability value of ,0.05 was considered statistically. According to China guideline in Practical Neonatology [16] and Practical Pediatrics [17], neonates were diagnosed as hyperbili- rubinemia when their maximum TcB exceeded 12.9 mg/dL (221 mmol/L) on day 3 or later before they were discharged. Once the infants developed high concentration of bilirubin before day 3 or the pathological cause of hyperbilirubinemia was confirme- d,infants would be transferred to the Neonatal Unit and excluded from our study. Otherwise, infants diagnosed as hyperbilirubin- emia would receive phototherapy and remain in our study as cases. Newborns whose peak TcB during birth hospitalization stay were less than or equal to 12.9 mg/dL served as controls. To avoid assumptions regarding the models of inheritance, additive, dominant, and recessive models for each bi-allelic polymorphism were assessed by SNPstats (http://bioinfo. iconcologia.net/SNPstats). Results Initially, we have enrolled 331 hyperbilirubinemic infants. After controlling the filter paper’s quality for further DNA analysis, only 170 hyperbilirubinemia participants left. After controlling the DNA quality, seven hundred and seventy-nine control participants were chosen at random within matching criteria of gender, delivery mode and birth season. The characteristics of the participants are summarized in Table 2. Cases and controls were comparable in gender, delivery mode, birth season, birth weight and gestational age. Peak TcB before discharge was significantly higher in hyperbilirubinemic infants than controls. The highest peak TcB was 18.0 mg/dL and 12.9 mg/dL in hyperbilirubine- mic and control groups, respectively. Each filter paper was placed in an Eppendorf tube, fully soaked in 16PBS (pH = 8.0), and incubated in boiled water for 5 minutes. After a quick spin, the supernatant was transferred to a new tube, washed with absolute ethanol, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was discarded. After a second wash with 70% ethanol, the precipitate was air dried. After resuspension in distilled H2O, the DNA extract was kept at 220uC until further use. All hyperbilirubinemic infants’ bilirubin values dropped to 12.9 mg/dL or even less after phototherapy. None received exchange transfusion, none developed encephalopathy. A final set of 11 genetic variants was selected for genotyping based on (1) functional significance, (2) tagging Single Nucleotide Polymorphisms (SNP) located at least 5 kb upstream and 2 kb downstream of HMOX1 (chr22: 34102087–34122194) and UGT1A1 (chr2:234328658..234348684), respectively [HapMap Data Release #27 (phase II+III), Feb 09, on NCBI B36 assembly, dbSNP b126], selected from the HapMap Han Chinese (CHB) population based on r2.0.8 and minor allele frequency (MAF). 0.1; (3) either G/A or C/T SNP combination due to the limitation of the SNPstream (Beckman Coulter) platform. These variants were able to capture 7 of 9 (78%) polymorphisms of HMOX1, 18 of 22 (82%) polymorphisms of UGT1A1. Information of the variants and the genotyping assays are listed in Table 1. The counts of genotypes at all variants are presented in Table 3. The average call rate was 95%. Among the 11 genotyped variants, rs1018124 (80%), rs4663972 (89%) and rs6717546 (94%)’s call rate was lower than 95%. None of the variants showed significant deviation from HWE, except for rs2071749 (p = 0.04). Additionally, we found 3 (TA)8 alleles in the (TA)n repeat. UGT1A1 Variants and Breast-Fed Hyperbilirubinemia of variants of the two genes and hyperbilirubinemia in breast-fed full-term Chinese infants. (Applied Biosystems, Foster City, USA) according to the manu- facturer’s manual. Nine SNPs were genotyped using a Genome- Lab SNPstream Genotyping System (Beckman Coulter Inc., Fullerton, California, USA) following the manufacturer’s standard procedure. To ensure the reliability and the reproducibility of the genotyping, about 3–5% samples were repeated for each assay. The overall genotyping call rate was 95%, and concordance rate was higher than 99%. Materials and Methods The best-fitting inheritance model for each polymorphism was selected based on the lowest Akaike information criteria (AIC) [19]. For multiple hypotheses test, False Discovery Rate (FDR) was applied. Blood samples for genotyping were obtained from surplus filter papers which were kept at 4uC for about one year after routine newborn screening. The study was approved by both Suzhou Municipal Hospital Reproductive Medicine Ethics Committee and the Ethics Com- mittee of Institutes of Biomedical Sciences. Since the data were analyzed anonymously, the filter paper was obtained from a standard screening procedure, and the TcB measurement was a completely noninvasive routine clinical assessment, both commit- tees approved a waiver of written consent. Introduction Neonatal jaundice is a physiological and generally benign phenomenon. However, excessively high bilirubin concentration may cause permanent neural damage in newborns, i.e., ‘‘chronic bilirubin encephalopathy’’ or kernicterus [1]. Genetic and environmental factors contribute to the development of hyperbil- irubinemia and the impact of genetic variation on this condition is increasingly recognized [2]. In particular, variants in the rate limiting enzymes in the bilirubin metabolism pathway may be associated with hyperbilirubinemia. Heme Oxygenase-1 (HMOX1) is another key enzyme in bilirubin metabolism pathway. It catalyzes the first and the rate- controlling step of heme degradation and generates biliverdin that is converted into bilirubin via biliverdin reductase [3]. A (GT)n dinucleotide repeat is located in the promoter region of HMOX1, and the repeat is associated with transcriptional activity [10]. However, little is known regarding the clinical impact of the HMOX1 variants on neonatal hyperbilirubinemia. Uridine Diphosphate Glucuronosyl Transferase 1A1 (UGT1A1) is the key enzyme for bilirubin conjugation while unconjugated bilirubin is the main cause of hyperbilirubinemia [3]. A (TA)n repeat located in the promoter region of the gene, UGT1A1, is found to be associated with the transcriptional activity [4]. In Caucasian newborns, the minor allele homozygote of this variant significantly increases bilirubin concentration during the first 2 days of life [5]. In addition, when the minor allele homozygote or heterozygote combined with Glucose-6-Posphate Dehydrogenase (G6PD) deficiency, the incidence of hyperbilirubinemia is Exclusive breastfeeding is defined as a risk factor for develop- ment of severe hyperbilirubinemia [11], but it is the reference normative standard for infant feeding and nutrition as well [12]. Since East Asian ethnicity is considered as a high risk factor [11], some studies have been carried out in Japan and China Taiwan to explore the genetic factors associated with breast-fed neonatal hyperbilirubinemia [13,14,15]. None was done in China main- land. Therefore, we performed an exploratory study on association 1 August 2014 \| Volume 9 \| Issue 8 \| e104251 PLOS ONE \| www.plosone.org Results Because of its low frequency and reported decreasing promoter activity with an increasing number of (TA)n repeat [20], we merged (TA)8 into (TA)7 for further analysis. Therefore, (TA)n repeat was considered as bi-allelic variant. LD results are shown in Table 4. Strong pairwise LD was observed in several pairs (each \|D9\|.0.8) within HMOX1 and UGT1A1. But higher r2 values (each r2.0.5) only existed between (TA)n repeat and (GT)n repeat were amplified by a duplex PCR whose forward primers were labeled with FAM respectively. The products were electrophoresed on a Genetics Analyzer 3730xl August 2014 \| Volume 9 \| Issue 8 \| e104251 PLOS ONE \| www.plosone.org 2 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Table 1. Genotyped variants and genotyping assays. Variant assay pirmers oligo(59-.39) UGT1A1 rs887829* SNPstream(GA) GA3U CAACAAGTGTTACCAGAGAGGAA GA3L TACAGTTGTGTTCTTTTCTTTCTAAAAG GA3S CGTGCCGCTCGTGATAGAATGTGAACAAGTTAGGCTTCTTTTCCA (TA)n * GeneScan(duplex) TA-F FAM-CCTTTGTGGACTGACAGCTTTTT TA-R TTGCTCCTGCCAGAGGTTCG rs4148323* SNPstream(GA) GA6U ACATGAAATAGTTGTCCTAGCAC GA6L ATTATGCCCGAGACTAACAAAA GA6S GGCTATGATTCGCAATGCTTTGACGCCTCGTTGTACATCAGAGAC rs1018124 SNPstream(CT) CT7U AGAAATGTAAGACATAAATTCAGTGTTC CT7L AGAAGTATCATTTTCTCTAAGAGACTCAA CT7S AGGGTCTCTACGCTGACGATTTTTTGGAGAAATACTTCTATTTAA rs6717546 SNPstream(GA) GA5U TTCATTGCGTGTGCATGC GA5L GTGTATTTGCCACAATTGTAACTG GA5S GCGGTAGGTTCCCGACATATGAGAAAAGAAAAATAACCAGTAATC rs11563250 SNPstream(GA) GA1U ACACCCTCATTAGCACAAAGTACT GA1L ATCAGCTTGAAAAGAAATACAATCTC GA1S ACGCACGTCCACGGTGATTTGAAAGCCTGTCAGTTTGATAGGAGA rs6719561 SNPstream(CT) CT9U TGGCTGGAACACATTCTGT CT8L TTGTAAACAAGAGTGCCTCAGT CT9S GACCTGGGTGTCGATACCTACTCTACATCCTTGAGGCTGTGCAGT rs4663972 SNPstream(CT) CT9U TGGCTGGAACACATTCTGT CT8L TTGTAAACAAGAGTGCCTCAGT CT8S GTGATTCTGTACGTGTCGCCAGGGACCCCAAGTTGCCATGACCTC HMOX1 (GT)n * GeneScan(duplex) GT-F FAM-CTTTCTGGAACCTTCTGGGACG GT-R GGGGTGGAGAGGAGCAGTCAT rs9607267 SNPstream(CT) CT10U TGGGGTTCAGAATAGGCC CT10L AACAGCTGAAATGAAAGTGCTT CT10S AGATAGAGTCGATGCCAGCTCAGGAAGGAGAATTGTGCCCTGTAG rs2071749 SNPstream(GA) GA7U TATCTGTAAAATAGGGATAATAATGGTACC GA7L TACAACTGATTCTCATGTCCCA GA7S AGGGTCTCTACGCTGACGATTCTTAGACTTATAAGGCTTGAGTGA These were (potential) functional variants from published (and unpublished) studies. doi:10.1371/journal.pone.0104251.t001 These were (potential) functional variants from published (and unpublished) studies. doi:10.1371/journal.pone.0104251.t001 between merged groups and neonatal hyperbilirubinemia (Ta- ble 3). rs887829 and (TA)n repeat (r2 = 0.864), rs6717546 and rs6719561 (r2 = 0.508) in UGT1A1. An LD block structure was observed within HMOX1, including rs9607267 and rs2071749 ((GT)n repeat was not analyzed). No block was found in UGT1A1. ) The associations of other variants are listed in Table 3. Distribution of genotypes of rs887829 (c-364t), rs4148323, rs6717546 (g+914a) and rs6719561 (t+2558c) in UGT1A1 showed significant differences between case and control groups. Allele distributions of these variants, plus (TA)n repeat, were associated with hyperbilirubinemia too. Discussion Breastfeeding has been shown to have several advantages for infants, mothers, and families [12]. However, there is a strong association between breastfeeding and an increased risk of hyperbilirubinemia [11]. Since the etiology of breast-fed hyper- bilirubinemia is unclear, associated factors should be systematically studied, especially in East Asian population, which is classified as a risk factor [11]. The result of best-fitting inheritance model analysis of associated variants was shown in Table 5. All of the five variants were associated with hyperbilirubinemia in its best-fitting inheritance model, and remained significantly after controlling gender, birth season, birth weight, delivery mode and gestational age and the FDR correction: the dominant effect of rs887829 (OR: 0.55; 95%CI: 0.34–0.89; p = 0.014), the additive effect of (TA)n repeat (OR: 0.59; 95%CI: 0.38–0.91; p = 0.017), the dominant effect of rs4148323 (OR: 2.02; 95%CI: 1.44–2.85; p = 5.061025), the recessive effect of rs6717546 (OR: 0.30; 95%CI: 0.11–0.83; p = 0.021) and rs6719561 (OR: 0.38; 95%CI: 0.20–0.75; p = 0.005) on neonatal jaundice. HMOX1 catalyzes the rate-limiting step in heme degradation. Based on the functional study, we suspected that shorter repeat carriers may have higher bilirubin level. Considering the distribution pattern of (GT)n repeat, we tried more bi-allele definitions (S and L) other than the Yamada classification [10], including: ,20 and $20, ,23 and $23, ,24 and $24 and ,27 and $27, #32 and .32, #35 and .35. Unfortunately we did not find any association between (GT)n repeat and hyperbilirubin- emia. We hypothesized that the ‘short’ and ‘long’ cut-off value of (GT)n repeat may be a grey zone instead of a single value. LD test data shown in Table 4 demonstrated low r2 between rs4148323 and any of the aforementioned protective variants (each r2,0.08). Correspondingly, PHASE results also showed the frequency that the minor alleles of rs4148323 and rs887829 located on the same chromosome was 0.0019, as low as those of rs4148323 and (TA)n repeat (frequency = 0.0018), rs4148323 and rs6717546 (frequency = 0.0188), and rs4148323 and rs6719561 (frequency = 0.0142). Furthermore, the frequency of all the minor alleles appearing on the same chromosome was 0. We speculated that the risk effect of the minor allele of rs4148323 and the protective effects of the minor alleles of the protective variants worked separately on the development of neonatal jaundice. The investigation of UGT1A1 showed that rs4148323 (211G. Results After applying the FDR adjustment, rs887829, (TA)n and rs4148323 retained statistical significance. rs6719561 (OR: 0.68; 95%CI: 0.52–0.89; p = 0.005) are two tag SNPs located in 914 and 2558 nucleotides downstream of UGT1A1, respectively. After applying the FDR adjustment for multiple testing, only rs887829 lost statistically significance. No variant in HMOX1 was found to be associated with neonatal hyperbilirubinemia, neither was the LD block found in this gene (data not shown). August 2014 \| Volume 9 \| Issue 8 \| e104251 Results After controlling gender, birth season, birth weight, delivery mode and gestational age, the minor allele of rs4148323 was found to be a risk factor of hyperbilirubinemia (Odds Ratio (OR): 1.75; 95% Confidence Interval (CI): 1.32–2.31; p = 8.061025); whereas, the minor alleles of the remaining variants were shown to be protective factors: rs887829 (OR: 0.61; 95%CI: 0.39–0.94; p = 0.027) and (TA)n repeat (OR: 0.58; 95%CI: 0.37–0.90; p = 0.015) are two variants at the promoter region and are 310 nucleotides apart from each other; rs6717546 (OR: 0.70; 95%CI: 0.53–0.93; p = 0.015) and The distributions of (GT)n repeat in HMOX1 promoter in both case and control groups are shown in Figure 1. Both groups had four modes, located at 23, 30, 34 and 36, respectively. Using the classification defined by Yamada et al., the allele was classified into three classes according to the repeat length, namely, short alleles (S: ,27 GT), middle alleles (M: 27–32 GT), and long alleles (L: $ 33 GT) [10]. The distributions of the genotypes are listed in Table 3. Neither genotypes nor allele distribution had significant difference between case and control groups. Based on previous publication, six genotypes could be merged into 2 groups: Group 1 (SS_SM) and Group 2 (SL_MM_ML_LL). Obviously, the HMOX1 expression of Group 1 was expected to be higher than that of Group 2 [21]. However, we did not find an association PLOS ONE \| www.plosone.org August 2014 \| Volume 9 \| Issue 8 \| e104251 3 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Table 2. Characteristics of hyperbilirubinemic patients and controls. Table 2. Characteristics of hyperbilirubinemic patients and controls. Variable Case Control p (n = 170) (n = 779) Gender,n(%) Male 89(52.4%) 421(54.0%) 0.69 Delivery mode,n(%) Vaginal 86(50.6%) 412(52.9%) 0.76 Cesarean 74(43.5%) 330(42.4%) Forceps 10(5.9%) 37(4.7%) Birth season,n(%) Winter (Feb,Mar) 21(12.4%) 96(12.3%) 0.77 Spring and Autumn (Apr,May,Sep,Oct) 106(62.4%) 465(59.7%) Summer (Jun,Jul,Aug) 43(25.3%) 218(28.0%) Birth weight,gram (mean6standard deviation)* 3437.46415.8 3423.86435.1 0.72 Gestational age, day (mean6standard deviation) 276.768.5 277.568.4 0.28 Peak TcB, mg/dL (mean6standard deviation) 14.761.1 10.462.0 2.16102143 Fifteen cases and 78 controls had missing data. doi:10.1371/journal.pone.0104251.t002 Fifteen cases and 78 controls had missing data. doi:10.1371/journal.pone.0104251.t002 difference was found in HMOX1 genotypes. After applying the FDR adjustment, rs887829, (TA)n and rs4148323 retained statistical significance. difference was found in HMOX1 genotypes. After applying the FDR adjustment, rs887829, (TA)n and rs4148323 retained statistical significance. difference was found in HMOX1 genotypes. Discussion ne Variant Case Control p(genotype) p(allele) OR(95%CI)a p(allele)b OR(95%CI)b m/m M/m M/M MAF HWE p m/m M/m M/M MAF HWE p T1A1 rs887829 2 20 141 0.074 0.2 8 162 597 0.116 0.49 0.035 0.025 0.61(0.39–0.94) 0.027 0.61(0.39–0.94) (TA)n 1 22 146 0.071 0.58 13 154 604 0.117 0.38 0.054 0.014 0.58(0.37–0.90) 0.015d 0.58(0.37–0.90) rs4148323 8 73 85 0.268 0.17 20 220 510 0.173 0.61 2.061024 7.061025 1.75(1.32–2.31) 8.061025d 1.75(1.32–2.31) rs1018124 1 41 94 0.158 0.2 23 206 394 0.202 0.62 0.137 0.095 0.74(0.52–1.06) 0.095 rs6717546 4 65 91 0.228 0.07 58 318 357 0.296 0.29 0.023 0.015 0.70(0.53–0.93) 0.015d 0.70(0.53–0.93) rs11563250 3 36 128 0.126 0.73 19 203 533 0.16 1 0.287 0.12 0.76(0.53–1.08) 0.12 rs6719561 10 71 77 0.288 0.33 111 330 301 0.372 0.21 0.01 0.005 0.68(0.52–0.89) 0.005d 0.68(0.52–0.89) rs4663972 1 38 115 0.13 0.47 13 188 491 0.155 0.38 0.43 0.271 0.82(0.57–1.17) 0.271 MOX1 rs9607267 39 79 48 0.473 0.54 157 389 217 0.461 0.51 0.647 0.686 1.05(0.83–1.33) 0.631 rs2071749 15 63 84 0.287 0.56 42 314 384 0.269 0.04 0.212 0.507 1.09(0.84–1.43) 0.507 (GT)n c SS 41 group1 0.08 SS 176 group1 0.08 SM 60 (SS_SM) SM 297 (SS_SM) 6-grouped: L:0.940 1.02(0.68–1.51) 0.94 SL 19 group2 SL 80 group2 0.931 M:0.727 0.96(0.74–1.23) 0.727 MM 30 (SL_MM_ MM 141 (SL_MM_ 2-grouped: S:/ 1 / ML 16 ML_LL) ML 65 ML_LL) 0.804 LL 1 LL 10 minor allele; M:major allele; MAF:minor allele frequency; HWE p:Hardy-Weinberg equilibrium test p value; OR: odds ratio; CI: confidence interval. ovided that OR of M allele was 1. djusted for gestational age, birth season, delivery mode, birth weight and gender. OR was provided when p value was below 0.05. T)n repeat was classified into 3 groups (S\M\L). Provided that OR of S allele was 1. etained significance after the FDR adjustment (,0.0564/11). :10.1371/journal.pone.0104251.t003 Table 3. Variations in UGT1A1 and HMOX1 genes in breast-fed newborns with hyperbilirubinemic patients and control subjects. August 2014 \| Volume 9 \| Issue 8 \| e104251 Discussion Gene Variant Case Control p(genotype) p(allele) OR(95%CI)a p(allele)b OR(95%CI)b m/m M/m M/M MAF HWE p m/m M/m M/M MAF HWE p UGT1A1 rs887829 2 20 141 0.074 0.2 8 162 597 0.116 0.49 0.035 0.025 0.61(0.39–0.94) 0.027 0.61(0.39–0.94) (TA)n 1 22 146 0.071 0.58 13 154 604 0.117 0.38 0.054 0.014 0.58(0.37–0.90) 0.015d 0.58(0.37–0.90) rs4148323 8 73 85 0.268 0.17 20 220 510 0.173 0.61 2.061024 7.061025 1.75(1.32–2.31) 8.061025d 1.75(1.32–2.31) rs1018124 1 41 94 0.158 0.2 23 206 394 0.202 0.62 0.137 0.095 0.74(0.52–1.06) 0.095 rs6717546 4 65 91 0.228 0.07 58 318 357 0.296 0.29 0.023 0.015 0.70(0.53–0.93) 0.015d 0.70(0.53–0.93) rs11563250 3 36 128 0.126 0.73 19 203 533 0.16 1 0.287 0.12 0.76(0.53–1.08) 0.12 rs6719561 10 71 77 0.288 0.33 111 330 301 0.372 0.21 0.01 0.005 0.68(0.52–0.89) 0.005d 0.68(0.52–0.89) rs4663972 1 38 115 0.13 0.47 13 188 491 0.155 0.38 0.43 0.271 0.82(0.57–1.17) 0.271 HMOX1 rs9607267 39 79 48 0.473 0.54 157 389 217 0.461 0.51 0.647 0.686 1.05(0.83–1.33) 0.631 rs2071749 15 63 84 0.287 0.56 42 314 384 0.269 0.04 0.212 0.507 1.09(0.84–1.43) 0.507 (GT)n c SS 41 group1 0.08 SS 176 group1 0.08 SM 60 (SS_SM) SM 297 (SS_SM) 6-grouped: L:0.940 1.02(0.68–1.51) 0.94 SL 19 group2 SL 80 group2 0.931 M:0.727 0.96(0.74–1.23) 0.727 MM 30 (SL_MM_ MM 141 (SL_MM_ 2-grouped: S:/ 1 / ML 16 ML_LL) ML 65 ML_LL) 0.804 LL 1 LL 10 m:minor allele; M:major allele; MAF:minor allele frequency; HWE p:Hardy-Weinberg equilibrium test p value; OR: odds ratio; CI: confidence interval. aProvided that OR of M allele was 1. bAdjusted for gestational age, birth season, delivery mode, birth weight and gender. OR was provided when p value was below 0.05. m:minor allele; M:major allele; MAF:minor allele frequency; HWE p:Hardy-Weinberg equilibrium test p value; OR: odds ratio; CI: confidence interval. aProvided that OR of M allele was 1. bAdjusted for gestational age, birth season, delivery mode, birth weight and gender. OR was provided when p value was below 0.05. c(GT)n repeat was classified into 3 groups (S\M\L). Provided that OR of S allele was 1. dRetained significance after the FDR adjustment (,0.0564/11). doi:10.1371/journal.pone.0104251.t003 m:minor allele; M:major allele; MAF:minor allele frequency; HWE p:Hardy-Weinberg equilibrium test p value; OR: odds ratio; CI: confidence interval. aProvided that OR of M allele was 1. bAdjusted for gestational age, birth season, delivery mode, birth weight and gender. OR was provided when p value was below 0.05. Discussion A, Gly71Arg), located in the first exon, was a risk factor in neonatal hyperbilirubinemia, and was associated with higher peak TcB during hospitalization stay. Previous functional studies have shown that UGT1A1 enzyme activity of the minor allele carriers was significantly lower than that of major allele carriers [22,23]. (TA)n repeat in the promoter of UGT1A1 is another extensively studied variant. Published functional studies have shown that the transcriptional activity of long repeat is significantly lower than that of short repeat [4,20]. But the effect of long repeat on neonatal jaundice is not consistent. Risk effect has been observed in some Caucasian populations but not in Asian populations [5,6,7,8,24].Our study found the association between (TA)n repeat and neonatal jaundice in an Asian population. Surprisingly, the long repeat appeared to be a protective factor and was associated Comparisons of peak TcB according to UGT1A1 genotypes were shown in Table 6. A general (additive) model was assumed for each polymorphism. The minor allele carriers (including both homozygous and heterozygous carriers) of rs4148323 had significant higher peak TcB than wild types (p = 2.961027). On the other hand, the minor allele carriers of rs887829, (TA)n repeat, rs6717546, or rs6719561 had significant lower peak TcB than wild types (p = 0.002, 0.001, 0.022 and 0.04, respectively). No August 2014 \| Volume 9 \| Issue 8 \| e104251 PLOS ONE \| www.plosone.org 4 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia able 3. Variations in UGT1A1 and HMOX1 genes in breast-fed newborns with hyperbilirubinemic patients and control subjects. UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Table 4. Linkage Disequilibrium. \|D9\| r2 HMOX1 rs9607267 rs2071749 rs9607267 0.976 rs2071749 0.308 UGT1A1 rs887829 (TA)n rs4148323 rs1018124 rs6717546 rs11563250 rs6719561 rs4663972 rs887829 0.937 1 1 0.588 0.394 0.642 0.582 (TA)n 0.864 0.829 0.686 0.608 0.663 0.641 0.472 rs4148323 0.028 0.02 0.728 0.751 0.737 0.766 0.925 rs1018124 0.029 0.014 0.032 0.701 0.547 0.696 0.285 rs6717546 0.017 0.018 0.054 0.286 0.936 0.852 0.785 rs11563250 0.003 0.01 0.023 0.216 0.377 0.789 1 rs6719561 0.088 0.088 0.077 0.195 0.508 0.199 0.92 rs4663972 0.007 0.005 0.035 0.058 0.259 0.031 0.239 Table 4. Linkage Disequilibrium. \|D9\| r2 HMOX1 rs9607267 rs2071749 rs9607267 0.976 rs2071749 0.308 UGT1A1 rs887829 (TA)n rs4148323 rs1018124 rs6717546 rs11563250 rs6719561 rs4663972 rs887829 0.937 1 1 0.588 0.394 0.642 0.582 (TA)n 0.864 0.829 0.686 0.608 0.663 0.641 0.472 rs4148323 0.028 0.02 0.728 0.751 0.737 0.766 0.925 rs1018124 0.029 0.014 0.032 0.701 0.547 0.696 0.285 rs6717546 0.017 0.018 0.054 0.286 0.936 0.852 0.785 rs11563250 0.003 0.01 0.023 0.216 0.377 0.789 1 rs6719561 0.088 0.088 0.077 0.195 0.508 0.199 0.92 rs4663972 0.007 0.005 0.035 0.058 0.259 0.031 0.239 with lower peak TcB during hospitalization stay, which was on the opposite of the functional study result. Indeed, one recent association study between UGT1A1 and neonatal jaundice in breast-fed Japanese newborns illustrated that heterozygous (TA)7 mutation significantly decreased the risk of hyperbilirubinemia (OR: 0.37; 95%CI: 0.15–0.89; p = 0.027) [15]. Another study in Taiwan showed the mean values of peak bilirubin in each (TA)n genotype were significantly different: LL, SL,SS (p = 0.040) [14]. Furthermore, two meta-analysis studies also showed the protective effect of long repeat in two East Asian populations: Chinese and Japanese. Yang et al. found the frequency of (7/7+6/7) in control group was significantly higher than that of the hyperbilirubinemic group in both Chinese and Japanese newborns. The OR (95%CI) were 0.59 (0.36–0.96) and 0.15 (0.04–0.51), respectively [25]. Later, Long et al. calculated the OR (95%CI) of (7/7+6/7) as 0.02 (0.14–0.42), the OR (95%CI) of (TA)7 as 0.13 (0.04–0.42) [26]. In fact, it was found that longer (TA)n repeat frequency was highest in African (49.5%), subsequently followed by European (38.7%) and Asian (16%) [20].And the hyperbilirubinemia guideline published by American Academy of Pediatrics (AAP) illustrated African origin was a protective factor whereas East Asian ethnicity was a risk factor for development of severe hyperbilirubinemia [11]. Discussion c(GT)n repeat was classified into 3 groups (S\M\L). Provided that OR of S allele was 1. 5 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Figure 1. Distribution of (GT)n repeat of HMOX1. doi:10.1371/journal.pone.0104251.g001 statistical analysis power, the power of this study might be limited due to the small case number. statistical analysis power, the power of this study might be limited due to the small case number. common problem worldwide, hospitalization stay is even shorter in some foreign countries. Therefore, some published papers adopted data on day 3 or even before day 3 [13,14]. (2) The diagnostic method our hospital used is different from AAP guidelines. Taking 12.9 mg/dL as the threshold from day 3 onwards is more stringent than the 95th centiles in Bhutani’s nomogram from 72 hours onwards. This is probably due to the high risk of the ethnicity. (3) Number of cases is small. Although our case number was higher than two recently published breast- fed hyperbilirubinemia association studies [14,15], and we recruited controls more than four times of cases to increase the UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Taking these two opinions into consid- eration, we argued that (TA)n longer repeat working as a protective factor for neonatal hyperbilirubinemia was more reasonable than a risk factor, although the mechanism (TA)n longer repeat protect newborns from hyperbilirubinemia may be totally different from the known functional study. Due to the unexplained contradiction, further case control studies in East Asian population and functional analysis may help to disclose the mechanism. Rs887829 (c-364t) is another variant located in the promoter of UGT1A1, 310 bp away from (TA)n repeat. Strong LD between these two sites was found in our population (D9 = 0.937, r2 = 0.864), similar to the observation in a Japanese population and an African population respectively [27,28]. There are no published functional studies on rs887829. One in vitro study showed that rs887829 did not affect the transcriptional activity of UGT1A1 (unpublished data). We proposed that the protective effect of rs887829 may be due to the strong LD between this site and (TA)n repeat. Additionally, two tag SNPs, rs6717546 (g+914a) and rs6719561 (t+2558c), both located in the 39 of UGT1A1, were associated with neonatal breast-fed hyperbilirubinemia too. So far there is no publication on these two sites. The only relative result we found was on rs4148329 (c+857t), which could be captured by rs6717546. That study showed significantly smaller bilirubin change before day 5 was observed for neonates who were homozygous for the minor allele versus those with wild-type (p, 0.003) [29].In other words, rs4148329 minor allele carriers’ bilirubin increased slowly so that they were at low risk for development of hyperbilirubinemia. That result of rs4148329 was consistent with ours on rs6717546. Unfortunately, no functional study was done on rs4148329. Therefore we proposed that the protective effects may be due to the strong LD with other potential functional sites. r2.0.5 was shown in bold; \|D9\|.0.8 was shown in bold. doi:10.1371/journal.pone.0104251.t004 This study has three limitations. (1) Due to short birth hospitalization stay, daily TcB measurement was normally stopped after day 4 for vaginal delivery and day 5 for cesarean delivery. We were not sure that some may become hyperbilirubinemia after discharge. But it is known that peak bilirubin normally reaches on day 4 and day 5, after that, the bilirubin either maintains or drops gradually [30,31,32]. August 2014 \| Volume 9 \| Issue 8 \| e104251 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Besides, short hospitalization stay is a PLOS ONE \| www.plosone.org August 2014 \| Volume 9 \| Issue 8 \| e104251 6 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Figure 1. Distribution of (GT)n repeat of HMOX1. doi:10.1371/journal.pone.0104251.g001 Author Contributions trials. In addition, further studies were required to investigate the mechanisms of the protective effects of the variants. Conceived and designed the experiments: YZ SW YC LJ. Performed the experiments: YZ WZ XW YL JS. Analyzed the data: YZ WZ QP. Contributed reagents/materials/analysis tools: HL SL. Contributed to the writing of the manuscript: YZ SW YC LJ. 14. Chou HC, Chen MH, Yang HI, Su YN, Hsieh WS, et al. (2011) 211 G to a variation of UDP-glucuronosyl transferase 1A1 gene and neonatal breastfeeding jaundice. Pediatr Res 69: 170–174. 15. Sato H, Uchida T, Toyota K, Kanno M, Hashimoto T, et al. (2013) Association of breast-fed neonatal hyperbilirubinemia with UGT1A1 polymorphisms: 211G.A (G71R) mutation becomes a risk factor under inadequate feeding. J Hum Genet 58: 7–10. We would like to thank the participants. We would like to thank the participants. Conclusions Our study provides important new information on the association between genetic factors and breast-fed hyperbilirubin- emia. Genotyping of UGT1A1 may be a supplementary method to predict the development of hyperbilirubinemia in breast-fed full-term Chinese infants. Because of exploratory nature of our study and relatively limited number of examined subjects and variants, our conclusions should be validated in larger prospective Table 5. Estimated effects of selected variants on hyperbilirubinemia. Variant Best-fitting inheritance model OR (95% CI) p-value OR (95% CI)a p-valuea rs887829 Dominant 0.55 (0.34–0.89) 0.0097b 0.55 (0.34–0.89) 0.014b (TA)n Additive 0.59 (0.38–0.91) 0.011b 0.59 (0.38–0.91) 0.017b rs4148323 Dominant 2.03 (1.44–2.85) 161024b 2.02 (1.44–2.85) 5.061025b rs6717546 Recessive 0.30 (0.11–0.83) 0.0065b 0.30 (0.11–0.83) 0.021b rs6719561 Recessive 0.38 (0.20–0.75) 0.0018b 0.38 (0.20–0.75) 0.005b aAdjusted for gender, birth season, birth weight, delivery mode and gestational age. bRetained significance after the FDR adjustment (,0.0565/11). doi:10.1371/journal.pone.0104251.t005 Table 5. Estimated effects of selected variants on hyperbilirubinemia. Variant Best-fitting inheritance model OR (95% CI) p-value OR (95% CI)a p-valuea rs887829 Dominant 0.55 (0.34–0.89) 0.0097b 0.55 (0.34–0.89) 0.014b (TA)n Additive 0.59 (0.38–0.91) 0.011b 0.59 (0.38–0.91) 0.017b rs4148323 Dominant 2.03 (1.44–2.85) 161024b 2.02 (1.44–2.85) 5.061025b rs6717546 Recessive 0.30 (0.11–0.83) 0.0065b 0.30 (0.11–0.83) 0.021b rs6719561 Recessive 0.38 (0.20–0.75) 0.0018b 0.38 (0.20–0.75) 0.005b aAdjusted for gender, birth season, birth weight, delivery mode and gestational age. bRetained significance after the FDR adjustment (,0.0565/11). doi:10.1371/journal.pone.0104251.t005 Table 5. Estimated effects of selected variants on hyperbilirubinemia. August 2014 \| Volume 9 \| Issue 8 \| e104251 7 PLOS ONE \| www.plosone.org 7 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Table 6. Comparison of peak TcB between different genotypes. Gene Variant n peak TcB, mg/dL p pa (mean±standard deviation) UGT1A1 rs887829 m/m+M/m 192 10.762.4 0.002 0.002b M/M 738 11.362.5 (TA)n m/m+M/m 190 10.762.4 0.001 0.001b M/M 750 11.362.5 rs4148323 m/m+M/m 321 11.862.3 2.461027 2.961027b M/M 595 10.962.5 rs1018124 m/m+M/m 271 10.962.6 0.052 0.055 M/M 488 11.362.5 rs6717546 m/m+M/m 445 11.062.6 0.02 0.022 M/M 448 11.462.4 rs11563250 m/m+M/m 261 11.162.5 0.229 0.246 M/M 661 11.362.5 rs6719561 m/m+M/m 522 11.162.5 0.048 0.04 M/M 378 11.462.4 rs4663972 m/m+M/m 240 11.162.6 0.268 0.323 M/M 606 11.362.5 HMOX1 (GT)n group1 574 11.262.5 0.893 0.77 group2 362 11.262.5 rs9607267 m/m+M/m 664 11.262.5 0.753 0.934 M/M 265 11.262.4 rs2071749 m/m+M/m 434 11.262.5 0.488 0.671 M/M 468 11.362.5 m:minor allele; M:major allele. aAdjusted for gestational age, birth season, delivery mode, birth weight and gender. bRetained significance after the FDR adjustment (,0.0563/11). doi:10.1371/journal.pone.0104251.t006 13. 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Pediatrics 103: 1224–1227. 8. Huang CS, Chang PF, Huang MJ, Chen ES, Hung KL, et al. (2002) Relationship between bilirubin UDP-glucuronosyl transferase 1A1 gene and neonatal hyperbilirubinemia. Pediatr Res 52: 601–605. 8. Huang CS, Chang PF, Huang MJ, Chen ES, Hung KL, et al. (2002) Relationship between bilirubin UDP-glucuronosyl transferase 1A1 gene and neonatal hyperbilirubinemia. Pediatr Res 52: 601–605. August 2014 \| Volume 9 \| Issue 8 \| e104251 PLOS ONE \| www.plosone.org 8 UGT1A1 Variants and Breast-Fed Hyperbilirubinemia Wei K (2000) Neonatal Jaundice. In: Wang M, editor. Pediatrics: Neonates and Neonatal Diseases. Beijing: People’s Medical Publishing House. pp. 117–119. 23. Aono S, Yamada Y, Keino H, Hanada N, Nakagawa T, et al. (1993) Identification of defect in the genes for bilirubin UDP-glucuronosyl-transferase in a patient with Crigler-Najjar syndrome type II. Biochem Biophys Res Commun 197: 1239–1244. 31. De Luca D, Jackson GL, Tridente A, Carnielli VP, Engle WD (2009) Transcutaneous bilirubin nomograms: a systematic review of population differences and analysis of bilirubin kinetics. Arch Pediatr Adolesc Med 163: 1054–1059. 24. Zhou YY, Lee LY, Ng SY, Hia CP, Low KT, et al. (2009) UGT1A1 haplotype mutation among Asians in Singapore. Neonatology 96: 150–155. 32. Yu ZB, Dong XY, Han SP, Chen YL, Qiu YF, et al. (2011) Transcutaneous bilirubin nomogram for predicting neonatal hyperbilirubinemia in healthy term and late-preterm Chinese infants. Eur J Pediatr 170: 185–191. August 2014 \| Volume 9 \| Issue 8 \| e104251 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9
https://openalex.org/W2623029357	https://repositorium.sdum.uminho.pt/bitstream/1822/43775/1/PhysRevLett.117.111802.pdf	English	null	Search for Higgs and<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>Z</mml:mi></mml:math>Boson Decays to<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:mi>ϕ</mml:mi><mml:mi>γ</mml:mi></mml:mrow></mml:math>with the ATLAS Detector	Physical review letters	2,016	cc-by	20,667	DOI: 10.1103/PhysRevLett.117.111802 Rare decays of the 125 GeV Higgs boson [1,2] H to a light meson and a photon γ have been suggested to present one viable probe of the Yukawa coupling of the Higgs boson to light (u, d, s) quarks [3–5]. While the Standard Model (SM) predicts these couplings to be small, sub- stantial modifications are predicted in several scenarios beyond the SM, which include the minimal flavor violation framework [6], the Froggatt-Nielsen mechanism [7], the Higgs-dependent Yukawa couplings model [8], the Randall-Sundrum family of models [9], and the possibility of the Higgs boson being a composite pseudo-Goldstone boson [10]. The light-quark Yukawa couplings are almost entirely unconstrained by existing data and the large multijet background at the Large Hadron Collider (LHC) severely inhibits the study of such couplings with inclusive H →q¯q decays. The decay of the Higgs boson to a ϕ meson and a photon would give access to the strange-quark Yukawa coupling and to potential deviations from the SM prediction. The expected SM branching fraction is BðH →ϕγÞ ¼ ð2.3 0.1Þ × 10−6 [4], and no direct exper- imental information about this decay mode currently exists. The analogous rare decays of the Higgs boson to a heavy quarkonium state and a photon offer sensitivity to the charm- and bottom-quark Yukawa couplings [11–13]. The Higgs boson decays to J=ψγ and ϒγ have already been searched for by the ATLAS Collaboration [14]. The former decay mode has also been searched for by the CMS Collaboration [15]. fractions much smaller than for Higgs boson decays to the same final state. The most precise prediction for the SM branching fraction is BðZ →ϕγÞ ¼ ð1.17 0.08Þ × 10−8 [16]. The decay Z →ϕγ has not yet been observed and is not well constrained by existing measurements of Z boson decays. This Letter describes a search for Higgs and Z boson decays to the exclusive final state ϕγ. The decay ϕ →KþK−is used to reconstruct the ϕ meson. The search is performed with a sample of pp collision data corre- sponding to an integrated luminosity of 2.7 fb−1 recorded at a center-of-mass energy ﬃﬃﬃs p ¼ 13 TeV with the ATLAS detector, described in detail in Ref. [18]. Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) Full author list given at the end of the article. DOI: 10.1103/PhysRevLett.117.111802 Higgs boson production is modeled using the POWHEG- BOX v2 Monte Carlo (MC) event generator [19–23] for the gluon fusion (ggH) and vector-boson fusion (VBF) proc- esses calculated up to next-to-leading order in αS with CT10 parton distribution functions [24]. Additional contributions from the associated production of a Higgs boson and a W or Z boson (denoted WH and ZH, respectively) are modeled by the PYTHIA 8.186 MC event generator [25,26] with NNPDF 2.3 parton distribution functions [27]. The pro- duction rates and dynamics for a SM Higgs boson with mH ¼ 125 GeV, obtained from Ref. [28], are assumed throughout this analysis. The ggH signal model is appro- priately scaled to account for the production of a Higgs boson in association with a t¯t or b¯b pair. The POWHEG-BOX v2 MC event generator, with the CTEQ6L1 parton distri- bution functions [29], is used to model Z boson production. The total cross section is obtained from the measurement in Ref. [30], with an uncertainty of 5.5%. The corresponding decay of the Z boson has also been considered from a theoretical perspective [16,17], as it offers a precision test of the SM and the predictions of the factorization approach in quantum chromodynamics [17]. Owing to the large Z boson production cross section at the LHC, rare Z boson decays can be probed at branching The Higgs and Z boson decays are simulated as a cascade of two-body decays. Effects of the helicity of the ϕ mesons on the K kinematics are found to modify the acceptance by at most 1% and this is corrected for in the Higgs boson case and treated as a systematic uncertainty in the Z boson case, due to the unknown Z boson polarization. PYTHIA 8.186 [25,26] with the AZNLO set of hadro- nization and underlying-event parameters [31] is used to simulate showering and hadronization. The simulated The Higgs and Z boson decays are simulated as a cascade of two-body decays. Effects of the helicity of the ϕ mesons on the K kinematics are found to modify the acceptance by at most 1% and this is corrected for in the Higgs boson case and treated as a systematic uncertainty in the Z boson case, due to the unknown Z boson polarization. PYTHIA 8.186 [25,26] with the AZNLO set of hadro- nization and underlying-event parameters [31] is used to simulate showering and hadronization. Full author list given at the end of the article. Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Search for Higgs and Z Boson Decays to ϕγ with the ATLAS Detector M. Aaboud et al.* (ATLAS Collaboration) (Received 14 July 2016; published 9 September 2016) A search for the decays of the Higgs and Z bosons to a ϕ meson and a photon is performed with a pp collision data sample corresponding to an integrated luminosity of 2.7 fb−1 collected at ﬃﬃﬃs p ¼ 13 TeV with the ATLAS detector at the LHC. No significant excess of events is observed above the background, and 95% confidence level upper limits on the branching fractions of the Higgs and Z boson decays to ϕγ of 1.4 × 10−3 and 8.3 × 10−6, respectively, are obtained. DOI: 10.1103/PhysRevLett.117.111802 DOI: 10.1103/PhysRevLett.117.111802 The total signal efficiency (kinematic acceptance, and trigger and reconstruction efficiencies) is 18% and 8% for the Higgs and Z boson decays, respectively. The difference in efficiencies primarily arises due to the softer pγ T and pKþK− T distributions in the case of Z →ϕγ production. The mKþK−γ resolution is around 1.8% for both the Higgs and Z boson decays. The mKþK−distribution for selected ϕγ candidates, with no mKþK−requirement applied, is shown in Fig. 1 and exhibits a clear peak at the ϕ meson mass. For this analysis, in the absence of particle identification capabilities in the relevant momentum range, every recon- structed charged particle satisfying the following require- ments is assumed to be a K meson. Events are selected if there are at least two tracks with pT > 400 MeV originat- ing from the primary vertex, which is defined as the vertex with the largest P p2 T in the event. The charged kaons are reconstructed from inner-detector tracks that satisfy quality requirements, including a requirement on the number of hits in the silicon detectors [35]. The K candidates are required to have pseudorapidity [36] jηj < 2.5 and pT > 15 GeV. The ϕ →KþK−decays are recon- structed from pairs of oppositely charged inner detector tracks. The higher-pT track in a pair, denoted the leading track, is required to have pT > 20 GeV. The experimental resolution in mKþK−is around 4 MeV, comparable to the natural width of the ϕ meson, Γϕ ¼ 4.266 0.031 MeV [34]. Track pairs with a mass mKþK−within 20 MeV of the ϕ meson mass [34] are selected as ϕ →KþK− candidates. Selected ϕ →KþK−candidates are required to satisfy an isolation requirement: the sum of the pT of the reconstructed inner detector tracks from the main vertex within ΔR ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΔϕÞ2 þ ðΔηÞ2 p ¼ 0.2 of the leading track (excluding both tracks constituting the ϕ →KþK−candi- date) is required to be less than 10% of the pT of the ϕ candidate, pKþK− T . [GeV] - K + K m 0.99 1.00 1.01 1.02 1.03 1.04 1.05 Candidates / 0.002 GeV 0 20 40 60 80 100 120 140 Data Fit Result - K + K → φ Combinatoric Background ATLAS -1 = 13 TeV, 2.7 fb s FIG. 1. DOI: 10.1103/PhysRevLett.117.111802 The simulated 111802-1 © 2016 CERN, for the ATLAS Collaboration 0031-9007=16=117(11)=111802(19) week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) multiple pp interactions per bunch crossing (pile-up) in this calculation are reduced by removing tracks that do not originate from the primary vertex. Additionally, the sum of the transverse momenta of all energy deposits in the calorimeters within ΔR ¼ 0.4 of the photon direction, excluding those associated with the reconstructed photon, is required to be less than ð2.45 GeV þ 0.022 × pγ TÞ. The calorimeter isolation measurements are also corrected for the effects of pile-up. events are passed through the detailed GEANT4 simulation of the ATLAS detector [32,33] and processed with the same software used to reconstruct data. events are passed through the detailed GEANT4 simulation of the ATLAS detector [32,33] and processed with the same software used to reconstruct data. The data sample used in this analysis was collected with a dedicated trigger, commissioned in September 2015, requiring an isolated photon with a transverse momentum pT greater than 35 GeV and an isolated pair of tracks with an invariant mass loosely consistent with the ϕ meson mass of 1019.5 MeV [34], one of which must have a transverse momentum greater than 15 GeV. The trigger efficiency for both the Higgs and Z boson signals is around 80% with respect to the offline selection. Events are retained for analysis if collected under stable LHC beam conditions and the detector was operating normally. Combinations of a ϕ →KþK−candidate and a photon, satisfying ΔϕðKþK−; γÞ > 0.5, are retained for further analysis. When multiple combinations are possible, the combination of the highest-pT photon and the ϕ →KþK− candidate with a mass closest to the ϕ meson mass is retained. The transverse momentum of ϕ →KþK−candi- dates is required to be greater than a threshold that varies as a function of the invariant mass of the three-body system, mKþK−γ. Thresholds of 40 GeVand 45 GeVare imposed for the regions mKþK−γ < 91 GeV and mKþK−γ ≥125 GeV, respectively. The threshold is varied from 40 GeV to 45 GeV as a linear function of mKþK−γ in the region 91 ≤mKþK−γ < 125 GeV. This approach ensures optimal sensitivity for both the Higgs and Z boson searches. PRL 117, 111802 (2016) Probability density functions (pdfs) that model the pKþK− T , pγ T, ΔηðKþK−; γÞ, and ΔϕðKþK−; γÞ distributions of this sample are con- structed using a Gaussian kernel density estimation [40]. Correlations between these variables and pγ T in the event were studied and accounted for in the background model by deriving separate pdfs in 13 exclusive regions of pγ T. In the case of the ϕ →KþK−and photon isolation variables, correlations are accounted for by using two- dimensional histograms derived in the same 13 exclusive regions of pγ T. Values of mKþK−are sampled from the corresponding distribution in the GR. The pdfs of these kinematic and isolation variables are sampled to generate an ensemble of pseudocandidates, each with a complete KþK−γ four-vector and an associated pair of ϕ →KþK− and photon isolation values. The nominal selection require- ments are imposed on the ensemble and the surviving pseudocandidates are used to construct templates for the mKþK−γ distribution. FIG. 2. The distribution of mKþK−γ in data compared to the prediction of the background model for a validation control sample defined by the GR selection with the addition of the nominal photon isolation requirement. The background model is normalized to the observed number of events within the region shown. The uncertainty band corresponds to the uncertainty envelope derived from variations in the background modeling procedure. threshold structures, and confirming that the background model describes the shapes of both mKþK−γ distributions. Further exclusive background contributions from Z →llγ decays have been studied but are found to represent a negligible contribution for the selection requirements and data set used in this analysis. Trigger and identification efficiencies for photons are determined from samples enriched with Z →eþe−events in data [37,41]. The systematic uncertainty on the expected signal yield associated with the trigger efficiency is estimated to be 2%. The photon identification efficiency uncertainties, for both the converted and unconverted photons, are estimated to be 2.4% and 2.6% for the Higgs and Z boson signals, respectively. An uncertainty of 6% is assigned to the track reconstruction efficiency and includes effects associated with the material budget of the inner detector and the behavior of the track reconstruction algorithm if a nearby track is present. The integrated luminosity of the data sample has an uncertainty of 5% derived using the method described in Ref. [42]. PRL 117, 111802 (2016) PRL 117, 111802 (2016) [GeV] γ - K + K m 0 50 100 150 200 250 300 Events / 5 GeV 0 20 40 60 80 100 120 140 160 180 200 -1 = 13 TeV, 2.7 fb s Data Background Model Model Shape Uncertainty ATLAS FIG. 2. The distribution of mKþK−γ in data compared to the prediction of the background model for a validation control sample defined by the GR selection with the addition of the nominal photon isolation requirement. The background model is normalized to the observed number of events within the region shown. The uncertainty band corresponds to the uncertainty envelope derived from variations in the background modeling procedure. 9 SEPTEMBER 2016 [GeV] γ - K + K m 0 50 100 150 200 250 300 Events / 5 GeV 0 20 40 60 80 100 120 140 160 180 200 -1 = 13 TeV, 2.7 fb s Data Background Model Model Shape Uncertainty ATLAS The main source of background to the search comes from events involving inclusive multijet or photon þ jet processes where a ϕ →KþK−candidate is reconstructed from tracks associated with a jet. The normalization of this inclusive background is extracted directly from a fit to data. from events involving inclusive multijet or photon þ jet processes where a ϕ →KþK−candidate is reconstructed from tracks associated with a jet. The normalization of this inclusive background is extracted directly from a fit to data. The selection criteria discussed earlier shape the mKþK−γ distribution for background such that it exhibits a threshold structure near 100 GeV, and falls then smoothly towards higher mass values. Given the nontrivial shape of this background, these processes are modeled with a nonpara- metric data-driven approach using templates to describe the kinematic distributions. A similar procedure was used in the search for Higgs and Z boson decays to J=ψγ and ϒðnSÞγ described in Ref. [14]. The approach exploits a sample of around 4000 KþK−γ candidate events passing all of the kinematic selection requirements described previously, except that the photon and ϕ→KþK−candidates are not required to satisfy the nominal isolation requirements. The events satisfying this selection are collected in a generation region (GR). The contamination of this sample from signal events is expected to be negligible and is verified not to affect the shape of the background model. DOI: 10.1103/PhysRevLett.117.111802 The mKþK−distribution of selected ϕγ combinations with the complete event selection applied (see text), apart from the requirement on mKþK−. The data are fitted with the con- volution of a Breit-Wigner distribution, using the ϕ width [34], and a Gaussian distribution to represent the experimental resolution, while the background is modeled with an analytical function, commonly used to describe a kinematic threshold [39]. [GeV] - K + K m 0.99 1.00 1.01 1.02 1.03 1.04 1.05 Candidates / 0.002 GeV 0 20 40 60 80 100 120 140 Data Fit Result - K + K → φ Combinatoric Background ATLAS -1 = 13 TeV, 2.7 fb s Photons are reconstructed from clusters of energy in the electromagnetic calorimeter. Clusters without matching tracks are classified as unconverted photon candidates while clusters matched to tracks consistent with the hypothesis of a photon conversion into an eþe−pair are classified as converted photon candidates [37]. Reconstructed photon candidates are required to have transverse momentum pγ T > 35 GeV, pseudorapidity jηγj < 2.37, excluding the barrel or endcap calorimeter transition region 1.37 < jηγj < 1.52, and to satisfy the “tight” photon identification criteria [38].Anisolation requirement isimposedtofurther suppress the contamination from jets. The sum of the transverse momenta of all tracks within ΔR ¼ 0.2 of the photon direction, excluding those associated with the reconstructed photon, is required to be less than 5% of pγ T. The effects of FIG. 1. The mKþK−distribution of selected ϕγ combinations with the complete event selection applied (see text), apart from the requirement on mKþK−. The data are fitted with the con- volution of a Breit-Wigner distribution, using the ϕ width [34], and a Gaussian distribution to represent the experimental resolution, while the background is modeled with an analytical function, commonly used to describe a kinematic threshold [39]. FIG. 1. The mKþK−distribution of selected ϕγ combinations with the complete event selection applied (see text), apart from the requirement on mKþK−. The data are fitted with the con- volution of a Breit-Wigner distribution, using the ϕ width [34], and a Gaussian distribution to represent the experimental resolution, while the background is modeled with an analytical function, commonly used to describe a kinematic threshold [39]. 111802-2 111802-2 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) Events/ 5 GeV 20 40 60 80 100 120 ATLAS -1 =13 TeV, 2.7 fb s Data σ 1 ± Background Fit Background -3 )=10 → B(H -6 )=10 γ φ γ φ → B(Z [GeV] γ - K + K m 0 50 100 150 200 250 300 Data/Fit 0 0.5 1 1.5 2 300 TABLE II. Expected and observed branching fraction limits at 95% C.L. for 2.7 fb−1 of pp collision data at ﬃﬃﬃs p ¼ 13 TeV. The 1σ intervals of the expected limits are also given. Branching fraction limit (95% C.L.) Expected Observed BðH →ϕγÞ½10−3 1.5þ0.7 −0.4 1.4 BðZ →ϕγÞ½10−6 4.4þ2.0 −1.2 8.3 TABLE II. Expected and observed branching fraction limits at 95% C.L. for 2.7 fb−1 of pp collision data at ﬃﬃﬃs p ¼ 13 TeV. The 1σ intervals of the expected limits are also given. TABLE II. Expected and observed branching fraction limits at 95% C.L. for 2.7 fb−1 of pp collision data at ﬃﬃﬃs p ¼ 13 TeV. The 1σ intervals of the expected limits are also given. Branching fraction limit (95% C.L.) Expected Observed BðH →ϕγÞ½10−3 1.5þ0.7 −0.4 1.4 BðZ →ϕγÞ½10−6 4.4þ2.0 −1.2 8.3 Events/ 5 GeV On the basis of the observed data, upper limits are set on the branching fractions for the Higgs and Z boson decays to ϕγ using the CLs modified frequentist formalism [44] with the profile-likelihood ratio test statistic [45]. The result of the background-only fit is shown in Fig. 3; a small excess of two standard deviations is observed in the Z boson mass region, estimated using the asymptotic approximation for the distribution of the test statistic. The expected SM production cross section is assumed for the Higgs boson while the ATLAS measurement of the inclusive Z boson cross section is used for the Z boson signal [30]. The results are summarized in Table II. The observed 95% confidence level (C.L.) upper limits on the branching fractions for H →ϕγ and Z →ϕγ decays are around 600 and 700 times the expected SM branching fractions, respectively. FIG. 3. The mKþK−γ distributions of the selected ϕγ candidates, along with the results of the maximum-likelihood fit with background-only model. The 1σ uncertainty band corresponds to the total uncertainty of the background model. The Higgs and Z boson contributions, expected for branching fraction values of 10−3 and 10−6, respectively, are also shown. PRL 117, 111802 (2016) In conclusion, a search for the decay of Higgs or Z bosons to ϕγ has been performed with a pp collision data sample at ﬃﬃﬃs p ¼ 13 TeV corresponding to an integrated luminosity of 2.7 fb−1 collected with the ATLAS detector at the LHC. No significant excess of events is observed above the background. Upper limits at the 95% C.L. are set on the branching fractions for the decay of the 125 GeV SM Higgs boson and the Z boson to ϕγ. The obtained limits are BðH →ϕγÞ < 1.4 × 10−3 and BðZ →ϕγÞ < 8.3 × 10−6. photon energy scale uncertainty, determined from Z → eþe−events and validated using Z →llγ events [43], is propagated through the simulated signal samples as a function of ηγ and pγ T. The uncertainty associated with the description of the photon energy scale in the simulation is found to be less than 0.3% of the three-body invariant mass while the uncertainty associated with the photon energy resolution is found to be negligible relative to the overall three-body invariant mass resolution. Similarly, the systematic uncertainty associated with the track momentum measurement is found to be negligible. We thank CERN for the very successful operation of the LHC, as well as the support staff from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR (Ministry of Industry and Trade) and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, HGF (Helmholtz Association), and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD (Ministry of Education, Science and Technological Development), Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and The uncertainty on the shape of the inclusive multijet and photon þ jet background is estimated through the study of variations in the background modeling procedure. PRL 117, 111802 (2016) The γ To validate this background model with data, the mKþK−γ distributions in several validation regions, defined by kin- ematic and isolation requirements looser than the nominal signal requirements, are used to compare the prediction of the background model with the data. The mKþK−γ distribu- tion in one of these validation regions, defined by the GR selection with the addition of the nominal photon isolation requirement, is shown in Fig. 2. The background model is found to describe the data well, and within the observed statistical uncertainties. A consistency test of the background modeling procedure has been performed with a sample of simulated photon þ jet events in place of the data; similarly good agreement is observed. The robustness of the back- ground model is further validated by splitting the data into high- and low-pKþK−γ T subsets, that exhibit different TABLE I. The number of observed events and the expected background yield for the two mKþK−γ ranges of interest. The Higgs and Z boson contributions expected for branching fraction values of 10−3 and 10−6, respectively, and estimated using Monte Carlo simulations are also shown. Observed (expected) background Expected signal Mass range [GeV] Z H All 81–101 120–130 B½10−6 B½10−3 1065 288 (266 9) 89 (87 3) 6.7 0.7 13.5 1.5 111802-3 111802-3 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) Events/ 5 GeV 20 40 60 80 100 120 ATLAS -1 =13 TeV, 2.7 fb s Data σ 1 ± Background Fit Background -3 )=10 → B(H -6 )=10 γ φ γ φ → B(Z [GeV] γ - K + K m 0 50 100 150 200 250 300 Data/Fit 0 0.5 1 1.5 2 FIG. 3. The mKþK−γ distributions of the selected ϕγ candidates, along with the results of the maximum-likelihood fit with background-only model. The 1σ uncertainty band corresponds to the total uncertainty of the background model. 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Bruni,22a 22 107 30 22 23 33 82 15 , g , , , , j , , , J. Brosamer,16 E. Brost,108 J. H Broughton,19 P. A. Bruckman de Renstrom,41 D. P H Y S I C A L R E V I E W L E T T E R S Cerda Alberich,166 B. C. Cerio,47 A. S. Cerqueira,26b A. Cerri,149 L. Cerrito,133a,133b F. Cerutti,16 M. Cerv,32 A. Cervelli,18 S. A. Cetin,20c A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30 D G Charlton 19 A Chatterjee 51 C C Chau 158 C A Chavez Barajas 149 S Che 111 S Cheatham 73 A Chegwidden 91 S. A. Cetin,20c A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30 D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S Chekanov 6 S V Chekulaev 159a G A Chelkov 66,k M A Chelstowska 90 C Chen 65 H Chen 27 K Chen 148 S Chen 35c D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91 S Chekanov 6 S V Chekulaev 159a G A Chelkov 66,k M A Chelstowska 90 C Chen 65 H Chen 27 K Chen 148 S Chen 35c S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130 135 27 7 136 49 124 124b 74 S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130 R. Cherkaoui El Moursli,135e V. Chernyatin,27,a E. Cheu,7 L. Chevalier,136 V. Chiarella,49 G. Chiarelli,124a,124b G. Chiodini,74a R. Cherkaoui El Moursli, V. Chernyatin, E. Cheu, L. Chevalier, V. Chiarella, G. Chiarelli, G. Chiodini, A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100 V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115 G. Ciapetti,132a,132b A. K. Ciftci,4a D. Cinca,45 V. Cindro,76 I. A. Cioara,23 C. Ciocca,22a,22b A. Ciocio,16 F. Cirotto,104a,104b A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100 V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115 Z. H. Citron,171 M. Citterio,92a M. Ciubancan,28b A. Clark,51 B. L. Clark,58 M. R. Clark,37 P. J. Clark,48 R. N. Clarke,16 C. Clement,146a,146b Y. Coadou,86 M. Cobal,163a,163c A. Coccaro,51 J. Cochran,65 L. Colasurdo,106 B. Cole,37 A. P. Colijn,107 J. Collot,57 T. Colombo,32 G. Compostella,101 P. Conde Muiño,126a,126b E. Coniavitis,50 S. H. Connell,145b I. A. Connelly,78 V. Consorti,50 S. Constantinescu,28b G. Conti,32 F. Conventi,104a,l M. Cooke,16 B. D. Cooper,79 A. M. P H Y S I C A L R E V I E W L E T T E R S Bruncko,144b R. Bruneliere,50 A. Bruni,22a G B i 22a L S B i 107 BH B t 30 M B hi 22a N B i 23 P B t 33 L B k 82 T B 15 G. Bruni, L. S. Bruni, BH Brunt, M. Bruschi, N. Bruscino, P. Bryant, L. Bryngemark, T. Buanes, Q. Buat,142 P. Buchholz,141 A. G. Buckley,55 I. A. Budagov,66 F. Buehrer,50 M. K. Bugge,119 O. Bulekov,98 D. Bullock,8 Q. Buat,142 P. Buchholz,141 A. G. Buckley,55 I. A. Budagov,66 F. Buehrer,50 M. K. Bugge,119 O. Bulekov,98 D. Bullock,8 H B kh 32 S B di 75 C D B d 50 B B h 108 K B k 41 S B k 131 I B i 45 J T P B 120 E. Busato,36 D. Büscher,50 V. Büscher,84 P. Bussey,55 J. M. Butler,24 C. M. Buttar,55 J. M. Butterworth,79 P. Butti,107 W. Buttinger,27 A. Buzatu,55 A. R. Buzykaev,109,d S. Cabrera Urbán,166 D. Caforio,128 V. M. Cairo,39a,39b O. Cakir,4a N. Calace,51 P. Calafiura,16 A. Calandri,86 G. Calderini,81 P. Calfayan,100 G. Callea,39a,39b L. P. Caloba,26a S. Calvente Lopez,83 D. Calvet,36 S. Calvet,36 T. P. Calvet,86 R. Camacho Toro,33 S. Camarda,32 P. Camarri,133a,133b D. Cameron,119 R. Caminal Armadans,165 C. Camincher,57 S. Campana,32 M. Campanelli,79 A. Camplani,92a,92b A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 M. D. M. Capeans Garrido,32 I. Caprini,28b M. Caprini,28b M. Capua,39a,39b R. Caputo,84 R. M. Carbone,37 R. Cardarelli,133a F. Cardillo,50 I. Carli,129 T. Carli,32 G. Carlino,104a L. Carminati,92a,92b S. Caron,106 E. Carquin,34b G. D. Carrillo-Montoya,32 30 126 126 19 13 i 13 162 145 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,16 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,162 E. Castaneda-Miranda,145a 107 107 166 126 j 32 119 32 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,162 E. Castaneda-Miranda,145a R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R. Catmore,119 A. Cattai,32 R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R R. Castelijn, A. Castelli, V. Castillo Gimenez, N. F. Castro, A. Catinaccio, J. R. Catmore, A. Cattai, J. Caudron,23 V. Cavaliere,165 E. Cavallaro,13 D. Cavalli,92a M. Cavalli-Sforza,13 V. Cavasinni,124a,124b F. Ceradini,134a,134b J. Caudron,23 V. Cavaliere,165 E. Cavallaro,13 D. Cavalli,92a M. Cavalli-Sforza,13 V. Cavasinni,124a,124b F. Ceradini,134a,134b L. P H Y S I C A L R E V I E W L E T T E R S Bagnaia,132a,132b 35 131 175 109 d 171 148 136 122 y g y M. J. Baca,19 H. Bachacou,136 K. Bachas,74a,74b M. Backes,148 M. Backhaus,32 P. Bagiacchi,132a,132b P. Bagnaia,132a,132b Y B i 35a J T B i 131 O K B k 175 E M B ldi 109,d P B l k 171 T B l t i 148 F B lli 136 W K B l 122 E. Banas,41 Sw. Banerjee,172,f A. A. E. Bannoura,174 L. Barak,32 E. L. Barberio,89 D. Barberis,52a,52b M. Barbero,86 T. Barillari,101 M-S Barisits,32 T. Barklow,143 N. Barlow,30 S. L. Barnes,85 B. M. Barnett,131 R. M 5 T. Barillari, M-S Barisits, T. Barklow, N. Barlow, S. L. Barnes, B. M. Barnett, R. M. Barnett, Z. Barnovska, A. Baroncelli,134a G. Barone,25 A. J. Barr,120 L. Barranco Navarro,166 F. Barreiro,83 J. Barreiro Guimarães da Costa,35a A. Baroncelli,134a G. Barone,25 A. J. Barr,120 L. Barranco Navarro,166 F. Barreiro,83 J. Barreiro Guimarães da Costa,35a R. Bartoldus,143 A. E. Barton,73 P. Bartos,144a A. Basalaev,123 A. Bassalat,117 R. L. Bates,55 S. J. Batista,158 J. R. Batley,30 M. Battaglia,137 M. Bauce,132a,132b F. Bauer,136 H. S. Bawa,143,g J. B. Beacham,111 M. D. Beattie,73 T. Beau,81 , , , , , , , y, M. Battaglia,137 M. Bauce,132a,132b F. Bauer,136 H. S. Bawa,143,g J. B. Beacham,111 M. D. Beattie,73 T. Beau,81 g A. J. Beddall,20d A. Beddall,20b V. A. Bednyakov,66 M. Bedognetti,107 C. P. Bee,148 L. J. Beemster,107 T. A. Beermann,32 A. J. Beddall,20d A. Beddall,20b V. A. Bednyakov,66 M. Bedognetti,107 C. P. Bee,148 L. J. Beemster,107 T. A. Beermann,32 M. Begel,27 J. K. Behr,44 C. Belanger-Champagne,88 A. S. Bell,79 G. Bella,153 L. Bellagamba,22a A. Bellerive,31 111802-6 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) M. Bellomo,87 K. Belotskiy,98 O. Beltramello,32 N. L. Belyaev,98 O. Benary,153 D. Benchekroun,135a M. Bender,100 K. Bendtz,146a,146b N. Benekos,10 Y. Benhammou,153 E. Benhar Noccioli,175 J. Benitez,64 D. P. Benjamin,47 J. R. Bensinger,25 S. Bentvelsen,107 L. Beresford,120 M. Beretta,49 D. Berge,107 E. Bergeaas Kuutmann,164 N. Berger,5 J. Beringer,16 S. Berlendis,57 N. R. Bernard,87 C. Bernius,110 F. U. Bernlochner,23 T. Berry,78 P. Berta,129 C. Bertella,84 G. Bertoli,146a,146b , , , , y, , F. Bertolucci,124a,124b I. A. Bertram,73 C. Bertsche,44 D. Bertsche,113 G. J. Besjes,38 O. 111802-7 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) M. Cristinziani,23 V. Croft,106 G. Crosetti,39a,39b A. Cueto,83 T. Cuhadar Donszelmann,139 J. Cummings,175 M. Curatolo,49 J. Cúth,84 H. Czirr,141 P. Czodrowski,3 G. D’amen,22a,22b S. D’Auria,55 M. D’Onofrio,75 M. Cristinziani, V. Croft, G. Crosetti, A. Cueto, T. Cuhadar Donszelmann, J. Cummings, M. Curatolo, J. Cúth,84 H. Czirr,141 P. Czodrowski,3 G. D’amen,22a,22b S. D’Auria,55 M. D’Onofrio,75 M. J. Da Cunha Sargedas De Sousa,126a,126b C. Da Via,85 W. Dabrowski,40a T. Dado,144a T. Dai,90 O. Dale,15 F. Dallaire,95 C. Dallapiccola,87 M. Dam,38 J. R. Dandoy,33 N. P. Dang,50 A. C. Daniells,19 N. S. Dann,85 M. Danninger,167 , , y, g, , , fmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C p y g M. Dano Hoffmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W 129 153 79 89 139 p y g g M. Dano Hoffmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C. David,168 T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 M. Dano Hoffmann, V. Dao, G. Darbo, S. Darmora, J. Dassoulas, A. Dattagupta, W. Davey, C. David, T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 R d A di 104a A D B d i 113 S D C 22a 22b S D C 81 N D G 106 P d J 107 H D l T 83 29 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. D y A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de Jong,107 H. De la T y de Asmundis,104a A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de J g F. De Lorenzi,65 A. De Maria,56 D. De Pedis,132a A. De Salvo,132a U. De Sanctis,149 A. De Santo,149 J. B. De Vivie De Regie,117 W. J. Dearnaley,73 R. Debbe,27 C. Debenedetti,137 D. V. Dedovich,66 N. Dehghanian,3 I. Deigaard,107 M. Del Gaudio,39a,39b J. Del Peso,83 T. Del Prete,124a,124b D. Delgove,117 F. Deliot,136 C. M. Delitzsch,51 vie De Regie,117 W. J. Dearnaley,73 R. Debbe,27 C. Debenedetti,137 D. V. Dedovich,66 N. Deh g g A. Dell’Acqua,32 L. Dell’Asta,24 M. 111802-7 Dell’Orso,124a,124b M. Della Pietra,104a,l D. della Volpe,51 M. Delmastro,5 P. A. Delsart,57 D. A. DeMarco,158 S. Demers,175 M. Demichev,66 A. Demilly,81 S. P. Denisov,130 D. Denysiu J. E. Derkaoui,135d F. Derue,81 P. Dervan,75 K. Desch,23 C. Deterre,44 K. Dette,45 P. O. Deviveiros,32 A. Dewhurst,131 S. Dhaliwal,25 A. Di Ciaccio,133a,133b L. Di Ciaccio,5 W. K. Di Clemente,122 C. Di Donato,132a B. Di Girolamo,32 B. Di Micco,134a,134b R. Di Nardo,32 A. Di Simone,50 R. Di Sipio,158 D. Di 158 48 34 90 17 86 B. Di Girolamo,32 B. Di Micco,134a,134b R. Di Nardo,32 A. Di Simone,50 R. Di Sipio,158 D. Di Valentino,31 C. Diaconu,86 M Di d 158 F A Di 48 M A Di 34a E B Di hl 90 J Di i h 17 S Di li 86 A Di i i k 14 J Di f ld 23 M. Diamond,158 F. A. Dias,48 M. A. Diaz,34a E. B. Diehl,90 J. Dietrich,17 S. Diglio,86 A. Dim P. Dita,28b S. Dita,28b F. Dittus,32 F. Djama,86 T. Djobava,53b J. I. Djuvsland,59a M. A. B. do Vale,2 P. Dita,28b S. Dita,28b F. Dittus,32 F. Djama,86 T. Djobava,53b J. I. Djuvsland,59a M. A. B. do Vale,26c D. Dobos,32 M. Dobre,28b 82 129 129 26d 124 124b 121 121b 36 131 C. Doglioni,82 J. Dolejsi,129 Z. Dolezal,129 M. Donadelli,26d S. Donati,124a,124b P. Dondero,121a,1 A. Doria,104a M. T. Dova,72 A. T. Doyle,55 E. Drechsler,56 M. Dris,10 Y. Du,35d J. Duarte-Campderros,153 E. Duchovni,171 A. Doria,104a M. T. Dova,72 A. T. Doyle,55 E. Drechsler,56 M. Dris,10 Y. Du,35d J. Duarte-C G. Duckeck,100 O. A. Ducu,95,n D. Duda,107 A. Dudarev,32 A. Chr. Dudder,84 E. M. Duffield,16 L. Duflot,117 M. Dührssen,32 M. Dumancic,171 M. Dunford,59a H. Duran Yildiz,4a M. Düren,54 A. Durglishvili,53b D. Duschinger,46 B. Dutta,44 M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16 M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16 T. Ekelof,164 M. El Kacimi,135c V. Ellajosyula,86 M. Ellert,164 S. Elles,5 F. Ellinghaus,174 A. A. Elliot,168 N. Ellis,32 T. Ekelof,164 M. El Kacimi,135c V. Ellajosyula,86 M. Ellert,164 S. Elles,5 F. Ellinghaus,174 A. A. Elliot,168 N. Ellis,32 J. Elmsheuser,27 M. Elsing,32 D. Emeliyanov,131 Y. Enari,155 O. C. Endner,84 J. S. Ennis,169 J. Erdmann,45 A. Ereditato,18 G. Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. Escobar,125 B. Esposito,49 g y G. 111802-7 Gomes , , , g, , , ç , J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166 13 51 32 97 27 105 32 J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166 G. Gonzalez Parra,13 S. Gonzalez-Sevilla,51 L. Goossens,32 P. A. Gorbounov,97 H. A. Gordon,27 I. Gorelov,105 B. Gorini,32 E. Gorini,74a,74b A. Gorišek,76 E. Gornicki,41 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,66 C. R. Goudet,117 5 5 5 5 5 g A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowska-Bold,40a P. O. J. G g D. Goujdami,135c A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowsk 22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. P. Grafström,22a,22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. Gray,32 g, , g , , , D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. G Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K E. Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. Griffiths,8 A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171 56 80 79 90 172 63 51 162 , , , g , g , , , , A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171 g g A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. G g g 137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162 O Gueta 153 E Guido 52a,52b T Guillemin 5 S Guindon 2 U Gul 55 C Gumpert 32 J Guo 35e Y Guo 35b,p R Gupta 42 J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R O. Gueta,153 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R. Gupta,42 S. Gupta,120 G. Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. 111802-7 Gutschow,46 C. Guyot,136 C. Gwenlan,120 5 6 5 6 , , , , , p , , , p , S. Gupta,120 G. Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. Gutschow,46 C. Guyot,136 C. Gwenlan,120 5 110 16 8 135 86 23 41 . Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. Gutschow,46 C. Guyot,136 C. G C. B. Gwilliam,75 A. Haas,110 C. Haber,16 H. K. Hadavand,8 N. Haddad,135e A. Hadef,86 S. Hageböck,23 Z. Hajduk,41 H. Hakobyan,176,a M. Haleem,44 J. Haley,114 G. Halladjian,91 G. D. Hallewell,86 K. Hamacher,174 P. Hamal,115 Hamano,168 A. Hamilton,145a G. N. Hamity,139 P. G. Hamnett,44 L. Han,35b K. Hanagaki,67,t K. H Hamilton,145a G. N. Hamity,139 P. G. Hamnett,44 L. Han,35b K. Hanagaki,67,t K. Hanawa,155 M. H B. Haney,122 S. Hanisch,32 P. Hanke,59a R. Hanna,136 J. B. Hansen,38 J. D. Hansen,38 M. C. Hansen,23 P. H. Hansen,38 6 6 Hard,172 T. Harenberg,174 F. Hariri,117 S. Harkusha,93 R. D. Harrington,48 P. F. Harrison,169 F. H K. Hara,160 A. S. Hard,172 T. Harenberg,174 F. Hariri,117 S. Harkusha,93 R. D. Harrington,48 P. F N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46 127 19 32 157 91 120 77 N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46 M. Havranek,127 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Hays,77 H S Hayward 75 S J Haywood 131 S J Head 19 T Heck 84 V Hedberg 82 L Heelan 8 S Heim 122 T Heim 16 N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46 M. Havranek,127 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Hays,77 H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 27 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Ha , , g , y , y , y , y , H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 y y g Heinemann,16 J. J. Heinrich,100 L. Heinrich,110 C. Heinz,54 J. Hejbal,127 L. Helary,32 S. Hellm J. Henderson,120 R. C. W. 111802-7 Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. Escobar,125 B. Esposito,49 A. I. Etienvre,136 E. Etzion,153 H. Evans,62 A. Ezhilov,123 F. Fabbri,22a,22b L. Fabbri,22a,22b G. Facini,33 R. M. Fakhrutdinov,130 S. Falciano,132a R. J. Falla,79 J. Faltova,32 Y. Fang,35a M. Fanti,92a,92b A. Farbin,8 A. Farilla,134a C. Farina,125 A. I. Etienvre,136 E. Etzion,153 H. Evans,62 A. Ezhilov,123 F. Fabbri,22a,22b L. Fabbri,22a,22b G. Facini,33 R. M. Fakhrutdinov,130 S. Falciano,132a R. J. Falla,79 J. Faltova,32 Y. Fang,35a M. Fanti,92a,92b A. Farbin,8 A. Farilla,134a C. Farina,125 E M Farina 121a,121b T Farooque 13 S Farrell 16 S M Farrington 169 P Farthouat 32 F Fassi 135e P Fassnacht 32 D. Fassouliotis,9 M. Faucci Giannelli,78 A. Favareto,52a,52b W. J. Fawcett,120 L. Fayard,117 O. L. Fedin,123,o W. Fedorko,167 S. Feigl,119 L. Feligioni,86 C. Feng,35d E. J. Feng,32 H. Feng,90 A. B. Fenyuk,130 L. Feremenga S. Fernandez Perez,13 J. Ferrando,55 A. Ferrari,164 P. Ferrari,107 R. Ferrari,121a D. E. Ferreira de Lima,59b A. Ferrer,166 D. Ferrere,51 C. Ferretti,90 A. Ferretto Parodi,52a,52b F. Fiedler,84 A. Filipčič,76 M. Filipuzzi,44 F. Filthaut,106 M. Fincke-Keeler,168 K. D. Finelli,150 M. C. N. Fiolhais,126a,126c L. Fiorini,166 A. Firan,42 A. Fischer,2 C. Fischer,13 J. Fischer,174 W. C. Fisher,91 N. Flaschel,44 I. Fleck,141 P. Fleischmann,90 G. T. Fletcher,139 R. R A. Floderus,82 L. R. Flores Castillo,61a M. J. Flowerdew,101 G. T. Forcolin,85 A. Formica,136 D. Fournier,117 H. Fox,73 S. Fracchia,13 P. Francavilla,81 M. Franchini,22a,22b D. Francis,32 L. Franconi,119 M. Franklin,58 M. Frate,162 M. Fraternali,121a,121b D. Freeborn,79 S. M. Fressard-Batraneanu,32 F. Friedrich,46 D. Froidevaux,32 J. A. Frost,120 C. Fukunaga,156 E. Fullana Torregrosa,84 T. Fusayasu,102 J. Fuster,166 C. Gabaldon,57 O. Gabizon,174 A. Gabrielli,22a,22b A. Gabrielli,16 G. P. Gach,40a S. Gadatsch,32 S. Gadomski,51 G. Gagliardi,52a,52b L. G. Gagnon,95 P. Gagnon,62 C. Galea,106 B. Galhardo,126a,126c E. J. Gallas,120 B. J. Gallop,131 P. Gallus,128 G. Galster,38 K. K. Gan,111 35b 86 48 143 48 166 166 16 g g g g P. F. Giraud,136 P. Giromini,58 D. Giugni,92a F. Giuli,120 C. Giuliani,101 M. Giulini,59b B. K. Gjelsten,119 S. Gkaitatzis,154 I. Gkialas,154 E. L. Gkougkousis,117 L. K. Gladilin,99 C. Glasman,83 J. Glatzer,50 P. C. F. Glaysher,48 A. Glazov,44 111802-8 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) M. Goblirsch-Kolb,25 J. Godlewski,41 S. Goldfarb,89 T. Golling,51 D. Golubkov,130 A. 111802-7 Henderson,73 Y. Heng,172 S. Henkelmann,167 A. M. Henriques Correia,32 S. Henrot-Versille,117 17 173 166 50 100 32 79 J. Henderson,120 R. C. W. Henderson,73 Y. Heng,172 S. Henkelmann,167 A. M. Henriques Correia,32 S. Henrot-Versille,117 G. H. Herbert,17 V. Herget,173 Y. Hernández Jiménez,166 G. Herten,50 R. Hertenberger,100 L. Hervas,32 G. G. Hesketh,79 N. P. Hessey,107 J. W. Hetherly,42 R. Hickling,77 E. Higón-Rodriguez,166 E. Hill,168 J. C. Hill,30 K. H. Hiller,44 S. J. Hillier,19 I. Hinchliffe,16 E. Hines,122 R. R. Hinman,16 M. Hirose,50 D. Hirschbuehl,174 J. Hobbs,148 N. Hod,159a M. C. Hodgkinson,139 N. P. Hessey,107 J. W. Hetherly,42 R. Hickling,77 E. Higón-Rodriguez,166 E. Hill,168 J. C. Hill,30 K. H. Hiller,44 S. J. Hillier,19 I Hi hliff 16 E Hi 122 R R Hi 16 M Hi 50 D Hi hb hl 174 J H bb 148 N H d 159a M C H d ki 139 P. Hodgson,139 A. Hoecker,32 M. R. Hoeferkamp,105 F. Hoenig,100 D. Hohn,23 T. R. Holmes,16 M. Homann,45 T. M. Hong,125 165 116 103 142 57 151 135a P. Hodgson,139 A. Hoecker,32 M. R. Hoeferkamp,105 F. Hoenig,100 D. Hohn,23 T. R. Holmes,16 M. Homann,45 T. M. Hong,125 B. H. Hooberman,165 W. H. Hopkins,116 Y. Horii,103 A. J. Horton,142 J-Y. Hostachy,57 S. Hou,151 A. Hoummada,135a J. Howarth,44 M. Hrabovsky,115 I. Hristova,17 J. Hrivnac,117 T. Hryn’ova,5 A. Hrynevich,94 C. Hsu,145c P. J. Hsu,151,u S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120 E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. Huth,58 G. Iacobucci,51 G. Iakovidis,27 I. Ibragimov,141 L. Iconomidou-Fayard,117 E. Ideal,175 Z. Idrissi,135e P. Iengo,32 O. Igonkina,107,v T. Iizawa,170 Y Ik i 67 M Ik 67 Y Il h k 11,w D Ili di 154 N Ili 143 T I 101 G I t i 121a,121b P I 9,a M I di 134a J. Howarth,44 M. Hrabovsky,115 I. Hristova,17 J. Hrivnac,117 T. Hryn’ova,5 A. Hrynevich,94 C. Hsu,145c P. J. Hsu,151,u S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120 E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. Huth,58 G. Iacobucci,51 G. Iakovidis,27 I. Ibragimov,141 L. Iconomidou-Fayard,117 E. Ideal,175 Z. Idrissi,135e P. Iengo,32 O. Igonkina,107,v T. Iizawa,170 G. Iakovidis, I. Ibragimov, L. Iconomidou Fayard, E. Ideal, Z. Idrissi, P. Iengo, O. Igonkina, T. Iizawa, Y. Ikegami,67 M. Ikeno,67 Y. 111802-7 Ilchenko,11,w D. Iliadis,154 N. Ilic,143 T. Ince,101 G. Introzzi,121a,121b P. Ioannou,9,a M. Iodice,134a Y. Ikegami,67 M. Ikeno,67 Y. Ilchenko,11,w D. Iliadis,154 N. Ilic,143 T. Ince,101 G. Introzzi,121a,121b P. Ioannou,9,a M. Iodice,134a K. Iordanidou,37 V. Ippolito,58 N. Ishijima,118 M. Ishino,155 M. Ishitsuka,157 R. Ishmukhametov,111 C. Issever,120 S. Istin,20a F. Ito,160 J. M. Iturbe Ponce,85 R. Iuppa,133a,133b W. Iwanski,41 H. Iwasaki,67 J. M. Izen,43 V. Izzo,104a S. Jabbar,3 S. Jiggins,79 J. Jimenez Pena,166 S. Jin,35a A. Jinaru,28b O. Jinnouchi,157 H. Jivan,145c P. Johansson,139 K. A. Johns,7 W. J. Johnson,138 K. Jon-And,146a,146b G. Jones,169 R. W. L. Jones,73 S. Jones,7 T. J. Jones,75 J. Jongmanns,59a P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117 H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. Kama,42 130 155 30 76 98 67 110 172 , , , , , , g , P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117 H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. Kama,42 130 155 30 76 98 67 110 172 A. Kamenshchikov,130 N. Kanaya,155 S. Kaneti,30 L. Kanjir,76 V. A. Kantserov,98 J. Kanzaki,67 B. Kaplan,110 L. S. Kaplan,172 A. Kapliy,33 D. Kar,145c K. Karakostas,10 A. Karamaoun,3 N. Karastathis,10 M. J. Kareem,56 E. Karentzos,10 M. Karnevskiy,84 S. N. Karpov,66 Z. M. Karpova,66 K. Karthik,110 V. Kartvelishvili,73 A. N. Ka L. Kashif,172 R. D. Kass,111 A. Kastanas,15 Y. Kataoka,155 C. Kato,155 A. Katre,51 J. Katzy,44 K. Kawagoe,71 T. Kawamoto,155 G. Kawamura,56 V. F. Kazanin,109,d R. Keeler,168 R. Kehoe,42 J. S. Keller,44 J. J. Kempster,78 K Kentaro,103 H. Keoshkerian,158 O. Kepka,127 B. P. Kerševan,76 S. Kersten,174 R. A. Keyes,88 M. Khader,165 F. Khalil-zada,12 A. Khanov,114 A. G. Kharlamov,109,d T. J. Khoo,51 V. Khovanskiy,97 E. Khramov,66 J. Khubua,53b,z S. Kido,68 C. R. Kilby,78 H. Y. Kim,8 S. H. Kim,160 Y. K. Kim,33 N. Kimura,154 O. M. Kind,17 B. T. King,75 M. King,166 S. B. King,167 J. Kirk,131 A. E. Kiryunin,101 T. Kishimoto,155 D. Kisielewska,40a F. Kiss,50 K. Kiuchi,160 O. Kivernyk,136 E. Kladiva,144b M. H. Klein,37 111802-9 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) PRL 117, 111802 (2016) M. Klein,75 U. Klein,75 K. Kleinknecht,84 P. Klimek,108 A. Klimentov,27 R. Klingenberg,45 J. A. Klinger,139 T. Klioutchnikova,32 E.-E. Kluge,59a P. Kluit,107 S. Kluth,101 J. Knapik,41 E. Kneringer,63 E. B. F. G. Knoops,86 A. Knue,55 A. Kobayashi,155 D. Kobayashi,157 T. Kobayashi,155 M. Kobel,46 M. Kocian,143 P. Kodys,129 N. M. Koehler,101 T. Koffas,31 E. Koffeman,107 T. Koi,143 H. Kolanoski,17 M. Kolb,59b I. Koletsou,5 A. A. Komar,96,a Y. Komori,155 T. Kondo,67 U. Kruchonak, H. Krüger, N. Krumnack, A. Kruse, M. C. Kruse, M. Kruskal, T. Kubota, H. Kucuk, S. Kuday,4b J. T. Kuechler,174 S. Kuehn,50 A. Kugel,59c F. Kuger,173 A. Kuhl,137 T. Kuhl,44 V. Kukhtin,66 R. Kukla,136 A. E. Loevschall-Jensen,38 K. M. Loew,25 A. Loginov,175,a T. Lohse,17 K. Lohwasser,44 M. Lokajicek,127 B. A. Long,24 J D L 165 R E L 73 L L 74a 74b K A L 111 L L 126a D L M 58 B L P d 139 g g g p p p p I. Lopez Paz,13 A. Lopez Solis,81 J. Lorenz,100 N. Lorenzo Martinez,62 M. Losada,21 P. J. Lösel,100 X. Lou,35a A. Lounis,117 g W. Lukas,63 L. Luminari,132a O. Lundberg,146a,146b B. Lund-Jensen,147 P. M. Luzi,81 D. Lynn,27 R. Lysak,127 E. Lytken,82 66 27 35d 35d 49 101 139 76 y , , , , , , , , J. Machado Miguens,122,126b D. Madaffari,86 R. Madar,36 H. J. Maddocks,164 W. F. Mader,46 A. Madsen,44 J. Maeda,68 15 27 99 56 107 117 26 101 S. Maeland,15 T. Maeno,27 A. Maevskiy,99 E. Magradze,56 J. Mahlstedt,107 C. Maiani,117 C. Maidantchik,26a A. A. Maier,101 T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41 123 57 64 6 143 10 32 166 S. Maeland,15 T. Maeno,27 A. Maevskiy,99 E. Magradze,56 J. Mahlstedt,107 C. Maiani,117 C. Maidantchik,26a A. A. Maier,101 T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41 V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166 G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. Manhaes de Andrade Filho,26b 159b 100 32 136 35 88 56 , , y, g , , , , , T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41 V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166 G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. PRL 117, 111802 (2016) Minashvili,66 A. M. Mineev,66 Y. Ming,172 L. M. Mir,13 K. P. Mistry,122 T. Mitani,170 J. Mitrevski,100 V. A. Mitsou,166 A. Miucci,51 P. S. Miyagawa,139 J. U. Mjörnmark,82 T. Moa,146a,146b K. Mochizuki,95 S. Mohapatra,37 S. Molander,146a,146b R. Moles-Valls,23 R. Monden,69 M. C. Mondragon,91 K. Mönig,44 J. Monk,38 E. Monnier,86 A. Montalbano,148 Valls,23 R. Monden,69 M. C. Mondragon,91 K. Mönig,44 J. Monk,38 E. Monnier,86 A. Montalb g g J. Montejo Berlingen,32 F. Monticelli,72 S. Monzani,92a,92b R. W. Moore,3 N. Morange,117 D. Moreno,21 M. Moreno Llácer,56 J. Montejo Berlingen,32 F. Monticelli,72 S. Monzani,92a,92b R. W. Moore,3 N. Morange,117 D P. Morettini,52a D. Mori,142 T. Mori,155 M. Morii,58 M. Morinaga,155 V. Morisbak,119 S. M 5 b G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. Motohashi,157 R. Mount,143 5 G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. E. Mountricha,27 S. V. Mouraviev,96,a E. J. W. Moyse,87 S. Muanza,86 R. D. Mudd,19 F. Mueller,101 J. Mueller,125 R. S. P. Mueller,100 T. Mueller,30 D. Muenstermann,73 P. Mullen,55 G. A. Mullier,18 F. J. Munoz Sanchez,85 B. P. Nachman,143 O. Nackenhorst,51 K. Nagai,120 R. Nagai,67,aa K. Nagano,67 Y. Nagasaka,60 K. Nagata,160 M. Nagel,50 E. Nagy,86 A. M. Nairz,32 Y. Nakahama,103 K. Nakamura,67 T. Nakamura,155 I. Nakano,112 H. Namasivayam,43 R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,123 T. Naumann,44 G. Navarro,21 R. Nayyar,7 S. Nemecek,127 P. Nemethy,110 A. A. Nepomuceno,26a M. Nessi,32,gg M. S. Neubauer,165 M. Neumann,174 R. M. Neves,110 2 19 6 95 120 136 13 A. Nikiforov,17 V. Nikolaenko,130,ff I. Nikolic-Audit,81 K. Nikolopoulos,19 J. K. Nilsen,119 P. Nilsson,27 Y. Ninomiya,155 A. Nisati,132a R. Nisius,101 T. Nobe,155 M. Nomachi,118 I. Nomidis,31 T. Nooney,77 S. Norberg,113 M. Nordberg,32 y g g N. Norjoharuddeen,120 O. Novgorodova,46 S. Nowak,101 M. Nozaki,67 L. Nozka,115 K. Ntekas,10 E. Nurse,79 F. Nuti,89 F. O’grady,7 D. C. O’Neil,142 A. A. O’Rourke,44 V. O’Shea,55 F. G. Oakham,31,e H. Oberlack,101 T. Obermann,23 J. Ocariz,81 A. Ochi,68 I. Ochoa,37 J. P. Ochoa-Ricoux,34a S. Oda,71 S. Odaka,67 H. Ogren,62 A. Oh,85 S. H. Oh,47 C. C. Ohm,16 H. Ohman,164 H. Oide,32 H. Okawa,160 Y. Okumura,155 T. Okuyama,67 A. Olariu,28b L. F. Oleiro Seabra,126a S. A. Olivares Pino,48 D. Oliveira Damazio,27 A. Olszewski,41 J. Olszowska,41 A. Onofre,126a,126e K. Onogi,103 P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr,158 B. Osculati,52a,52b R. Ospanov,85 G. PRL 117, 111802 (2016) Otero y Garzon,29 H. Otono,71 M. Ouchrif,135d F. Ould-Saada,119 A. Ouraou,136 K. P. Oussoren,107 Q. Ouyang,35a M. Owen,55 R. E. Owen,19 V. E. Ozcan,20a N. Ozturk,8 K. Pachal,142 A. Pacheco Pages,13 136 13 50 16 27 87 119 L. Pacheco Rodriguez,136 C. Padilla Aranda,13 M. Pagáčová,50 S. Pagan Griso,16 F. Paige,27 P. Pais,87 K. Pajchel,119 5 g , , g , g , g , , j , G. Palacino,159b S. Palazzo,39a,39b S. Palestini,32 M. Palka,40b D. Pallin,36 E. St. Panagiotopoulou,10 C. E. Pandini,81 J. G. Panduro Vazquez,78 P. Pani,146a,146b S. Panitkin,27 D. Pantea,28b L. Paolozzi,51 Th. D. Papadopoulou,10 K. Papageorgiou,154 A. Paramonov,6 D. Paredes Hernandez,175 A. J. Parker,73 M. A. Parker,30 K. A. Parker,139 F. Parodi,52a,52b J. A. Parsons,37 U. Parzefall,50 V. R. Pascuzzi,158 E. Pasqualucci,132a S. Passaggio,52a Fr. Pastore,78 G. Pásztor,31,hh S. Pataraia,174 J. R. Pater,85 T. Pauly,32 J. Pearce,168 B. Pearson,113 L. E. Pedersen,38 M. Pedersen,119 S. Pedraza Lopez,166 R. Pedro,126a,126b S. V. Peleganchuk,109,d O. Penc,127 C. Peng,35a H. Peng,35b J. Penwell,62 B. S. Peralva,26b M. M. Perego,136 D. V. Perepelitsa,27 E. Perez Codina,159a L. Perini,92a,92b H. Pernegger,32 S Perrella 104a,104b R Peschke 44 V D Peshekhonov 66 K Peters 44 R F Y Peters 85 B A Petersen 32 T C Petersen 38 g g g g j G. Palacino,159b S. Palazzo,39a,39b S. Palestini,32 M. Palka,40b D. Pallin,36 E. St. Panagiotopoulou,10 C. E. Pandini,81 J. G. Panduro Vazquez,78 P. Pani,146a,146b S. Panitkin,27 D. Pantea,28b L. Paolozzi,51 Th. D. Papadopoulou,10 K. Papageorgiou,154 A. Paramonov,6 D. Paredes Hernandez,175 A. J. Parker,73 M. A. Parker,30 K. A. Parker,139 F. Parodi,52a,52b J. A. Parsons,37 U. Parzefall,50 V. R. Pascuzzi,158 E. Pasqualucci,132a S. Passaggio,52a Fr. Pastore,78 G. Pásztor,31,hh S. Pataraia,174 J. R. Pater,85 T. Pauly,32 J. Pearce,168 B. Pearson,113 L. E. Pedersen,38 M. Pedersen,119 B. S. Peralva,26b M. M. Perego,136 D. V. Perepelitsa,27 E. Perez Codina,159a L. Perini,92a,92b H. Pernegger,32 S. Perrella,104a,104b R. Peschke,44 V. D. Peshekhonov,66 K. Peters,44 R. F. Y. Peters,85 B. A. Petersen,32 T. C. Petersen,38 E. Petit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134b N. E. Pettersson,87 etit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134 A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143 E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3 A. Pingel,38 S. Pires,81 H. PRL 117, 111802 (2016) Manhaes de Andrade Filho,26b J. Manjarres Ramos,159b A. Mann,100 A. Manousos,32 B. Mansoulie,136 J. D. Mansour,35a R. Mantifel,88 M. Mantoani,56 S. Manzoni,92a,92b L. Mapelli,32 G. Marceca,29 L. March,51 G. Marchiori,81 M. Marcisovsky,127 M. Marjanovic,14 D. E. Marley,90 F. Marroquim,26a S. P. Marsden,85 Z. Marshall,16 S. Marti-Garcia,166 B. Martin,91 T. A. Martin,169 V. J. Martin,48 B. Martin dit Latour,15 M. Martinez,13,s V. I. Martinez Outschoorn,165 S. Martin-Haugh,131 V. S. Martoiu,28b A. C. Martyniuk,79 M. Marx,138 A. Marzin,32 L. Masetti,84 T. Mashimo,155 R. Mashinistov,96 J. Masik,85 A. L. Maslennikov,109,d I. Massa,22a,22b L. Massa,22a,22b P. Mastrandrea,5 A. Mastroberardino,39a,39b T. Masubuchi,155 P. Mättig,174 J. Mattmann,84 J. Maurer,28b S. J. Maxfield,75 D. A. Maximov,109,d R. Mazini,151 S. M. Mazza,92a,92b N. C. Mc Fadden,105 G. Mc Goldrick,158 S. P. Mc Kee,90 A. McCarn,90 R. L. McCarthy,148 T. G. McCarthy,101 T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41 V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166 G M i i 49 B M d lli 32 L M d lli 92a I M dić 76 J M i 126a 126b L M h d A d d Filh 26b J. Manjarres Ramos,159b A. Mann,100 A. Manousos,32 B. Mansoulie,136 J. D. Mansour,35a R. Mantifel,88 M. Mantoani,56 S. Manzoni,92a,92b L. Mapelli,32 G. Marceca,29 L. March,51 G. Marchiori,81 M. Marcisovsky,127 M. Marjanovic,14 D. E. Marley,90 F. Marroquim,26a S. P. Marsden,85 Z. Marshall,16 S. Marti-Garcia,166 B. Martin,91 T. A. Martin,169 V. J. Martin,48 B. Martin dit Latour,15 M. Martinez,13,s V. I. Martinez Outschoorn,165 S. Martin-Haugh,131 V. S. Martoiu,28b A. C. Martyniuk,79 M. Marx,138 A. Marzin,32 L. Masetti,84 T. Mashimo,155 R. Mashinistov,96 J. Masik,85 A L Maslennikov 109,d I Massa 22a,22b L Massa 22a,22b P Mastrandrea 5 A Mastroberardino 39a,39b T Masubuchi 155 111802-10 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) L. I. McClymont,79 E. F. McDonald,89 J. A. Mcfayden,79 G. Mchedlidze,56 S. J. McMahon M. Medinnis,44 S. Meehan,138 S. Mehlhase,100 A. Mehta,75 K. Meier,59a C. Meineck,100 B. C. Meyer,122 J-P. Meyer,136 J. Meyer,107 H. Meyer Zu Theenhausen,59a F. Miano,149 R. P. Middle L. Mijović,48 G. Mikenberg,171 M. Mikestikova,127 M. Mikuž,76 M. Milesi,89 A. Milic,63 D A. Milov,171 D. A. Milstead,146a,146b A. A. Minaenko,130 Y. Minami,155 I. A. PRL 117, 111802 (2016) Riegel,174 J. Rieger,56 O. Rifki,113 g g M. Rijssenbeek,148 A. Rimoldi,121a,121b M. Rimoldi,18 L. Rinaldi,22a B. Ristić,51 E. Ritsch,32 I. Riu,13 F. Rizatdinova,114 E. Rizvi,77 C. Rizzi,13 S. H. Robertson,88,m A. Robichaud-Veronneau,88 D. Robinson,30 J. E. M C. Roda,124a,124b Y. Rodina,86 A. Rodriguez Perez,13 D. Rodriguez Rodriguez,166 S. Roe,32 C. S. Rogan,58 O. Røhne,119 98 22 22b 36 166 138 50 81 a,124b Y. Rodina,86 A. Rodriguez Perez,13 D. Rodriguez Rodriguez,166 S. Roe,32 C. S. Rogan,5 98 22 22b 36 166 138 A. Romaniouk,98 M. Romano,22a,22b S. M. Romano Saez,36 E. Romero Adam,166 N. Rompotis,1 A. Romaniouk,98 M. Romano,22a,22b S. M. Romano Saez,36 E. Romero Adam,166 N. Rompotis,138 M. Ronzani,50 L. Roos,81 E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rossetti,146a,146b E. Rossi,104a,104b p E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rossetti,146a,146b E. Rossi,104a,104b L. P. Rossi,52a J. H. N. Rosten,30 R. Rosten,138 M. Rotaru,28b I. Roth,171 J. Rothberg,138 D. Ro A. Rozanov,86 Y. Rozen,152 X. Ruan,145c F. Rubbo,143 M. S. Rudolph,158 F. Rühr,50 A. Ruiz-Martinez,31 Z. Rurikova,50 66 100 138 7 32 123 165 N. A. Rusakovich,66 A. Ruschke,100 H. L. Russell,138 J. P. Rutherfoord,7 N. Ruthmann,32 Y. F. Ryabov,123 M. Rybar,165 G. Rybkin,117 S. Ryu,6 A. Ryzhov,130 G. F. Rzehorz,56 A. F. Saavedra,150 G. Sabato,107 S. Sacerdoti,29 y y G. Rybkin,117 S. Ryu,6 A. Ryzhov,130 G. F. Rzehorz,56 A. F. Saavedra,150 G. Sabato,107 S. Sacerdoti,29 H. F-W. Sadrozinski,137 R. Sadykov,66 F. Safai Tehrani,132a P. Saha,108 M. Sahinsoy,59a M. Saimpert,136 T. Saito,155 H. Sakamoto,155 Y. Sakurai,170 G. Salamanna,134a,134b A. Salamon,133a,133b J. E. Salazar Loyola,34b D. Salek,107 H. F W. Sadrozinski, R. Sadykov, F. Safai Tehrani, P. Saha, M. Sahinsoy, M. Saimpert, T. Saito, H. Sakamoto,155 Y. Sakurai,170 G. Salamanna,134a,134b A. Salamon,133a,133b J. E. Salazar Loyola,34b D. Salek,107 P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Sa P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Salvatore,149 A. Salvucci,61a A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sanchez Martinez,166 A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sanchez Martinez,166 H. Sandaker, R. L. Sandbach, H. G. Sander, M. Sandhoff, C. Sandoval, R. Sandstroem, D. P. C. Sankey, M. Sannino,52a,52b A. Sansoni,49 C. Santoni,36 R. Santonico,133a,133b H. Santos,126a I. Santoyo Castillo,149 K. Sapp,125 y M. Sannino,52a,52b A. Sansoni,49 C. Santoni,36 R. Santonico,133a,133b H. PRL 117, 111802 (2016) Pirumov,44 M. Pitt,171 L. Plazak,144a M.-A. Pleier,27 V. Pleskot,84 E. Plotnikova,66 P. Plucinski,91 D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158 A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143 E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3 A. Pingel,38 S. Pires,81 H. Pirumov,44 M. Pitt,171 L. Plazak,144a M.-A. Pleier,27 V. Pleskot,84 E. Plotnikova,66 P. Plucinski,91 D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158 A. Polini,22a C. S. Pollard,55 V. Polychronakos,27 K. Pommès,32 L. Pontecorvo,132a B. G. Pope,91 G. A. Popeneciu,28c A. Poppleton,32 S. Pospisil,128 K. Potamianos,16 I. N. Potrap,66 C. J. Potter,30 C. T. Potter,116 G. Poulard,32 J. Poveda,32 A. Peyaud, R. Pezoa, P. W. Phillips, G. Piacquadio, E. Pianori, A. Picazio, E. Piccaro, M. Piccinini, M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3 111802-11 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) S. Prince,88 K. Prokofiev,61c F. Prokoshin,34b S. Protopopescu,27 J. Proudfoot,6 M. Przybyc p p y y M. Purohit,27,jj P. Puzo,117 J. Qian,90 G. Qin,55 Y. Qin,85 A. Quadt,56 W. B. Quayle,163a,163b M. Queitsch-Maitland,85 D Quilty 55 S Raddum 119 V Radeka 27 V Radescu 120 S K Radhakrishnan 148 P Radloff 116 P Rados 89 F Ragusa 92a,92b y G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. Ratti,92a,92b F. Rauscher,100 84 55 171 32 119 75 74 74b S. Rave,84 T. Ravenscroft,55 I. Ravinovich,171 M. Raymond,32 A. L. Read,119 N. P. Read D. M. Rebuzzi,121a,121b A. Redelbach,173 G. Redlinger,27 R. Reece,137 K. Reeves,43 L. Rehnisch,17 J. Reichert,122 H. Reisin,29 C. Rembser,32 H. Ren,35a M. Rescigno,132a S. Resconi,92a O. L. Rezanova,109,d P. Reznicek,129 R. Rezvani,95 R. Richter,101 S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. Riegel,174 J. Rieger,56 O. Rifki,113 C. Rembser, H. Ren, M. Rescigno, S. Resconi, O. L. Rezanova, P. Reznicek, R. Rezvani, R. Richter, S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. PRL 117, 111802 (2016) Santos,126a I. Santoyo Castillo,149 K. Sapp,125 A. Sapronov,66 J. G. Saraiva,126a,126d B. Sarrazin,23 O. Sasaki,67 Y. Sasaki,155 K. Sato,160 G. Sauvage,5,a E. Sauvan,5 G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79 p , , , , , , g , , G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79 162 150 39a 39b 171 101 100 p g G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79 D A Scannicchio 162 M Scarcella 150 V Scarfone 39a,39b J Schaarschmidt 171 P Schacht 101 B M Schachtner 100 D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. Schäfer,84 A. C. Schaffer,117 D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. S D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. Schäfer,84 A. C. Schaffer,117 100 148 59a 123 129 162 52a 52b D. Schaile,100 R. D. Schamberger,148 V. Scharf,59a V. A. Schegelsky,123 D. Scheirich,129 M. Sc S. Schier,137 C. Schillo,50 M. Schioppa,39a,39b S. Schlenker,32 K. R. Schmidt-Sommerfeld,101 K. Schmieden,32 C. Schmitt,84 S. Schmitt,44 S. Schmitz,84 B. Schneider,159a U. Schnoor,50 L. Schoeffel,136 A. Schoening,59b B. D. Schoenrock,91 E. Schopf,23 M. Schott,84 J. Schovancova,8 S. Schramm,51 M. Schreyer,173 N. Schuh,84 A. Schulte,84 M. J. Schultens,23 H.-C. Schultz-Coulon,59a H. Schulz,17 M. Schumacher,50 B. A. Schumm,137 Ph. Schune,136 A. Schwartzman,143 T. A. Schwarz,90 H. Schweiger,85 Ph. Schwemling,136 R. Schwienhorst,91 J. Schwindling,136 T. Schwindt,23 G. Sciolla,25 F. Scuri,124a,124b F. Scutti,89 J. Searcy,90 P. Seema,23 S. C. Seidel,105 A. Seiden,137 F. Seifert,128 J. M. Seixas,26a G. Sekhniaidze,104a K. Sekhon,90 S. J. Sekula,42 D. M. Seliverstov,123,a N. Semprini-Cesari,22a,22b C. Serfon,119 L. Serin,117 L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A. Sfyrla,51 E. Shabalina,56 N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b . Se , . Sessa, . Seuste , . Seve , . S goj, . S o a, . S y a, . S aba a, N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b 85 146 146b 149 9 151 kk 68 162 S. M. Shaw,85 A. PRL 117, 111802 (2016) Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,kk S. Shimizu,68 C. O. Shimmin,162 M Shi ji 102 M Shi k 66,ll A Sh l 96 D Sh l h S di 95 M J Sh h t 33 S Sh j ii 92a,92b S Sh th 111 S. M. Shaw,85 A. Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,kk S. Shimizu,68 C. O. Shimmin,162 M. Shimojima,102 M. Shiyakova,66,ll A. Shmeleva,96 D. Shoaleh Saadi,95 M. J. Shochet,33 S. Shojaii,92a,92b S. Shrestha,111 j y j E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D. Sidorov,114 A. Sidoti,22a,22b E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D F. Siegert,46 Dj. Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S F. Siegert,46 Dj. Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S. Simion,117 E. Simioni,84 B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Siragusa,173 S. Yu. Sivoklokov,99 J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161 R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98 L. N. Smirnova,99,mm O. Smirnova,82 M. N. K. Smith,37 R. W. Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Snesarev,96 S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128 E. Yu. Soldatov,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V. Solovyev,123 P. Sommer,50 H. Son,161 H. Y. Song,35b,nn A. Sood,16 A. Sopczak,128 V. Sopko,128 V. Sorin,13 D. Sosa,59b C. L. Sotiropoulou,124a,124b R. Soualah,163a,163c A. M. Soukharev,109,d D. South,44 B. C. Sowden,78 S. Spagnolo,74a,74b M. Spalla,124a,124b B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Siragusa,173 S. Yu. Sivoklokov,99 B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Si J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161 R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98 J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161 R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98 L. N. Smirnova,99,mm O. Smirnova,82 M. N. K. Smith,37 R. W. PRL 117, 111802 (2016) Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Snesarev,96 S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128 98 166 130 66 130 123 50 E. Yu. Soldatov,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V H. Son,161 H. Y. Song,35b,nn A. Sood,16 A. Sopczak,128 V. Sopko,128 V. Sorin,13 D. Sosa,59b C. L. Sotiropoulou,124a,124b R. Soualah,163a,163c A. M. Soukharev,109,d D. South,44 B. C. Sowden,78 S. Spagnolo,74a,74b M. Spalla,124a,124b 111802-12 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) N. Taiblum,153 H. Takai,27 R. Takashima,70 T. Takeshita,140 Y. Takubo,67 M. Talby,86 A. A. Talyshev,109,d K. G. Tan,89 J. Tanaka,155 M. Tanaka,157 R. Tanaka,117 S. Tanaka,67 B. B. Tannenwald,111 S. Tapia Araya,34b S. Tapprogge,84 S. Tarem,152 G. F. Tartarelli,92a P. Tas,129 M. Tasevsky,127 T. Tashiro,69 E. Tassi,39a,39b A. Tavares Delgado,126a,126b Y. Tayalati,135e A. C. Taylor,105 G. N. Taylor,89 P. T. E. Taylor,89 W. Taylor,159b F. A. Teischinger,32 P. Teixeir 142 32 151 118 1 4 6 D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,101 49 158 86 19 8 3 D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,101 M Testa 49 R J Teuscher 158,m T Theveneaux Pelzer 86 J P Thomas 19 J Thomas Wilsker 78 E N Thompson 37 M. Testa,49 R. J. Teuscher,158,m T. Theveneaux-Pelzer,86 J. P. Thomas,19 J. Thomas-Wilsker,78 E. N. Thompson,37 P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16 R. E. Ticse Torres,86 V. O. Tikhomirov,96,oo Yu. A. Tikhonov,109,d S. Timoshenko,98 P. Tipton,175 S. Tisserant,86 P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16 86 96 109 d 98 175 86 p K. Todome,157 T. Todorov,5,a S. Todorova-Nova,129 J. Tojo,71 S. Tokár,144a K. Tokushuku,67 E. Tolley,58 L. Tomlinson,85 j M. Tomoto,103 L. Tompkins,143,pp K. Toms,105 B. Tong,58 E. Torrence,116 H. Torres,142 E. To F. Touchard,86 D. R. Tovey,139 T. Trefzger,173 A. Tricoli,27 I. M. Trigger,159a S. Trincaz-Duvoid,81 M. F. Tripiana,13 W. Trischuk,158 B. Trocmé,57 A. Trofymov,44 C. Troncon,92a M. Trottier-McDonald,16 M. Trovatelli,168 L. Truong,163a,163c M. Trzebinski,41 A. PRL 117, 111802 (2016) Trzupek,41 J. C-L. Tseng,120 P. V. Tsiareshka,93 G. Tsipolitis,10 N. Tsirintanis,9 S. Tsiskaridze,13 V. Tsiskaridze,50 E. G. Tskhadadze,53a K. M. Tsui,61a I. I. Tsukerman,97 V. Tsulaia,16 S. Tsuno,67 D. Tsybychev,148 Y. Tu,61b y y A. Tudorache,28b V. Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Turecek,128 D. Turgeman,171 R Turra 92a,92b A J Turvey 42 P M Tuts 37 M Tyndel 131 G Ucchielli 22a,22b I Ueda 155 M Ughetto 146a,146b F Ukegawa 160 y y A. Tudorache,28b V. Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Turecek,128 D. Turgeman,171 R. Turra,92a,92b A. J. Turvey,42 P. M. Tuts,37 M. Tyndel,131 G. Ucchielli,22a,22b I. Ueda,155 M. Ugh G. Unal,32 A. Undrus,27 G. Unel,162 F. C. Ungaro,89 Y. Unno,67 C. Unverdorben,100 J. Urban,144b P. Urquijo,89 P. Urrejola,84 G. Usai,8 A. Usanova,63 L. Vacavant,86 V. Vacek,128 B. Vachon,88 C. Valderanis,100 E. Valdes Santurio,146a,146b N. Valencic,107 S. Valentinetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. Van Den Wollenberg,107 5 N. Valencic,107 S. Valentinetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. Van Den Wollenberg,107 P C Van Der Deijl 107 H van der Graaf 107 N van Eldik 152 P van Gemmeren 6 J Van Nieuwkoop 142 I van Vulpen 107 P. C. Van Der Deijl,107 H. van der Graaf,107 N. van Eldik,152 P. van Gemmeren,6 J. Van Nieuw M. C. van Woerden,32 M. Vanadia,132a,132b W. Vandelli,32 R. Vanguri,122 A. Vaniachine,130 P. Vankov,107 G. Vardanyan,176 132 7 42 81 8 150 175 36 R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36 T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a 74 74b 168 158 25 121 132 132b 107 R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36 T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107 J C V l 107 A V 46 rr M C V li 142 e O Vi l 82 I Vi h 165 a T Vi k 139 O E Vi k B i 139 q g A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107 J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. PRL 117, 111802 (2016) Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56 C. Weiser,50 H. Weits,107 P. S. Wells,32 T. Wenaus,27 T. Wengler,32 S. Wenig,32 N. Wermes,23 M. Werner,50 M. D. Werner,65 P. Werner,32 M. Wessels,59a J. Wetter,161 K. Whalen,116 N. L. Whallon,138 A. M. Wharton,73 A. White,8 M. J. White,1 R. White,34b D. Whiteson,162 F. J. Wickens,131 W. Wiedenmann,172 M. Wielers,131 P. Wienemann,23 C. Wiglesworth,38 L. A. M. Wiik-Fuchs,23 A. Wildauer,101 F. Wilk,85 H. G. Wilkens,32 H. H. Williams,122 S. Williams,107 C. Willis,91 S. Willocq,87 J. A. Wilson,19 I. Wingerter-Seez,5 F. Winklmeier,116 O. J. Winston,149 B. T. Winter,23 M. Wittgen,143 R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30 D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85 D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85 B. M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56 50 107 32 27 32 32 23 50 65 p , , , , , , , B. M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56 111802-13 PRL 117, 111802 (2016) Viazlo,82 I. Vichou,165,a T. Vickey,139 O. E. Vickey Boeriu,139 120 16 120 22a 22b 92a 92b 49 31 g M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke, A. Ventura, M. Venturi, N. Venturi, A. Venturini, V. Vercesi, M. Verducci, W. Verkerke, J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. Viazlo,82 I. Vichou,165,a T. Vickey,139 O. E. Vickey Boeriu,139 120 16 120 22 22b 92 92b 49 31 J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. Viazlo,82 I. Vichou,165,a T. Vickey,139 O G. H. A. Viehhauser,120 S. Viel,16 L. Vigani,120 M. Villa,22a,22b M. Villaplana Perez,92a,92b E. V g p V. B. Vinogradov,66 C. Vittori,22a,22b I. Vivarelli,149 S. Vlachos,10 M. Vlasak,128 M. Vogel,174 P. Vokac,128 G. Volpi,124a,124b M. Volpi,89 H. von der Schmitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75 14 14 127 107 32 33 128 M. Volpi,89 H. von der Schmitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75 N. Vranjes,14 M. Vranjes Milosavljevic,14 V. Vrba,127 M. Vreeswijk,107 R. Vuillermet,32 I. Vukotic,33 Z. Vykydal,128 mitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75 j , j j , , j , , , y y , P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141 V. Wallangen,146a,146b C. Wang,35c C. Wang,35d,86 F. Wang,172 H. Wang,16 H. Wang,42 J. Wang,44 J. Wang,150 K. Wang,88 R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30 D R Wardrope 79 A Washbrook 48 P M Watkins 19 A T Watson 19 M F Watson 19 G Watts 138 S Watts 85 j , j j , , j , , , y y , P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141 V W ll 146a,146b C W 35c C W 35d,86 F W 172 H W 16 H W 42 J W 44 J W 150 K W 88 g , g, g, g, g, g, g, g, g, R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30 D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85 B. M. Waugh,79 S. 111802-13 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) J. Wittkowski,100 T. M. H. Wolf,107 M. W. Wolter,41 H. Wolters,126a,126c S. D. Worm,131 B. K. Wosiek,41 J. Wotschack,32 M. J. Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48 S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67 S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15 W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58 E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8 J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,ss B. Zabinski,41 R. Zaidan,35d A. M. Zaitsev,130,ff N. Zakharchuk,44 J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,nn H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,tt X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84 S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b M zur Nedden 17 and L Zwalinski32 M. zur Nedden,17 and L. Zwalinski32 J. Wittkowski,100 T. M. H. Wolf,107 M. W. Wolter,41 H. Wolters,126a,126c S. D. Worm,131 B. K. Wosiek,41 J. Wotschack,32 M. J. Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48 S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67 S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15 W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58 E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8 J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,ss B. Zabinski,41 R. Zaidan,35d A. M. Zaitsev,130,ff N. Zakharchuk,44 J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,nn H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,tt X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84 S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b M zur Nedden 17 and L Zwalinski32 (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, The University of Texas at Austin, Austin, Texas, USA 12Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain 14Institute of Physics, University of Belgrade, Belgrade, Serbia 15Department for Physics and Technology, University of Bergen, Bergen, Norway 16Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA 17Department of Physics, Humboldt University, Berlin, Germany 18Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20aDepartment of Physics, Bogazici University, Istanbul, Turkey 20bDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 20cIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 20dBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 21Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22aINFN Sezione di Bologna, Italy 22bDipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 23Physikalisches Institut, University of Bonn, Bonn, Germany 24Department of Physics, Boston University, Boston, Massachusetts, USA 25Department of Physics, Brandeis University, Waltham, Massachusetts, USA 26aUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 26bElectrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil 26cFederal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil 26dInstituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 27Physics Department, Brookhaven National Laboratory, Upton, New York, USA 28aTransilvania University of Brasov, Brasov, Romania, Romania 28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 1Department of Physics, University of Adelaide, Adelaide, Australia 2 1Department of Physics, University of Adelaide, Adelaide, Australia 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, The University of Texas at Austin, Austin, Texas, USA 12Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain 14Institute of Physics, University of Belgrade, Belgrade, Serbia 15Department for Physics and Technology, University of Bergen, Bergen, Norway 16Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA 17Department of Physics, Humboldt University, Berlin, Germany 18Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20aDepartment of Physics, Bogazici University, Istanbul, Turkey 20bDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 20cIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 20dBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 21Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22aINFN Sezione di Bologna, Italy 22bDipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 23Physikalisches Institut, University of Bonn, Bonn, Germany 24Department of Physics, Boston University, Boston, Massachusetts, USA 25Department of Physics, Brandeis University, Waltham, Massachusetts, USA 26aUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 26bElectrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil 26cFederal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil 26dInstituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 27Physics Department, Brookhaven National Laboratory, Upton, New York, USA 28aTransilvania University of Brasov, Brasov, Romania, Romania 28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey al Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 2aINFN Sezione di Bologna, Italy 28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 111802-14 111802-14 52aINFN Sezione di Genova, Italy 52bDipartimento di Fisica, Università di Genova, Genova, Italy shvili Institute of Physics, Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia b 53bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54 54II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 111802-15 44DESY, Hamburg and Zeuthen, Germany 45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46 45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United 49 49INFN Laboratori Nazionali di Frascati, Frascati, Italy 50 50Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 51 51Section de Physique, Université de Genève, Geneva, Switzerland 52 111802-14 Andronikashvili Institute of Physics, Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia 53bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 57Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 58Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 59aKirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 59bPhysikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 59cZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 60Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 61aDepartment of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 61bDepartment of Physics, The University of Hong Kong, Hong Kong, China epartment of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, C 62Department of Physics, Indiana University, Bloomington, Indiana, USA 63Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 64University of Iowa, Iowa City, Iowa, USA 65Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 66Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 67KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 68Graduate School of Science, Kobe University, Kobe, Japan L 117, 111802 (2016) 9 SEPTEMBER 28cNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca, Romania 28d 36Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37Nevis Laboratory, Columbia University, Irvington, New York, USA 38 38Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 9aINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 39b 39bDipartimento di Fisica, Università della Calabria, Rende, Italy 0aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 40aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41 41Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 42 42Physics Department, Southern Methodist University, Dallas, Texas, USA 3 42Physics Department, Southern Methodist University, Dallas, Texas, USA 3 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA 44 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA 44 111802-14 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) 28cNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca, Romania 28dUniversity Politehnica Bucharest, Bucharest, Romania 28eWest University in Timisoara, Timisoara, Romania 29Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 30Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31Department of Physics, Carleton University, Ottawa, Ontario, Canada 32CERN, Geneva, Switzerland 33Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA 34aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 34bDepartamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 35aInstitute of High Energy Physics, Chinese Academy of Sciences, Beijing, China 35bDepartment of Modern Physics, University of Science and Technology of China, Anhui, China 35cDepartment of Physics, Nanjing University, Jiangsu, China 35dSchool of Physics, Shandong University, Shandong, China 35eDepartment of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University, Shanghai; (also affiliated with PKU-CHEP), China 35fPhysics Department, Tsinghua University, Beijing 100084, China 36Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37Nevis Laboratory, Columbia University, Irvington, New York, USA 38Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 39aINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 39bDipartimento di Fisica, Università della Calabria, Rende, Italy 40aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 42Physics Department, Southern Methodist University, Dallas, Texas, USA 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA 44DESY, Hamburg and Zeuthen, Germany 45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49INFN Laboratori Nazionali di Frascati, Frascati, Italy 50Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 51Section de Physique, Université de Genève, Geneva, Switzerland 52aINFN Sezione di Genova, Italy 52bDipartimento di Fisica, Università di Genova, Genova, Italy 53aE. P H Y S I C A L R E V I E W L E T T E R S P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) 69Faculty of Science, Kyoto University, Kyoto, Japan 70Kyoto University of Education, Kyoto, Japan 71Department of Physics, Kyushu University, Fukuoka, Japan 72Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 73Physics Department, Lancaster University, Lancaster, United Kingdom 74aINFN Sezione di Lecce, Italy 74bDipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 75Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 76Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia 77School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 78Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 79Department of Physics and Astronomy, University College London, London, United Kingdom 80Louisiana Tech University, Ruston, Louisiana, USA 81Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, Fr 82Fysiska institutionen, Lunds universitet, Lund, Sweden 83Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 84Institut für Physik, Universität Mainz, Mainz, Germany 85School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 86CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 87Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 88Department of Physics, McGill University, Montreal, Québec, Canada 89School of Physics, University of Melbourne, Victoria, Australia 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA 91Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 92aINFN Sezione di Milano, Italy 92bDipartimento di Fisica, Università di Milano, Milano, Italy 93B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 94National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus 95Group of Particle Physics, University of Montreal, Montreal, Québec, Canada 96P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 97Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 98National Research Nuclear University MEPhI, Moscow, Russia 99D.V. Skobeltsyn Institute of Nuclear Physics, M.V. P H Y S I C A L R E V I E W L E T T E R S Lomonosov Moscow State University, Moscow, Russia 100Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 102Nagasaki Institute of Applied Science, Nagasaki, Japan 103Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 104aINFN Sezione di Napoli, Italy 104bDipartimento di Fisica, Università di Napoli, Napoli, Italy 105Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 106Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherland 107Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 109Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 110Department of Physics, New York University, New York, New York, USA 111Ohio State University, Columbus, Ohio, USA 112Faculty of Science, Okayama University, Okayama, Japan 113Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 115Palacký University, RCPTM, Olomouc, Czech Republic 116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 117LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 118Graduate School of Science, Osaka University, Osaka, Japan 119Department of Physics, University of Oslo, Oslo, Norway 120Department of Physics, Oxford University, Oxford, United Kingdom 121aINFN Sezione di Pavia, Italy 121bDipartimento di Fisica, Università di Pavia, Pavia, Italy 122Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Ru 124aINFN Sezione di Pisa, Italy PRL 117, 111802 (2016) P H Y S I C A L R E V I E W L E T T E R S week ending 9 SEPTEMBER 70Kyoto University of Education, Kyoto, Japan 81Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 82 de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 84 cs and Astronomy, University of Manchester, Manchester, United Kingdom 88Department of Physics, McGill University, Montreal, Québec, Canada 89 89School of Physics, University of Melbourne, Victoria, Australia 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA rtment of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 92 Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 92 111802-15 week ending 9 SEPTEMBER 2016 92aINFN Sezione di Milano, Italy ento di Fisica, Università di Milano, Milano, Italy 92bDipartimento di Fisica, Università di Milano, Milano, Italy 100Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 101 101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 102 102Nagasaki Institute of Applied Science, Nagasaki, Japan 3Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 104a Casablanca, Morocco 35bCentre National de l’Energie des Sciences Techniques Nucleaires, Rabat, Morocco 5 135cFaculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocc 135d 5dFaculté des Sciences, Université Mohamed Premier and LPTPM, Oujda, Morocco 135 135eFaculté des sciences, Université Mohammed V, Rabat, Morocco sciences, Université Mohammed V, Rabat, Morocco erches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et 136DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commis aux Energies Alternatives), Gif-sur-Yvette, France aux Energies Alternatives), Gif-sur-Yvette, France aux Energies Alternatives), Gif-sur-Yvette, France g f ute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA g f Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 138 137Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, Cal 138 ticle Physics, University of California Santa Cruz, 138Department of Physics, University of Washington, Seattle, Washington, USA 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 140 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom rtment of Physics, Shinshu University, Nagano, Jap 141Fachbereich Physik, Universität Siegen, Siegen, Germany 141Fachbereich Physik, Universität Siegen, Siegen, Germany of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 143SLAC National Accelerator Laboratory, Stanford, California, USA 144aFaculty of Mathematics, Physics & Informatics, Comenius University, Bratislava, Slovak Republic 144b p f y f p y f y f p 145aDepartment of Physics, University of Cape Town, Cape Town, South Africa 145b 145bDepartment of Physics, University of Johannesburg, Johannesburg, South Africa 145 145cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 146 46aDepartment of Physics, Stockholm University, Sweden 146b 46bThe Oskar Klein Centre, Stockholm, Sweden 147Physics Department, Royal Institute of Technology, Stockholm, Sweden 149Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 150 150School of Physics, University of Sydney, Sydney, Australia 151 151Institute of Physics, Academia Sinica, Taipei, Taiwan 152Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 154Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece for Elementary Particle Physics and Department of Physics, The University of Tokyo, T uate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 157 157Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 8 158Department of Physics, University of Toronto, Toronto, Ontario, Canada 159 159aTRIUMF, Vancouver, British Columbia, Canada 59bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering 159bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, p f y y y 160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 111802-17 4aINFN Sezione di Napoli, Italy 104bDipartimento di Fisica, Università di Napoli, Napoli, Italy 5Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA r Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Ne l Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 109 e of Nuclear Physics, SB RAS, Novosibirsk, Russia 110Department of Physics, New York University, New York, New York, USA 111 111Ohio State University, Columbus, Ohio, USA 112Faculty of Science, Okayama University, Okayama, Japan 113Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 114D f Ph i Okl h S U i i S ill Okl h USA dge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 4 114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 115 115Palacký University, RCPTM, Olomouc, Czech Republic 116 115Palacký University, RCPTM, Olomouc, Czech Republic 116 cký University, RCPTM, Olomouc, Czech Republic 116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 117 117LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 118 118Graduate School of Science, Osaka University, Osaka, Japan 119 119Department of Physics, University of Oslo, Oslo, Norway 120Department of Physics, Oxford University, Oxford, United Kingdom 121 122Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 124aINFN Sezione di Pisa, Italy 111802-16 week ending 9 SEPTEMBER 2016 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) y 132bDipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 133 133aINFN Sezione di Roma Tor Vergata, Italy 133bDipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 134 bDipartimento di Matematica e Fisica, Università R p , , , y 135aFaculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Uni 135aFaculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, P H Y S I C A L R E V I E W L E T T E R S wAlso at Department of Physics, The University of Texas at Austin, Austin TX, US x te of Theoretical Physics, Ilia State University, Tbil xAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. retical Physics, Ilia State University, Tbilisi, Georgi yAlso at CERN, Geneva, Switzerland. yAlso at CERN, Geneva, Switzerland. zAlso at Georgian Technical University (GTU),Tbilisi, Georgia. zAlso at Georgian Technical University (GTU),Tbilisi, Georgia. zAlso at Georgian Technical University aaAlso at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. bb aaAlso at Ochadai Academic Production, Ochanomizu University, To bb bbAlso at Manhattan College, New York NY, USA. bbAlso at Manhattan College, New York NY, USA. cAlso at Hellenic Open University, Patras, Greece. d p y ddAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. eeAlso at School of Physics, Shandong University, Shandong, China. ff eeAlso at School of Physics, Shandong University, Shandong, China. ff ffAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Ru ggAlso at Section de Physique, Université de Genève, Geneva, Switzerland. hh o at Section de Physique, Université de Genève, Ge hhAlso at Eotvos Lorand University, Budapest, Hungary. jjAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, USA. kk jjAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, USA. kk Department of Physics and Astronomy, University o kkAlso at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ll y g g, y, g , llAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Sofia, Bulgaria. mmAlso at Faculty of Physics M VLomonosov Moscow State University Moscow Russia llAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Aca mmAlso at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. 111802-18 P H Y S I C A L R E V I E W L E T T E R S fAlso at Department of Physics & Astronomy, University of Louisville, Louisville, KY, USA. hysics, California State University, Fresno CA, USA hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i iAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. j iAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barce j jAlso at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portug k jAlso at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Unive k jAlso at Departamento de Fisica e Astronomia, Fac k kAlso at Tomsk State University, Tomsk, Russia. kAlso at Tomsk State University, Tomsk, Russia. lAlso at Universita di Napoli Parthenope, Napoli, I mAlso at Institute of Particle Physics (IPP), Canada mAlso at Institute of Particle Physics (IPP), Canada. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania oAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Pe oAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russi pAlso at Department of Physics, The University of Michigan, Ann Arbor MI, USA. pAlso at Department of Physics, The University of Michigan, Ann Arbor MI, USA. qAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Afr Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. rAlso at Louisiana Tech University, Ruston LA, USA. rAlso at Louisiana Tech University, Ruston LA, USA. rAlso at Louisiana Tech University, Ruston LA, USA. Also at Louisiana Tech University, Ruston LA, USA y sAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. t tAlso at Graduate School of Science, Osaka University, Osaka, Japan. tAlso at Graduate School of Science, Osaka University, Osaka, Japan. uAlso at Department of Physics, National Tsing Hua University, Taiwan. uAlso at Department of Physics, National Tsing Hua University, Taiwa vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud Universi Netherlands. 111802-17 week ending 9 SEPTEMBER 2016 P H Y S I C A L R E V I E W L E T T E R S PRL 117, 111802 (2016) 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 163bICTP, Trieste, Italy 163cDipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 164Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 165Department of Physics, University of Illinois, Urbana, Illinois, USA 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 167Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada 168Department of Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada 169Department of Physics, University of Warwick, Coventry, United Kingdom 170Waseda University, Tokyo, Japan 171Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 172Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 173Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 174Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 175Department of Physics, Yale University, New Haven, Connecticut, USA 176 165Department of Physics, University of Illinois, Urbana, Illinois, USA 66 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Department of Physics, University of Warwick, Coventry, United Kingdom 170 71Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 172 172Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 173 173Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany thematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal 175 f f g pp y g pp 175Department of Physics, Yale University, New Haven, Connecticut, USA 176 175Department of Physics, Yale University, New Haven, Connecticut, USA 176 176Yerevan Physics Institute, Yerevan, Armenia 76Yerevan Physics Institute, Yerevan, Armenia 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. d dAlso at Novosibirsk State University, Novosibirsk, Russia. dAlso at Novosibirsk State University, Novosibirsk, Russia. eAlso at TRIUMF, Vancouver BC, Canada. f eAlso at TRIUMF, Vancouver BC, Canada. f fAlso at Department of Physics & Astronomy, University of Louisville, Louisville, KY, USA. 111802-18 week ending 9 SEPTEMBER 2016 PRL 117, 111802 (2016) nnAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. ooAlso at National Research Nuclear University MEPhI, Moscow, National Research Nuclear University MEPhI, Mos ppAlso at Department of Physics, Stanford University, Stanford CA, USA. at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary qqAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physi rrAlso at Flensburg University of Applied Sciences, Flensburg, Germany. ssAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. tt ssAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. tt ttAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. 111802-19 111802-19 ttAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. p y , y, , qqAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. rr
https://openalex.org/W4281567814	https://zenodo.org/records/6583555/files/DIS012.pdf	Swahili	null	FIZIKA TA`LIMIDA KOMPETENSIYALARNI RIVOJLANTIRISHGA QARATILGAN OMILLAR	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	950	DEVELOPMENT AND INNOVATIONS IN SCIENCE International scientific-online conference tug`dirishi mumkin bo`lgan ishlarni robot texnikasi tomonidan bajarilishi yuzasidan qator ilmiy izlanish ishlari olib borilmoqda. Internet tarmog`ida ko`plab manbalarda mehnat bozorining kelgusidagi rivojlanishiga doir tadqiqotlar e`lon qilinib, ayrim kasblar yaqin 15-20 yildan keyin yo`qolib ketishi yoki ularga talab keskin kamayishi, va aksincha, ba`zi kasblar va faoliyat turlariga ehtiyoj oshishi yoki ular talabgor bo`lishi kutilmoqda. Shubxasiz, kelajakda aksariyat jarayonlar va xizmatlar Internet tarmog`i, axborot tizimlari va ularga bog`liq yechimlar orqali amalga oshirilishi yoshlarimizdan va hozirgi maktab o`quvchilaridan XXI asrga doir bilim va ko`nikmalarni egallashini taqozo etadi. Bu borada qator davlatlarda ta`lim jarayonlarida yaqin istiqbolda kerak bo`ladigan bilim va ko`nikmalarni shakllantirishga doir yechimlar amaliyotga joriy etilmoqda. tug`dirishi mumkin bo`lgan ishlarni robot texnikasi tomonidan bajarilishi yuzasidan qator ilmiy izlanish ishlari olib borilmoqda. Internet tarmog`ida ko`plab manbalarda mehnat bozorining kelgusidagi rivojlanishiga doir tadqiqotlar e`lon qilinib, ayrim kasblar yaqin 15-20 yildan keyin yo`qolib ketishi yoki ularga talab keskin kamayishi, va aksincha, ba`zi kasblar va faoliyat turlariga ehtiyoj oshishi yoki ular talabgor bo`lishi kutilmoqda. Shubxasiz, kelajakda aksariyat jarayonlar va xizmatlar Internet tarmog`i, axborot tizimlari va ularga bog`liq yechimlar orqali amalga oshirilishi yoshlarimizdan va hozirgi maktab o`quvchilaridan XXI asrga doir bilim va ko`nikmalarni egallashini taqozo etadi. Bu borada qator davlatlarda ta`lim jarayonlarida yaqin istiqbolda kerak bo`ladigan bilim va ko`nikmalarni shakllantirishga doir yechimlar amaliyotga joriy etilmoqda. Hozirgi vaqtda butun dunyo miqiyosida fizika ta`limini takomillashtirish bo`yicha intensiv ishlar amalga oshirilmoqda: fizika o`qitishning maqsadlari, o`quv materiali mazmunini tanlash tamoyillari aniqlashtirilayapti, darsliklar va boshqa o`quv vositalarini modernizatsiya qilish ishlari olib borilmoqda, o`qitishning samarali shakllari va metodlari ishlab chiqilmoqda. Bu jarayon hozirgi vaqtdagi fanning, fan va texnika o`rtasidagi o`zaro aloqalarning rivojlanishi, jamiyatning barcha sohalariga yangi axborot texnologiyalarining keng joriy etilishiga asoslangan ilmiy-texnik revolyusiyaning natijalarini o`z ichiga qamrab oladi. Shu bilan bog`liq ravishda maktabda fizika o`qitishning maqsadlari ham o`zgarmoqda. Tegishli axborotlarni egallashning kuchayishi esa o`quv materiallari strukturasini takomillashtirishni talab etmoqda. Bu esa o`quvchilarning fizikaning umumiy prinsiplari va qonunlarini o`zlashtirish darajalariga va ularni nazariy fikrlash metodlari asosida egallashlariga o`z ta`sirini ko`rsatadi. Ko`pgina mamlakatlarda fizika o`qitishga qo`yiladigan maqsadlar deyarli bir xil: o`quv fani bo`yicha ilmiy va texnik savodxonlik asoslarini shakllantirish; ijodiy qobiliyatlarni rivojlantirishni ta`minlash, ilmiy fikrlashni shakllantirish, turli manbalar (adabiyotlar, eksperimentlar va h.k) yordamida mustaqil bilim egallash ko`nikmalarini rivojlantirish. Deyarli barcha mamlakatlarda o`qituvchilar faoliyati xarakterini o`zgartirish harakatlari yaqqoli ko`zga tashlanmoqda: bilimlarni oddiy usulda berishdan bilish jarayonini boshqarishga o`rgatishga o`tilmoqda. Asosiy e`tibor o`quvchilar bilish faoliyatlarini ta`minlovchi darsliklar mazmunini o`zgartirishga (eksperimentlar, turlicha mustaqil topshiriqlar va boshqalarni ko`paytirish), o`quv jarayonini tashkil qilishning shakllari, usullari va metodlariga qaratilmoqda. O'ktamova Sevara G'ayrat qizi G'ijduvon tuman 1-son kasb hunar maktabi Fizika fani o'qituvchisi https://doi.org/10.5281/zenodo.6583555 p // g/ / Annotatsiya: fizika ta`limini takomillashtirish bo`yicha olib borilayotgan intensiv ishlar, fizika o`qitishning maqsad va vazifalari keltirilgan. Kalit so`zlar: modernizatsiya, kompetentsiya, axborot tizimi, intellektual salohiyatni, ta`lim oluvchi, avtomatlashtirish. y Umumiy o’rta ta’lim maktablari o’quvchilarining ilmiy dunyoqarashi, mantiqiy fikrlashi, kasbiy tayyorgarligi va shaxsiy o’sishlarida fizika fani o’ziga xos o’rin tutadi.XXI asrda dunyoning rivojlangan davlatlarida ta`lim mazmunini modernizatsiya qilishning asosiy yo`nalishlaridan biri sifatida ta`limda kompetensiyaviy yondashuvni joriy etish masalasiga asosiy e`tibor qaratilmoqda. Kompetensiyaviy yondashuvga asoslangan ta`lim o`quvchilarda egallangan bilim, ko`nikma va malakalarini o`z shaxsiy, kasbiy va ijtimoiy faoliyatlarida amaliy qo`llay olish layoqatlarini shakllantirishga yo`naltirilgan ta`lim hisoblanadi. Mazkur masala bo`yicha Yevropa Kengashi 1996 yilda «Yevropa uchun tayanch kompetensiyalar» mavzusidagi Simpozium tashkil etib, unda Yevropa Ittifoqiga a`zo davlatlarning uzluksiz ta`lim tizimi uchun dastlabki tayanch kompetensiyalari belgilangan. Amaliyotda foydalanilayotgan tizimli faoliyatli yondashuvga asoslangan davlat ta`lim standartlari jamiyatning ijtimoiy-ma`naviy va madaniy taraqqiyoti hamda O`zbekistonning 2030 yilgacha barqaror rivojlantirish strategiyasi talablariga to`la javob bermaydi4 . Mazkur masalani kompleks hal etish maqsadida umumiy o`rta va o`rta maxsus, kasbhunar ta`limida uzviy o`qitiladigan umumta`lim fanlari bo`yicha kompetensiyaviy yondashuvga asoslangan davlat ta`lim standartlari va o`quv dasturlarini ishlab chiqib, amaliyotga joriy etish lozim. Kompetensiya so`zi lotin tilidagi «competo», «competentia» so`zlaridan olingan bo`lib, erishaman, mos kelaman, loyiq kabi ma`nolarni bildiradi. Mazkur atamaning mazmun-mohiyati esa muvaffaqiyatlilik, natijalilik, yutuqlilik kabi tushunchalar bilan tavsiflanadi. Aksariyat foydalanuvchilar yangilik va boshqa o`quv materiallarini nafaqat an`anaviy tarzda kitob, darslik, gazeta, jurnallardan olishi, balkim ancha tez va qulay tarzda global tarmoqdan topishi va tanishib chiqishi mumkin bo`lmoqda. Shu bilan birga, axborotlar oqimi ko`paymoqda, ularni saralash va faqat kerakli ma`lumotlarni tanlab olish zarurati tug`ilmoqda. Bundan tashqari, ko`plab ishlab chiqarish va boshqa jarayonlarni avtomatlashtirish, inson tomonidan bajariladigan va intellektual salohiyatni talab qilmaydigan yoki bajarish jarayonida xavf-xatar 57 1. Axmedov X, Doniyev M, Husanov Z.Fizikadan ma’ruza matni 2018 yil 2. Mirzaxmedov B., G‘ofurov N. va boshqalar. Fizika o‘qitish nazariyasi va metodikasi Toshtent.: 2010 3. Urazova M.B., Eshpulatov Sh.N. Bo`lajak o`qituvchining loyihalash faoliyati. // Metodik qo`llanma. – T.: TDPU, 2014 yil. DEVELOPMENT AND INNOVATIONS IN SCIENCE International scientific-online conference Modellardan ongli va keng foydalanish,ilmiy 58 DEVELOPMENT AND INNOVATIONS IN SCIENCE International scientific-online conference tadqiqot metodlarini o`rganish,tarixiylik va boshqalar metodologik bilimlar sifatida muhim ahamiyat kasb etadi. tadqiqot metodlarini o`rganish,tarixiylik va boshqalar metodologik bilimlar sifatida muhim ahamiyat kasb etadi. Dunyoning ko`pgina davlatlariga xos bo`lgan fizika kursining strukturasi sifatida quyidagi o`qitish tizimini qarab chiqishimiz mumkin: boshlang`ich maktab, o`rta maktabning birinchi bosqichi, o`rta maktabning ikkinchi bosqichi, o`rta maktabning yuqori bosqichi, to`liqsiz o`rta maktab (ta`limni birinchi, ikkinchi va uchinchi bosqichlari; har bir bosqich uch sinfdan), gimnaziya (gumanitar, tabiiy- ilmiy bo`lim va boshqalar; odatda 2- 3 yillik ta`lim). 50% vaqt tayanch fanlarni (til, adabiyot, matematika va boshqalar) o`qitishga ajratiladi. Integrallashgan kurslardan foydalanish keng tarqalgan, yuqori sinflarda esa – maxsus kurslar ko`proq o`qitiladi.Boshlang`ich maktabda ko`pincha fizika, tabiiy fanlar doirasida o`rganiladi. Masalan, Shvetsiyada birinchi bosqichda fizika elementlari o`lkashunoslik, qo`l mehnati, ikkinchi bosqichda tabiatshunoslik, uchinchi bosqichda alohida kurs yoki tabiatshunoslik doirasida ko`rib chiqiladi. Gimnaziyada tabiiy-ilmiy bo`limda fizika uch yil davomida yetarlicha yuqori darajada o`qitiladi Mexanika, elektr, atom fizikasi va boshqa fizikaning bo`limlari o`qitiladi. Demak fizika ta`lim samaradorligini oshirishda fanlararo bog`liqlikni amalga oshirgan holda ta`lim olib borilsa o`quvchining dunyoqarashi va tafakkuri keng bo`lar ekan. Bundan tashqari o`quvchini erkin fikrlashga undash va mustaqil ta`limni kuchaytirish orqali ham yuqori natija olish mumkin. 59
https://openalex.org/W2256062582	https://europepmc.org/articles/pmc4730757?pdf=render	English	null	Long- versus short-interval follow-up of cytologically benign thyroid nodules: a prospective cohort study	BMC medicine	2,016	cc-by	5,976	* Correspondence: ekalexander@partners.org The Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis Street, PBB-B4. Room 417, Boston, MA 02115, USA © 2016 Medici 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. Long- versus short-interval follow-up of cytologically benign thyroid nodules: a prospective cohort study oyun Liu, Norra Kwong, Trevor E. Angell, Ellen Marqusee, Matthew I. Kim and Erik K. Alexander* arco Medici, Xiaoyun Liu, Norra Kwong, Trevor E. Angell, Ellen Marqusee, Matthew I. Kim and Er Marco Medici, Xiaoyun Liu, Norra Kwong, Trevor E. Angell, Ellen Marqusee, Matthew I. Kim and Erik K. Alexander* Please see related commentary article: http://dx.doi.org/10.1186/s12916-016-0559-9 and research article: http://dx.doi.org/10.1186/s12916-015-0419-z. Medici et al. BMC Medicine (2016) 14:11 DOI 10.1186/s12916-016-0554-1 Medici et al. BMC Medicine (2016) 14:11 DOI 10.1186/s12916-016-0554-1 Methods We reviewed the medical records of all patients referred to the thyroid biopsy clinic of the Brigham and Women’s Hospital, Boston, MA, between January 1999 and January 2010. We identified all euthyroid patients with a clinically- relevant thyroid nodule (>1 cm) found to have initial be- nign cytology. Previous analysis confirmed that this clinic evaluates >95 % of all patients seeking thyroid nodule evaluation in our healthcare system, thus limiting referral bias. The Brigham and Women’s Hospital catchment gen- erally includes the greater Boston area and southwestern Massachusetts, including some patients from Rhode Island and Connecticut. US evaluation was performed by one of four radiologists with expertise in thyroid evaluation, using a 6–15 mHz transducer (GE Logic 9, GE Healthcare, Milwaukee, WI). The length, width and depth of each nodule was documented, in addition to its solid or cystic content. FNA was performed by one of four thyroidologists under US guidance. A 25-gauge needle was typically used to obtain three samples per nod- ule, and FNA cytology was evaluated by a Brigham and Women’s Hospital cytopathologist. Although much of the study predates the Bethesda System for Reporting Thyroid Cytopathology [15, 16], all Brigham and Women’s Hospital cytopathologists were already using identical criteria later adopted by the Bethesda System for Reporting Thyroid Cytopathology. All thyroid FNAs were classified into one of the following categories: non-diagnostic, negative for malignant cells (benign), atypical cells of undetermined sig- nificance (AUS), suggestive of a follicular or Hurthle cell neoplasm, suspicious for malignancy, or positive for malig- nancy. The vast majority of cytologically benign nodules were followed conservatively, with regular sonographic as- sessments. Patients were typically asked to return for repeat thyroid assessment in 1 year. However, the empirical evidence for this recommenda- tion remains weak, as there are little data on the optimal time interval for such a repeat evaluation, while other investigations have suggested a negligible risk of disease- specific mortality regardless of follow-up strategy [7]. Current guidelines recommend repeat evaluation after 1–2 years; however, this is based solely on expert opinion [5, 6]. Furthermore, it is unclear what specific findings at the time of repeat evaluation should prompt concern or further investigation, which has led to confusion and variability in clinical practice. The importance of establishing improved guidance should not be underestimated. Thyroid nodules afflict millions of patients worldwide, and most prove benign. Background this recommendation varied. Thus, some patients returned for repeat sonographic assessment in less than 1 year, while others were reassessed after much longer periods. Though non-randomized, such variation is free of substantial refer- ral bias. We therefore used this opportunity to determine differences between short- and long-interval follow-up strategies after initial benign cytology. g Thyroid nodules are increasingly common, affecting 5–20 % of the adult population [1–3]. Once detected, ultrasound (US) is recommended, often in conjunction with fine needle aspiration (FNA). A benign cytological result is obtained in 65–75 % of cases, most often leading to a recommendation for conservative (i.e. non-surgical) management. However, cytological examination is not flawless and many studies confirm a modest, though non-negligible 1–10 % risk of a false-negative cytology (i.e. malignancy) [4]. Because of this, physicians have long recommended continued follow-up of cytologically benign nodules [5, 6]. Abstract Background: Thyroid nodules are common, and most are benign. Given the risk of false-negative cytology (i.e. malignancy), follow-up is recommended after 1–2 years, though this recommendation is based solely on expert opinion. Sonographic appearance may assist with planning, but is limited by large inter-observer variability. We therefore compared the safety and efficacy of long- versus short-interval follow-up after a benign initial aspiration, regardless of sonographic appearance. Methods: This study evaluated all patients referred to the Brigham and Women’s Hospital Thyroid Nodule Clinic, between 1999 and 2010, with a cytologically benign nodule >1 cm and who had returned for follow-up sonographic evaluation. Despite standard clinical recommendations, variation in patient compliance resulted in variable follow-up intervals from time of initial aspiration to the first repeat evaluation. Main outcome measures included nodule growth, repeat fine needle aspiration (FNA), thyroidectomy, malignancy, and disease-specific mortality. Results: We evaluated 1,254 patients with 1,819 cytologically benign nodules, with a median time to first follow-up of 1.4 years (range, 0.5–14.1 years). The longer the follow-up interval, the more nodules grew and the more repeat FNAs were performed (P <0.001). The most clinical meaningful endpoints of malignancy or mortality, however, did not differ between the various follow-up intervals. The risk of a thyroidectomy (usually because of compressive symptoms) increased when time to first follow-up exceeded >3 years (4.9 % vs. 1.2 %, P = 0.0001), though no difference in malignancy risk was identified (0.2–0.8 %, P = 0.77). No (0 %) thyroid cancer-specific deaths were identified in either cohort. Conclusions: While expert opinion currently recommends repeat evaluation of a cytologically benign nodule at 1–2 years, these are the first data to demonstrate that this interval can be safely extended to 3 years without increased mortality or patient harm. Nodule growth can be expected, though detection of malignancies is unchanged. While replication of these data in large prospective multicenter studies is needed, this extension in follow-up interval would reduce unnecessary visits and medical interventions for millions of affected patients worldwide, leading to healthcare savings. Please see related commentary article: http://dx.doi.org/10.1186/s12916-016-0559-9 and research article http://dx.doi.org/10.1186/s12916-015-0419-z. Keywords: Benign thyroid nodule, Follow-up, Thyroid cancer, Thyroidectomy Medici et al. BMC Medicine (2016) 14:11 Page 2 of 9 Page 2 of 9 Results subject with a follow-up interval defined as the time be- tween first benign aspiration and their first repeat sono- graphic evaluation, regardless if subsequent ultrasounds were performed thereafter. This is because management decisions made at subsequent follow-up visits are likely influenced by previous visits, while our goal was to in- vestigate the clinical outcomes as a function of various time intervals through the first follow-up visit. Patients whose first follow-up was less than 6 months after initial benign aspiration were excluded, as such scenarios were deemed unique and likely influenced by separate factors. Over a 10 year period, there were 34 patients with follow- up periods <6 months, and 88 patients who had initial surgery as the nodule was large (mostly >5 cm). There were no thyroidectomies because of suspicious US features or family history of thyroid carcinoma. In total, 1,372 pa- tients with 2,006 cytologically benign thyroid nodules met entry criteria and were analyzed. We excluded patients aged <20 years (n = 43 nodules), given the uniqueness of this population and potential selection bias [18]. Overall, 144 separate nodules were excluded because initial follow-up was less than 6 months. This resulted in a final population of 1,819 nodules, as shown in Table 1. As ex- pected, the population was predominantly female (89.8 %) with a mean age of 52.5 years. Thyroid nodule size aver- aged 2.1 cm. These baseline characteristics were not asso- ciated with follow-up time (all P >0.05). Nodule measurements were documented at baseline and at the follow-up visit. Other follow-up variables were also collected, including if a repeat FNA or if thyroidectomy were performed. We used our Electronic Medical Record system to identify whether each patient was presently living or deceased (and if so, the cause of death) to determine if the detection of a malignancy at a delayed follow-up period was associated with increased disease-specific mortality. This protocol was approved by the Investigational Re- view Board of the Brigham and Women’s Hospital (IDs 1999P002899 and 2000P000167). Individual written con- sent was not required given the lack of intervention and reporting of data in an individually non-identifiable way. The proportion of nodules identified in each follow-up quintile is as follows: 0.5–1 year follow-up, n = 489 nodules; >1–2 years follow-up, n = 715 nodules; >2–3 years follow-up, n = 249 nodules; >3–4 years follow-up, n = 143 nodules; >4 years follow-up, n = 223 nodules. Methods Despite this, follow-up and repeat assessment are ex- tended over many years, with some undergoing lifelong follow-up. Nodule growth can be expected over time, though most often it does not signal malignant concern [8, 9]. While US is superior to physical examination of the neck [10, 11], such technology also exacerbates concern regarding small changes in nodule size or par- enchyma. In an era of increasing healthcare cost and intervention, some have argued that overtreatment and overdiagnosis of nodular thyroid disease is occurring [2, 12, 13]. It is estimated that over 500,000 thyroid nodule aspirates occur annually in the United States alone (Medicare data) [14]; as 300,000–350,000 will prove be- nign, millions of repeat assessments are mandated during the years thereafter. Determining the optimal time interval for such evaluations would reduce unnecessary anxieties, while likely leading to fewer US examinations, repeat aspi- rations, and thyroidectomies. Ultimately, patient morbidity may be decreased while healthcare savings are realized. For the purpose of this study, we took advantage of the known variability in patient compliance with the above recommendation, specifically acknowledging that some patients return for repeat assessment in less than 1 year, while others were reassessed after much longer periods. We therefore identified all patients with cyto- logically benign thyroid nodules who pursued repeat sonographic evaluation at some point following the diag- nosis of a cytologically benign thyroid nodule, regardless of time interval. Each patient was analyzed as a single At Brigham and Women’s Hospital, a prospective data- base has been maintained from 1995 onward, tracking all patients evaluated for nodular thyroid disease using US examination and US-guidance for FNA. This affords a unique opportunity to address this important follow- up question. Specifically, while each patient with ini- tially benign cytology was asked to return for repeat thyroid assessment in 1 year, patient compliance with Medici et al. BMC Medicine (2016) 14:11 Medici et al. BMC Medicine (2016) 14:11 Page 3 of 9 Medici et al. BMC Medicine (2016) 14:11 Page 3 of 9 Results Comparisons of the risk of nodule growth, repeat FNA or thyroidectomy, identification of a thyroid cancer (i.e. false-negative cytology), and thyroid cancer-attributable mortality are shown in Table 2. With longer follow-up intervals, more nodules demonstrated growth, using both the 15 % and 50 % volumetric cut-offs. This resulted in a greater number of repeat aspirations, though there were no differences in cytology results, even when follow-up duration was lengthened. The number of thyroidectomies Statistical analyses The follow-up time between the two visits was rounded to the nearest whole month. We grouped subjects accord- ing to follow-up time into one of five clinically relevant quintiles as follows: 0.5–1 year, >1–2 years, >2–3 years, >3–4 years, and >4 years. To determine change in size with time, nodule volume was calculated by using the formula for a rotational ellipsoid (length × width × depth × π/6) [17]. We employed two commonly used criteria for defining sonographic growth over time. The first was a volume in- crease of 15 %, while the second was a volume increase of 50 % [5, 6]. Each group was assessed for statistically signifi- cant nodule growth, repeat FNA, thyroidectomy, and risk of malignancy using χ2 tests and logistic regression analyses. These analyses were corrected for age and sex. Table 1 Baseline patient and nodule characteristics Table 1 Baseline patient and nodule characteristics Patients, n 1,254 Women, % 89 Age, years 52.5 (13.1) Thyroid nodules, n 1,819 Maximum diameter, cm 2.1 (1.0) Volume, cm3 3.7 (7.2) Nodule characteristics, %: <50 % Cystic 88.5 ≥50 % Cystic 11.5 Years from initial benign aspirate to first follow-up: Median (interquartile range) 1.4 (1.0–2.5) years Range 0.5–14.1 years 0.5–1 year, n 489 1–2 years, n 715 2–3 years, n 249 3–4 years, n 143 >4 years, n 223 Age, maximum diameter and volume are shown as mean (SD) Finally, acknowledging that growth was an important reason for repeat FNA and thyroidectomy, we sought to identify the determinants of benign nodule growth. To do so, the effects of patient (age, sex) and nodule charac- teristics (single versus multinodular, size, and solid versus cystic parenchyma) on growth were tested using linear and logistic regression analyses, correcting for follow- up time. Sensitivity analyses were performed for the continuous variables to identify the cut-off with the strongest effects. Statistical analysis was performed using SPSS version 22 (SPSS IBM, New York), and P values <0.05 were considered significant. Statistical analyses Table 2 Time interval until first follow-up of a benign thyroid nodule and the risk of growth, repeat FNAs, thyroidectomies, malignancies and mortality Follow-up time, years n 15 % Growth, % (n) 50 % Growth, % (n) Repeat FNAs, % (n) Outcomes of repeat FNAs Thyroidectomies, % (n) Indication for thyroidectomy Malignancies, % (n) Disease- related mortality, % (n) 0.5–1 489 30.3 (148) 8.6 (42) 5.1 (25) 21 Benign 1 AUS 3 Non-diagnostica 0.8 (4) 3 US Large size/growth 1 Compressive symptoms 0.2 (1) 0 (0) >1–2 715 34.8 (249) 15.1 (108) 5.6 (40) 29 Benign 4 AUS 1 Susp. foll. neopl. 1 Susp. PTC 5 Non-diagnosticb 0.8 (6) 4 Abnormal repeat FNA 2 US Large size/growth 0.3 (2) 0 (0) >2–3 249 40.2 (100) 19.7 (49) 8.8 (22) 18 Benign 1 AUS 1 Susp. foll. neopl. 1 Malignant 1 Non-diagnosticc 1.2 (3) 3 Abnormal repeat FNA 0.8 (2) 0 (0) >3–4 143 50.3 (72) 34.3 (49) 18.9 (27) 22 Benign 3 AUS 2 Susp. foll. neopl. 4.9 (7) 3 Abnormal repeat FNA 2 Compressive symptoms 1 US Large size/growth 1 Afirma GEC positive 0.7 (1) 0 (0) >4 (range 4.0–14.1) 223 52.5 (117) 35.0 (78) 19.3 (43) 35 Benign 3 AUS 1 Susp. Hurthle cell neopl. 1 Malignant 3 Non-diagnosticd 4.0 (9) 5 Compressive symptoms 2 Abnormal repeat FNA 1 US Large size/growth 1 Afirma GEC positive 0.4 (1) 0 (0) P value <0.0001 <0.0001 <0.0001 0.0001 0.77 – aAll nodules were >75 % cystic and had therefore a negligible low risk of malignancy and were not rebiopsied bThree nodules were >75 % cystic and had therefore a negligible low risk of malignancy and were not rebiopsied. One nodule did not change in size during follow-up, and was therefore not rebiopsied. One nodule was surgically removed (lobectomy) due to its large size (4.4 cm) and histological diagnosis confirmed a 3.3 cm follicular variant PTC (see Table 3 subject no. 3) cNodule did not change in size during follow-up, and was therefore not rebiopsied dOne nodule >75 % cystic and another 50–75 % cystic, which had therefore a negligible low risk of malignancy. The third nodule underwent total thyroidectomy as this patient had another nodule diagnosed with malignant cytology. Statistical analyses All were follicular variants of papillary thyroid carcinoma (fvPTC). One patient (subject no. 1) underwent a thyroidectomy for a symptomatic nodule, and a fvPTC with focal capsular invasion and one scapular bone metas- tasis was diagnosed. The metastasis showed an excellent response to radioactive iodine treatment, and the patient is currently disease free. All other malignancies demon- strated very low risk characteristics. Specifically, all were encapsulated or partially-encapsulated/well-circumscribed, with only one nodule demonstrating extensive capsular in- vasion. None demonstrated evidence of lymphovascular invasion or extrathyroidal extension, and no lymph node or distant metastases were identified. All patients are pres- ently alive and considered disease free. y To further guide optimal follow-up strategies after a benign aspiration, we sought to identify determinants of benign nodule growth (Fig. 1). Sensitivity analyses were performed to identify the cut-off with the strongest effects. There were no effects of sex, multinodularity, or nodule size on the risk of growth when using the 15 % volume in- crease cut-off, nor when using the 50 % cut-off. However, an age <50 years was associated with a higher risk of nodule growth, both when using the 15 % cut-off (odd ratio (OR), 1.48; confidence interval (CI), 1.22–1.81; P <0.0001) as well as the 50 % cut-off (OR, 1.61; CI, 1.26–2.07; P = 0.0002). A solid parenchyma (<50 % cystic content) was also associ- ated with a higher risk of growth, again both when using the 15 % (OR, 2.91; CI, 2.02–4.18; P <0.0001) and 50 % (OR, 1.91; CI, 1.21–3.02; P = 0.005) cut-offs. In order to provide more insight, Fig. 2 depicts the absolute risk of growth attributable to these factors. Given this important role for growth, we also sought to find determinants of benign nodule growth in hopes that this may further guide the clinician in personalizing any follow-up interval using simple patient and US char- acteristics. Age under 50 years and a solid parenchyma were both associated with a substantially higher risk of growth. While our results support a broad recommen- dation for a 3 year follow-up interval, these sub-analyses suggest that a longer interval may be considered for older patients with more cystic nodules, or conversely slightly shorter intervals for young patients with solid nodules. Individual assessment incorporating all available data, including patient preferences, remains paramount. Statistical analyses Histopathology confirmed a 1.1 cm follicular variant PTC, while the nodule with the non-diagnostic biopsy was histologically confirmed to be benign FNA, Fine needle aspiration; AUS, Atypical cells of undetermined significance; PTC, Papillary thyroid carcinoma; GEC, Gene expression classifier. All malignancies were determined by histopathology and the malignancy percentage indicates the rate of malignancies for the respective follow-up time group dule and the risk of growth, repeat FNAs, thyroidectomies, malignancies and mortality Medici et al. BMC Medicine (2016) 14:11 Page 5 of 9 Page 5 of 9 similarly increased, most notably when the follow-up inter- val was longer than 3 years. Compressive symptoms were the predominant indication for thyroidectomy. Only seven malignancies were identified among 1,819 nodules, with no significant difference in malignancy risk between the cohorts. There were no (0 %) thyroid cancer related deaths during a median follow-up of 7.7 (interquartile range, 5.5–10.5) years. malignancies or adverse consequences. The risk of false- negative cytology was similar among all cohorts, and malignancy-related mortality was 0 % in the entire study population. Together, these data show that most thyroid nodules with initial benign cytology can be safely recom- mended for repeat evaluation at a 3 year interval without increased risk or likelihood of harm. Little data are available on the optimal time interval for repeat evaluation of a cytologically benign nodule. Negro et al. [19] described 249 patients with benign nodular disease who were followed for 5 years and documented the timing of relevant events, including the risk of re- peat FNA, thyroidectomy or newly diagnosed thyroid malignancy. Unfortunately, due to the limited number of patients, this study was underpowered to stratify for follow-up time and did not investigate the outcomes at various time intervals. Separately, we have previously studied the disease-specific mortality attributable to thyroid malignancies in a pilot population of patients with cytologically benign nodules [7]. There were no malignancy-related deaths, while thyroidectomies were performed and malignancies identified at an average of 4.5 years. However, the design of that study was critically different from the present one as it did not investigate the outcomes of various follow-up intervals. It also did not in- vestigate the risk of nodule growth, which is the driving factor for most relevant interventions, including repeat FNAs and thyroidectomies, as confirmed by the results of the present study. Table 3 shows the characteristics of the seven malig- nancies. Statistical analyses In this context it is of interest to note that the growth rates of the nodules which turned out to be histologi- cally malignant did not differ from the growth rates of the nodules which were either cytologically (at repeat FNA) or histologically benign, with mean increases of 119.5 % (45.8) (mean (SE)) for the malignant and 108.5 % (10.3) for the benign nodules (P = 0.82). How- ever, a borderline significant difference in their largest diameter at first visit was detected (3.1 (0.4) (mean (SE)) vs. 2.5 (0.9) cm, P = 0.08). Importantly, these data need to be interpreted with caution as only seven malignancies were detected in our cohort. aWith focal capsular invasion bWith extensive capsular invasion cMetastasis showed excellent reaction to radioactive iodine treatment PTC, Papillary thyroid carcinoma; LVI, Lymphovascular invasion Discussion BMC Medicine (2016) 14:11 Page 7 of 9 0 0.5 1 1.5 2 2.5 3 3.5 Sex (Female) Age (<50 yrs) Multinodularity Nodule size (>3 cm) Volume (>3 cm3) Nodule volume increase Odds Ratio Nodule volume increase Sex (Female) Age (<50 yrs) Multinodularity Nodule size (>3 cm) Volume (>3 cm3) Solid Parenchyma 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Odds Ratio Solid Parenchyma 50 15 (<50 cystic) (<50 cystic) Fig. 1 Determinants of benign nodule growth. Growth was calculated as the increase in volume between the baseline and first follow-up ultrasounds. All analyses were corrected for follow-up time, as well as for age and cystic content, as these factors were associated with growth in univariate analyses 0 0.5 1 1.5 2 2.5 3 3.5 Nodule volume increase Odds Ratio 50 Fig. 1 Determinants of benign nodule growth. Growth was calculated as the increase in volume between the baseline and first follow-up ultrasounds. All analyses were corrected for follow-up time, as well as for age and cystic content, as these factors were associated with growth in univariate analyses Recently, some studies have suggested that the presence of certain sonographic characteristics (such as hypoecho- genicity, irregular margins or microcalcifications) should be used to determine the follow-up strategy of cytologic- ally benign nodules [20, 21]. However, these studies are retrospective and based on a limited number of nodules with suspicious US characteristics (n = 93 and n = 102) [20, 21], while others were not able to replicate these find- ings [22]. Furthermore, various studies have shown that interobserver agreement for most of these characteristics is typically limited [23–26]. Thus, while helpful, it may therefore be difficult to use these specific US characteris- tics exclusively as a guide to optimal benign nodule follow-up. The exact role of these specific US characteris- tics in nodule care should therefore be determined in future studies. Strengths of this study include its large sample size and the variation in time to first follow-up, which pro- vides a robust comparison. Discussion This is the first study to investigate the optimal time interval for repeat evaluation of a benign thyroid nodule. While a longer time interval is associated with greater nodule growth, no detriments in health outcomes were identified. Nodule growth led to a higher risk of repeat FNA as well as thyroidectomy, but not an increased risk of malignancy. As an increased rate of thyroidectomy was only noted when follow-up exceeded 3 years, it appears that symptomatic concerns related to nodule growth were not clinically concerning with shorter follow-up intervals. Importantly, follow-up intervals exceeding even 4 years were not associated with a higher risk of detecting harmful Medici et al. BMC Medicine (2016) 14:11 Table 3 Description of the seven patients with false benign malignancies Table 3 Description of the seven patients with false benign malignancies Subject no. Time until first follow-up, years Nodule size at initial aspiration, cm Nodule size at first follow-up, cm Thyroidectomy indication Histopathology Encapsulated Lymphovascular invasion/Extrathyroidal extension Lymph node/ Distant metastases Clinical status 1 1.0 5.2 6.1 Compressive symptoms PTC follicular variant 5.0 cm Encapsulated a 1 Focus suspicious for LVI 1 Scapular metastasisc Disease free with no recurrence 2 1.3 4.0 4.5 Abnormal repeat cytology PTC follicular variant 4.3 cm Encapsulated No No Disease free with no recurrence 3 1.4 2.9 4.4 Compressive symptoms PTC follicular variant 3.3 cm Encapsulated No No Disease free with no recurrence 4 2.3 2.0 2.9 Abnormal repeat cytology PTC follicular variant 3.8 cm Encapsulated b No No Disease free with no recurrence 5 2.7 1.2 1.6 Abnormal repeat cytology PTC follicular variant 1.1 cm Encapsulated No No Disease free with no recurrence 6 3.7 2.2 2.7 Abnormal repeat cytology PTC follicular variant 2.5 cm Encapsulated No No Disease free with no recurrence 7 4.4 4.5 6.1 Abnormal repeat cytology PTC follicular variant 5.5 cm Partially-encapsulated/ well-circumscribed No No Disease free with no recurrence aWith focal capsular invasion bWith extensive capsular invasion cMetastasis showed excellent reaction to radioactive iodine treatment PTC, Papillary thyroid carcinoma; LVI, Lymphovascular invasion Medici et al. Discussion We provide detailed data on sonographic growth, repeat FNAs, thyroidectomies, Absolute risk Absolute risk 0 5 10 15 20 25 30 35 40 45 50 15% Volume increase 50% Volume increase Age <50 yrs P < 0.0001 P = 0.0002 0 5 10 15 20 25 30 35 40 45 50 15% Volume increase 50% Volume increase <50% Cystic Age 50 yrs 50% Cystic P = 0.005 P < 0.0001 Fig. 2 Effects of age <50 years and <50 % cystic content on the absolute risk of nodule growth. Growth was calculated as the increase in volume between the baseline and first follow-up ultrasounds. Age analyses were corrected for follow-up time, cystic content and sex, and cystic content analyses were corrected for follow-up time, age and sex Absolute risk 0 5 10 15 20 25 30 35 40 45 50 15% Volume increase 50% Volume increase <50% Cystic 50% Cystic P = 0.005 P < 0.0001 Absolute risk 0 5 10 15 20 25 30 35 40 45 50 15% Volume increase 50% Volume increase Age <50 yrs P < 0.0001 P = 0.0002 Age 50 yrs Age <50 yrs Age 50 yrs Age <50 yrs Age 50 yrs <50% Cystic 50% Cystic Fig. 2 Effects of age <50 years and <50 % cystic content on the absolute risk of nodule growth. Growth was calculated as the increase in volume between the baseline and first follow-up ultrasounds. Age analyses were corrected for follow-up time, cystic content and sex, and cystic content analyses were corrected for follow-up time, age and sex Medici et al. BMC Medicine (2016) 14:11 Medici et al. BMC Medicine (2016) 14:11 Page 8 of 9 Page 8 of 9 histopathological results and mortality, and additionally performed sub-analyses on determinants of benign nodule growth. However, we similarly acknowledge limitations to our study, including that it was performed in a single center. Despite this, our clinic captures more than 95 % of patients undergoing thyroid evaluation in our healthcare system, and all have been registered in our database, which improves the generalizability of our findings. While prospective, this study also does not pursue a randomized intervention. The main con- cern, therefore, would be that our follow-up intervals were not randomly determined but rather influenced by nodule-specific characteristics. However, this is unlikely given that all patients with benign thyroid nodules were provided similar recommendations for routine 1-year follow-up. Acknowledgments This study was supported by Brigham and Women’s hospital funds (to TEA, EM, MIK, EKA) and by the NIH T32 DK007529 training grant (to NK). Received: 25 July 2015 Accepted: 7 January 2016 Received: 25 July 2015 Accepted: 7 January 2016 Received: 25 July 2015 Accepted: 7 January 2016 Authors’ contributions MM d d h d MM designed the study, analyzed the data, and drafted the manuscript. XL, NK, TEA, EM, and MIK acquired and interpreted the data. EKA designed the study, acquired and interpreted the data, and drafted the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Availability of data and materials These data are not publicly available as these concern patient-related data. References 1. Sosa JA, Hanna JW, Robinson KA, Lanman RB. Increases in thyroid nodule fine-needle aspirations, operations, and diagnoses of thyroid cancer in the United States. Surgery. 2013;154:1420–6. 2. Davies L, Welch HG. Current thyroid cancer trends in the United States. JAMA Otolaryngol Head Neck Surg. 2014;140:317–22. 3. Leenhardt L, Bernier MO, Boin-Pineau MH, Conte Devolx B, Maréchaud R, Niccoli-Sire P, et al. Advances in diagnostic practices affect thyroid cancer incidence in France. Eur J Endocrinol. 2004;150:133–9. 4. Wang CC, Friedman L, Kennedy GC, Wang H, Kebebew E, Steward DL, et al. A large multicenter correlation study of thyroid nodule cytopathology and histopathology. Thyroid. 2011;21:243–51. 5. American Thyroid Association Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009;19:1167–214. y 6. Gharib H, Papini E, Paschke R, Duick DS, Valcavi R, Hegedüs L, et al. American Association of Clinical Endocrinologists, Associazione Medici Endocrinologi, and European Thyroid Association medical guidelines for clinical practice for the diagnosis and management of thyroid nodules. Endocr Pract. 2010;16 Suppl 1:1–43. 9. Durante C, Costante G, Lucisano G, Bruno R, Meringolo D, Paciaroni A, et al. The natural history of benign thyroid nodules. JAMA. 2015;313:926–35. Funding This study was supported by Brigham and Women’s hospital funds (to TEA, EM, MIK, EKA) and by the NIH T32 DK007529 training grant (to NK). The funders were not involved in the study design, conduct, or manuscript preparation. Discussion Furthermore, long interval follow-ups were mostly attributable to patient compli- ance and organizational delays – factors unlikely to significantly bias our findings. Importantly, follow-up time intervals were not associated with nodule size, cystic content, age, or sex. It is therefore unlikely that the variation in follow-up intervals was related to nodule-specific factors. Furthermore, we cannot ex- clude that part of the patients with faster growth pre- sented earlier. However, as previously discussed, growth did not seem to be a reliable indicator of malig- nancy risk. Finally, we intentionally only included re- sults from the first follow-up visit. This is because management decisions made at subsequent follow-up visits are likely influenced by previous visits, while our goal was to investigate the clinical outcomes as a func- tion of various time intervals through the first follow- up visit. While this was therefore the correct design for our main study outcomes, no recommendations on timing of further follow-up visits could be made. 10. Danese D, Sciacchitano S, Farsetti A, Andreoli M, Pontecorvi A. Diagnostic accuracy of conventional versus sonography-guided fine-needle aspiration biopsy of thyroid nodules. Thyroid. 1998;8:15–21. 7. Nou E, Kwong N, Alexander LK, Cibas ES, Marqusee E, Alexander EK. Determination of the optimal time interval for repeat evaluation after a benign thyroid nodule aspiration. J Clin Endocrinol Metab. 2014;99:510–6. 8. Alexander EK, Hurwitz S, Heering JP, Benson CB, Frates MC, Doubilet PM, et al. Natural history of benign solid and cystic thyroid nodules. Ann Intern Med. 2003;138:315–8. Conclusions In summary, current guidelines recommend repeat evaluation of a benign thyroid nodule after 1–2 years, which is only based on expert opinion. This study sug- gests that this recommendation can be safely extended to 3 years without increased malignancy and mortality risk, or likelihood of harm. Nodule growth can be ex- pected, and therefore young patients, those experiencing possible structural symptoms, or those with larger nod- ules at baseline may be candidates for modified recom- mendations. Conversely, even longer follow-up intervals should be considered for older patients with predomin- antly cystic nodules. While replication of these data in large prospective multicenter studies is needed, this extension in follow-up interval would reduce unneces- sary visits and medical interventions for millions of affected patients worldwide, leading to healthcare savings. 7. Nou E, Kwong N, Alexander LK, Cibas ES, Marqusee E, Alexander EK. Determination of the optimal time interval for repeat evaluation after a benign thyroid nodule aspiration. J Clin Endocrinol Metab. 2014;99:510–6. 8. Alexander EK, Hurwitz S, Heering JP, Benson CB, Frates MC, Doubilet PM, et al. Natural history of benign solid and cystic thyroid nodules. Ann Intern Med. 2003;138:315–8. 9. Durante C, Costante G, Lucisano G, Bruno R, Meringolo D, Paciaroni A, et al. The natural history of benign thyroid nodules. JAMA. 2015;313:926–35. 10. Danese D, Sciacchitano S, Farsetti A, Andreoli M, Pontecorvi A. Diagnostic accuracy of conventional versus sonography-guided fine-needle aspiration biopsy of thyroid nodules. Thyroid. 1998;8:15–21. Page 9 of 9 Medici et al. BMC Medicine (2016) 14:11 15. Cibas ES. Ali SZ; NCI Thyroid FNA State of the Science Conference. The Bethesda System for Reporting Thyroid Cytopathology. Am J Clin Pathol. 2009;132:658–65. 16. Ali SZ, Cibas ES, editors. The Bethesda System for Reporting Thyroid Cytopathology. New York: Springer; 2009. 16. Ali SZ, Cibas ES, editors. The Bethesda Sys Cytopathology. New York: Springer; 2009. Cytopathology. New York: Springer; 2009. 17. Brunn J, Block U, Ruf G, Bos I, Kunze WP. Scriba PC. Dtsch Med Wochenschr. 1981;106:1338–40. Volumetric analysis of thyroid lobes by real-time ultrasound (author’s transl), [In German]. 18. Gupta A, Ly S, Castroneves LA, Frates MC, Benson CB, Feldman HA, et al. A standardized assessment of thyroid nodules in children confirms higher cancer prevalence than in adults. J Clin Endocrinol Metab. 2013;98:3238–45. 19. Negro R. What happens in a 5-year follow-up of benign thyroid nodules. J Thyroid Res. 2014;2014:459791. 20. 26. Choi SH, Kim EK, Kwak JY, Kim MJ, Son EJ. Interobserver and intraobserver variations in ultrasound assessment of thyroid nodules. Thyroid. 2010;20:167–72. Conclusions Kwak JY, Koo H, Youk JH, Kim MJ, Moon HJ, Son EJ, et al. Value of US correlation of a thyroid nodule with initially benign cytologic results. Radiology. 2010;254:292–300. 21. Rosario PW, Purisch S. Ultrasonographic characteristics as a criterion for repeat cytology in benign thyroid nodules. Arq Bras Endocrinol Metabol. 2010;54:52–5. 22. Illouz F, Rodien P, Saint-André JP, Triau S, Laboureau-Soares S, Dubois S, et al. Usefulness of repeated fine-needle cytology in the follow-up of non-operated thyroid nodules. Eur J Endocrinol. 2007;156:303–8. 23. Park SH, Kim SJ, Kim EK, Kim MJ, Son EJ, Kwak JY. Interobserver agreement in assessing the sonographic and elastographic features of malignant thyroid nodules. AJR Am J Roentgenol. 2009;193:W416–23. 24. Park SJ, Park SH, Choi YJ, Kim DW, Son EJ, Lee HS, et al. Interobserver variability and diagnostic performance in US assessment of thyroid nodule according to size. Ultraschall Med. 2012;33:E186–90. 25. Park CS, Kim SH, Jung SL, Kang BJ, Kim JY, Choi JJ, et al. Observer variability in the sonographic evaluation of thyroid nodules. J Clin Ultrasound. 2010;38:287–93. 26. Choi SH, Kim EK, Kwak JY, Kim MJ, Son EJ. Interobserver and intraobserver variations in ultrasound assessment of thyroid nodules. Thyroid. 2010;20:167–72. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2766035540	https://www.scielo.br/j/rsbmt/a/5HYbMFX783HrfBCqSjS3LDq/?lang=en&format=pdf	English	null	Prevalence and antibiogram of methicillin resistant Staphylococcus aureus isolated from medical device-related infections; a retrospective study in Lahore, Pakistan	Revista da Sociedade Brasileira de Medicina Tropical	2,017	cc-by	3,366	Abstract Introduction: With the advancement of medicine and surgery, various types of medical devices have become part of treatment strategies. Methods: Identification and antimicrobial sensitivity testing were done according to CLSI guidelines following standard microbiological practices. Results: Urinary catheter infections (31%) were most frequent followed by central venous catheter (18%) and orthopedic implants (15%). Methicillin resistant Staphylococcus aureus (MRSA) was a major cause of device-related infection after Escherichia coli (21%); other pathogens were Klebsiella pneumoniae (14%), Pseudomonas spp. (10%), Acinetobacter spp. (8%) and Candida species (7%). None of MRSA was resistant to vancomycin (MIC ≥16µg/mL). Resistance rates were 98% and 97% for ofloxacin and ciprofloxacin, respectively. Conclusions: Escherichia coli and MRSA are major pathogens of medical device-related infections. Keywords: Prosthetic devices. MRSA. Escherichia coli. Vancomycin. Antibiogram. Healthcare-associated infection is a major problem of modern health care management; it is suggested that 60% of these infections are related to prosthetic devices. Advancements in medicine, surgery and bioengineering have paved the way for the use of prosthetic and medical devices in healthcare. This marvelous achievement proved a turning point in medicine that led to better treatment options. Patients with prosthetic devices, catheters or implants are more vulnerable to infections. Device- related infections are caused by Staphylococcus aureus and Staphylococcus epidermidis that have the potential for biofilm formation. This makes them difficult to eradicate even after removal of the prosthetic device and leads to chronic infection. Central venous catheters (CVCs) are essential devices used for medication and nutrition to critically ill patients. Infection of CVCs dramatically increases the morbidity (35%), hospital stay and cost to the health care setting along with medical complications. Bacteremia is an outcome of CVC infections in newborn infants and the risk increases with the duration of hospital stay. CVC infections are the most (90%) common cause of catheter-related infections and increase the mortality by 2.27 folds1. Infection of catheters depends on multiple factors, including retention time, the procedure of catheterization, sterilization status of the healthcare setting, immunocompetence status of the patient, and nutritional status. Urinary tract infections (UTIs) are very common, and 80% of UTIs are due to urinary catheterization, which makes patients vulnerable to uropathogens. Catheterization is a common procedure (15-20%) among hospitalized patients worldwide. Bacterial translocation is the main source of infection due to endotracheal intubation. Rev Soc Bras Med Trop 50(5):680-684, September-October, 2017 doi: 10.1590/0037-8682-0352-2016 Muhammad Sohail[1] and Zakia Latif[1] Muhammad Sohail[1] and Zakia Latif[1] [1]. Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan. Corresponding author: Muhammad Sohail. e-mail: drsohailmmg@gmail.com Received 31 August 2016 Accepted 30 June 2017 Abstract The endotracheal tube (ETT) is an independent risk factor for developing ventilator-associated pneumonia (VAP); which is reported in 15-20% of mechanically ventilated patients. Catheter-related infection is the major complication and drawback of peritoneal dialysis, which is associated with a 3.5-10% mortality rate2. For the management of hydrocephalus, ventriculoperitoneal (VP) shunt is preferred to ventriculoatrial (VA) shunt because it is associated with fewer complications and is a relatively straightforward surgery. VA/VP shunt infections were reported as 4.2% to 11.3%, which is very high for a complicated medical condition. The control of prosthetic and healthcare-associated infection is a challenge today, and there is an urgent need to take precautionary measures against it. This study aimed to determine the prevalence and identification of bacteria causing indwelling device-related infections and to evaluate the antimicrobial sensitivity pattern of methicillin resistant Staphylococcus aureus (MRSA). Six major catheter or indwelling devices including CVCs, urinary catheters, ETT, peritoneal dialysis catheter, and VA/VP shunts were included in this study. This study was conducted in the Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, 680 Sohail M and Latif Z - Prevalence and antibiogram of device-related MRSA difference among proportions of resistance in different years among samples with isolated MRSA. Pakistan from March 2012 to March 2016. During this period, samples were taken from outpatients and in-patients with prosthetic device-related infection. The specimens included the prosthetic devices themselves, part of the prosthetic device, or the pus and urine from the infection site. Among 6,242 prosthetic device-related infection samples, 79% (4,902) were positive for bacterial growth. Among the infected patients 65% (3,186) were male and 35% (1,716) were female. The age of the participants ranged from 10 days to 80 years, with 44 being the median age for males and 33 years for females. Urinary catheter infections (31%) were prominent among the device-related infections, followed by CVCs (18%) and orthopedic implants (15%). The χ2 analysis revealed that the number of patients with prosthetic device infection increased significantly with time [χ2 (2N=4,902)] = 18, p=0.04). Overall, Escherichia coli (21%) was most common cause of device-related infections followed by MRSA (15%), Klebsiella pneumoniae (14%), coagulase negative Staphylococcus (CONS) (11%), Pseudomonas spp. (10%), MSSA (10%), Acinetobacter spp. (8%), Candida spp. (7%), and Streptococcus species (4%) (Figure 1). All the specimens were processed aseptically according to standard microbiological protocols. Abstract Pus specimens were inoculated on sheep blood agar, MacConkey and chocolate agar plates, whereas urine samples were inoculated on cystine lactose electrolyte deficient (CLED) agar plates. All inoculated plates were incubated overnight aerobically at 37C°. Prosthetic devices and catheters were incubated in brain heart infusion for two hours and then inoculated on agar plates to maximize the recovery of pathogens. The guidelines of the Clinical and Laboratory Standards Institute (CLSI) were followed to declare infection especially urine catheter and ETT infections. Following the CLSI guidelines, antimicrobial activity of the isolates were determined by the disk diffusion method3. MRSA was confirmed by the cefoxitin interpretation criteria per the CLSI recommendations: S. aureus having an inhibition zone ≥22mm was confirmed as MRSA. The minimum inhibitory concentration (MIC) of vancomycin was determined by the E-test strip (Etest® bioMérieux, USA) method by following the procedure of the manufacturer and interpretation criteria of the CLSI3. MRSA was most common pathogen after E. coli, isolated from patients with indwelling device-related infection. MRSA was isolated from 30% of orthopedic implants followed by CVCs (27%), peritoneal dialysis catheters (14%), VP/VA shunts (8%), ETTs (7%) and urinary catheters (5%). Age- wise prevalence of MRSA was determined among patients with prosthetic device-related infection. The most affected age group was 31-40 years (29.9%), followed by 41-50 years (23.3%) and 21-30 years (19.7%). The present study showed that prosthetic device infection was less (4.5%) prevalent among individuals younger than 10 years of age. Statistical analysis was conducted using Statistical Package for the Social Sciences (SPSS) version 20 (IBM Corp., Armonk, NY, USA). Chi square (χ2) test was performed to analyze the E . c o l i M RS A M S S A Ac i n eto b ac ter CONS K . p n eu m o n i a e P seu d o m o n as S tr ep to c o cc i C an d i d a 1 2 Prevalence (%) 8 16 32 64 Central venous catheter Endotracheal tube Urinary catheter Orthopedic implants Peritoneal dialysis catheter VP/VA shunt FIGURE 1 - Prevalence of pathogens in device-related infections. VP/VA: ventriculoperitoneal/ventriculoatrial; MRSA: methicillin resistant Staphylococcus aureus; MSSA: methicillin-sensitive Staphylococcus aureus; CONS: coagulase negative Staphylococcus. FIGURE 1 - Prevalence of pathogens in device-related infections. VP/VA: ventriculoperitoneal/ventriculoatrial; MRSA: methicillin resistant Staphylococcus aureus; MSSA: methicillin-sensitive Staphylococcus aureus; CONS: coagulase negative Staphylococcus. Abstract 681 Rev Soc Bras Med Trop 50(5):680-684, September-October, 2017 The antibiogram of MRSA was determined with 14 antibiotics recommended by the CLSI3. Most of the MRSA isolated from prosthetic devices were resistant to ofloxacin (98%), ciprofloxacin (97%), followed by tobramycin (92%), macrolides (88%), and gentamycin (87%); as shown in Table 1. Linezolid (1.1% resistance) and vancomycin were the most effective against MRSA isolated from the patients with prosthetic device-related infections. The resistance pattern of MRSA isolated from the patients with prosthetic device-related infection is shown in Figure 2. prosthetic device-related infections. The resistance pattern of MRSA isolated from the patients with prosthetic device-related infection is shown in Figure 2. None of the MRSA was fully resistant to vancomycin (MIC ≥16µg/mL); 5.75% isolates had intermediate resistance (MIC, 4-8µg/mL). A total of 31.5 % of MRSA were inhibited by 1µg/mL of vancomycin and 25% by 0.5µg/mL (Figure 2). None of the MRSA was fully resistant to vancomycin (MIC ≥16µg/mL); 5.75% isolates had intermediate resistance (MIC, 4-8µg/mL). A total of 31.5 % of MRSA were inhibited by 1µg/mL of vancomycin and 25% by 0.5µg/mL (Figure 2). 682 0.16 0.32 0.64 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 0 5 10 15 20 25 30 35 40 45 50 2013 2014 2015 2016 0 10 20 30 40 50 60 70 80 90 100 2013 2014 2015 2016 Resistance (%) Fusidic Acid Doxycydine Amikacin Gentamicin Tobramycin Azithromycine Erythromycine Ciproﬂoxacin Oﬂoxacin Trime/Sulphamethoxazole Clindamycin Percentage (%) resistance FIGURE 2 - Antibiotic resistance patterns of MRSA against different antibiotics isolated from different device-related infections. MRSA: methicillin resistant Staphylococcus aureus. Drugs Sensitive resistance number percentage (%) number percentage (%) Fusidic Acid 709 78 635 22 Doxycycline 494 55 411 45 Amikacin 300 33 605 67 Gentamicin 120 13 785 87 Tobramycin 70 8 835 92 Azithromycine 111 12 795 88 Erythromycine 111 12 795 88 Ciprofloxacin 30 3 875 97 Ofloxacin 20 2 885 98 Trime/Sulphamethoxazole 250 28 655 72 Clindamycin 270 30 196 70 TABLE 1 Antibiogram of MRSA isolated from prosthetic device related infections. MRSA: methicillin resistant Staphylococcus aureus. (98%), ciprofloxacin (97%), followed by tobramycin (92%), macrolides (88%), and gentamycin (87%); as shown in Table 1. Linezolid (1.1% resistance) and vancomycin were the most effective against MRSA isolated from the patients with None of the MRSA was fully resistant to vancomycin (MIC ≥16µg/mL); 5.75% isolates had intermediate resistance (MIC, 4-8µg/mL). Abstract A total of 31.5 % of MRSA were inhibited by 1µg/mL of vancomycin and 25% by 0.5µg/mL (Figure 2). Drugs Sensitive resistance number percentage (%) number percentage (%) Fusidic Acid 709 78 635 22 Doxycycline 494 55 411 45 Amikacin 300 33 605 67 Gentamicin 120 13 785 87 Tobramycin 70 8 835 92 Azithromycine 111 12 795 88 Erythromycine 111 12 795 88 Ciprofloxacin 30 3 875 97 Ofloxacin 20 2 885 98 Trime/Sulphamethoxazole 250 28 655 72 Clindamycin 270 30 196 70 TABLE 1 Antibiogram of MRSA isolated from prosthetic device related infections. MRSA: methicillin resistant Staphylococcus aureus. TABLE 1 Antibiogram of MRSA isolated from prosthetic device related infections. MRSA: methicillin resistant Staphylococcus aureus. MRSA: methicillin resistant Staphylococcus aureus. 0.16 0.32 0.64 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 0 5 10 15 20 25 30 35 40 45 50 2013 2014 2015 2016 0 10 20 30 40 50 60 70 80 90 100 2013 2014 2015 2016 Resistance (%) Fusidic Acid Doxycydine Amikacin Gentamicin Tobramycin Azithromycine Erythromycine Ciproﬂoxacin Oﬂoxacin Trime/Sulphamethoxazole Clindamycin Percentage (%) resistance FIGURE 2 - Antibiotic resistance patterns of MRSA against different antibiotics isolated from different device-related infections. MRSA: methicillin resistant Staphylococcus aureus. 0.16 0.32 0.64 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 0 5 10 15 20 25 30 35 40 45 50 2013 2014 2015 2016 Resistance (%) FIGURE 2 - Antibiotic resistance patterns of MRSA against different antibiotics isolated from different device-related infections. MRSA: methicillin resistant Staphylococcus aureus. FIGURE 2 - Antibiotic resistance patterns of MRSA against different antibiotics isolated from different device-related Staphylococcus aureus. 682 Sohail M and Latif Z - Prevalence and antibiogram of device-related MRSA In this study, the prevalence of bacterial pathogens causing device-related infection was determined and the recent trend in the antibiogram of MRSA was evaluated. The prevalence of prosthetic device-related infection was 76% which was in accordance with a study conducted in Turkey. The pathogens identified from prosthetic device-related infections were similar to many studies conducted in the past. E. coli (21%) was the commonest cause of catheter-related infections, followed by MRSA (14.5%) and K. pneumoniae (14.3%). E. coli and Pseudomonas spp. were dominant in Europe while Streptococcus spp. and Candida spp. were the dominant causes of device-related infections in the USA, depending on the health care facility. Abstract The prevalent pathogens infecting CVCs were K. pneumoniae and Candida spp. (10% each) followed by Streptococcus spp. (9%), Pseudomonas spp. (7%), E. coli (5%) and Acinetobacter spp. (4%); these prevalence rates are in accordance with the results reported in a study conducted from 2011 to 20134. Our study also supported the retrospective survey conducted in Italy except for CONS which was reported at 34% and the result of our study showed 16%; that retrospective study was conducted on oncological patients which were more prone to CONS infections5. of infected orthopedic implants, followed by CONS (20%), MSSA and K. pneumoniae (15% each), and Candida spp., Pseudomonas spp., Acinetobacter spp. and E. coli (5% each). A previous study showed an increase in Acinetobacter spp. (16%) isolated in orthopedic infections, while the prevalence of other pathogens was the same9. Staphylococcus spp. (28%) and Pseudomonas spp. (8%) were the major causes of peritoneal dialysis catheter infection; similar results were obtained in Senegal and Sudan10. The results of this study strongly agreed with the results of a retrospective study conducted internationally. Shunt infections are caused by CONS (22%), followed by Pseudomonas spp. (15%) and E. coli (12%). Similar results were obtained in a retrospective study conducted in Switzerland and Canada, whereas some other studies related to pediatric CSF reported Staphylococcus spp. as the most common cause of infection (75%)11. MRSA isolated from prosthetic devices showed a 67% resistance to amikacin and 87% resistance to gentamycin; these results disagreed with those of a previous study. The antibiogram of azithromycin (88%), erythromycin (88%), ciprofloxacin (97%), trimethoprim/sulphamethoxazole (71%) was quite similar to that reported in a study conducted in Nepal12; similar results were also reported in Iran except for fusidic acid13. Some studies reported a high prevalence of resistance to fusidic acid (60%) and clindamycin (71%) which strongly agreed with the results of the present study14. The resistance of MRSA to aminoglycosides was very high compared to a study conducted in Germany, which reported a 23% resistance to amikacin and 24% to gentamycin15. The susceptibility of vancomycin was almost 100% which strongly agreed with studies conducted in Spain, India, China, and Nepal. This study showed an MIC of <16ug/ml for vancomycin but some studies reported MIC an greater than 16ug/ml as well16. In the present study the difference in the antibiogram was due to MRSA isolated from six different types of prosthetic device-related infection. Abstract Other studies considered one or two types of device-related infection. There was a gradual increase in the acquisition of resistance by MRSA as shown in Figure 2, but this difference in resistance was not statistically significant (p> 0.05). For catheter-related UTI, our results were similar to a retrospective study conducted in India except for E.coli, Klebsiella and Pseudomonas spp; our results were 71%, 8% and 3% as compared to 35%, 21% and 17%, respectively6. This study was in agreement with the results of a study in Saudi Arabia in 2010 except for E. coli (71% vs 40%). This difference was due to the varying demographic and health care settings. Different studies reported different prevalence rates of uropathogens, Candida spp. (28.2%); K. pneumoniae (44%) and E. coli (70%); depending on the nature of sample, immune status and the underlying disease of the patient. E. coli was not only most common cause of urinary catheter-related infection, but most device related-infections were due to E. coli as claimed in the present study. The prevalence of pathogens in ETT infection was not in agreement with a study published in the United States except for Klebsiella spp. because only VAP was included in the study7. Yagmurdur reported the prevalence of Acinetobacter spp. (51%) and Pseudomonas spp. (17%); the prevalence of Acinetobacter spp. was greater because it was a major cause of VAP8 which was the theme of that study. K. pneumoniae (30%) was the major cause of ETT infection followed by Acinetobacter spp. (25%), followed by Pseudomonas spp. (22%), Candida spp. and MRSA (7% each), MSSA (4%), E. coli and Streptococcus spp. (2% each), and CONS (1%). The prevalence of pathogens isolated from ETTs was the same as that reported in a study conducted in Poland in 2014. Prosthetic device-related infections are a major concern today; due to advancements in medicine and surgery, prosthetic devices are used frequently for better health care facilities. This study demonstrated the prevalence of pathogens from six different types of prosthetic devices and catheters. This revealed the recent trend of antibiogram of MRSA which is emerging as a superbug of device related infections. This data will help clinicians to manage device-related infections effectively. A study conducted in Iran demonstrated same the prevalence of pathogens especially MRSA. Orthopedic implant-related infections were worse and it is difficult to eradicate the causative pathogen because of low drug penetration in bone tissues. Staphylococcus spp. Acknowledgements We highly acknowledge the managements of Chughtais Lab, Lahore for supporting this study. This study received funding from the Higher Education Commission (HEC), Pakistan under pin no. 112-22691-2BM1-376. Abstract are notorious for causing bone and joint infections and form a biofilm on implants. Half (45-55%) of bone and joint infections were due to S. aureus especially related to orthopedic implants. MRSA was the most common (30%) cause Financial support This study received funding from the Higher Education Commission (HEC), Pakistan under pin no. 112-22691-2BM1-376. 683 Rev Soc Bras Med Trop 50(5):680-684, September-October, 2017 REFERENCES 10. Niang A, Cisse MM, Mahmoud SMOM, Lemrabott ATO, Ka el HF, Diouf B. Pilot experience in senegal with peritoneal dialysis for end-stage renal disease. Perit Dial Int. 2014;34(5):539-43. 1. Stevens V, Geiger K, Concannon C, Nelson RE, Brown J, Dumyati G. Inpatient costs, mortality and 30-day re-admission in patients with central-line-associated bloodstream infections. Clin Microbiol Infect. 2014;20(5):O318-24. 11. Odio C, McCracken Jr GH, Nelson JD. CSF shunt infections in pediatrics. A seven-year experience. Am J Dis Child. 1984;138(12):1103-8. 2. Mujais S. Microbiology and outcomes of peritonitis in North America. Kidney Int. 2006;70(Suppl 103):S55-62. 12. Bhatta DR, Cavaco LM, Nath G, Gaur A, Gokhale S, Bhatta DR. Threat of multidrug resistant Staphylococcus aureus in Western Nepal. Asian Pac J Trop Dis. 2015;5(8):617-21. 3. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: 26th Informational supplement. In: document M100-S26. Wayne, PA: CLSI; 2016. 13. Moghadam SO, Pourmand MR, Aminharati F. Biofilm formation and antimicrobial resistance in methicillin-resistant Staphylococcus aureus isolated from burn patients, Iran. J Infect Dev Ctries. 2014;8(12):1511-7. 4. Bouzad C, Duron S, Bousquet A, Arnaud FX, Valbousquet L, Weber-Donat G, et al. Peripherally inserted central catheter-related infections in a cohort of hospitalized adult patients. J Cardiovasc Intervent Radiol. 2016;39(3):385-93. 14. Girgis SA, El Din Gomaa HE, Saad NE, Salem MM. A comparative study for detection of methicillin resistance Staphylococci by polymerase chain reaction and phenotypic methods. Life Sci J. 2013;10(4):3711-8. 5. Wisplinghoff H, Seifert H, Wenzel RP, Edmond MB. Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. Clin Infect Dis. 2003;36(9):1103-10. 6. Nandini MS, Madhusudan K. Bacteriological profile of catheter associated urinary tract infection and its antimicrobial susceptibility pattern in a tertiary care hospital. J Pharm Sci Res. 2016;8(4):204-7. 15. Hu Y, Liu A, Vaudrey J, Vaiciunaite B, Moigboi C, McTavish SM, et al. Combinations of β-Lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus. PLoS One. 2015;10(2):e0117664. 7. Apisarnthanarak A, Holzmann-Pazgal G, Hamvas A, Olsen MA, Fraser VJ. Ventilator-associated pneumonia in extremely preterm neonates in a neonatal intensive care unit: characteristics, risk factors, and outcomes. Pediatrics. 2003;112(6 Pt 1):1283-9. 16. Hasan R, Acharjee M, Noor R. Prevalence of vancomycin resistant Staphylococcus aureus (VRSA) in methicillin resistant S. aureus (MRSA) strains isolated from burn wound infections. Ci Ji Yi Xue Za Zhi. 2016;28(2)49-53. 8. Conflict of interest lavage cultures in ventilator-associated pneumonia diagnosis. Niger J Clin Pract. 2016;19(1):46-51. The authors declare that there is no conflict of interest. 9. Helal S, El Anany M, Ghaith D, Rabeea S. The Role of MDR- Acinetobacter baumannii in orthopedic surgical site infections. Surg Infect (Larchmt). 2015;16(5):518-22. REFERENCES Yagmurdur H, Tezcan AH, Karakurt O, Leblebici F. The efficiency of routine endotracheal aspirate cultures compared to bronchoalveolar 684
https://openalex.org/W4254699523	https://europepmc.org/articles/pmc7863265?pdf=render	English	null	Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection, intestinal permeability, and cecal microbiota composition in broiler chickens	Research Square (Research Square)	2,020	cc-by	9,906	RESEARCH Open Access Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection, intestinal permeability, and cecal microbiota composition in broiler chickens Anaisa A. Leyva-Diaz1, Daniel Hernandez-Patlan2, Bruno Solis-Cruz2, Bishnu Adhikari3, Young Min Kwon3, Juan D. Latorre3, Xochitl Hernandez-Velasco1, Benjamin Fuente-Martinez4, Billy M. Hargis3, Raquel Lopez-Arellano2 and Guillermo Tellez-Isaias3* Raquel Lopez-Arellano2 and Guillermo Tellez-Isaias3* * Correspondence: gtellez@uark.edu * Correspondence: gtellez@uark.edu * Correspondence: gtellez@uark.edu 3Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, 1260 W. Maple, POSC 0-114, Fayetteville, AR 72704, USA USA Full list of author information is available at the end of the article Full list of author information is available at the end of the article (2021) 12:23 (2021) 12:23 Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 https://doi.org/10.1186/s40104-021-00545-7 Leyva-Diaz et al. Journal of Animal Science and Biotechnology https://doi.org/10.1186/s40104-021-00545-7 Abstract Background: Interest in the use of natural feed additives as an alternative to antimicrobials in the poultry industry has increased in recent years because of the risk of bacterial resistance. One of the most studied groups are polyphenolic compounds, given their advantages over other types of additives and their easy potentiation of effects when complexes are formed with metal ions. Therefore, the objective of the present study was to evaluate the impact of dietary supplementation of copper acetate (CA), curcumin (CR), and their combination (CA-CR) against Salmonella Typhimurium colonization, intestinal permeability, and cecal microbiota composition in broiler chickens through a laboratory Salmonella infection model. S. Typhimurium recovery was determined on day 10 post-challenge by isolating Salmonella in homogenates of the right cecal tonsil (12 chickens per group) on Xylose Lysine Tergitol-4 (XLT-4) with novobiocin and nalidixic acid. Intestinal integrity was indirectly determined by the fluorometric measurement of fluorescein isothiocyanate dextran (FITC-d) in serum samples from blood obtained on d 10 post-S. Typhimurium challenge. Finally, microbiota analysis was performed using the content of the left caecal tonsil of 5 chickens per group by sequencing V4 region of 16S rRNA gene. Results: The results showed that in two independent studies, all experimental treatments were able to significantly reduce the S. Typhimurium colonization in cecal tonsils (CT, P < 0.0001) compared to the positive control (PC) group. However, only CA-CR was the most effective treatment in reducing S. Typhimurium counts in both independent studies. Furthermore, the serum fluorescein isothiocyanate dextran (FITC-d) concentration in chickens treated with CR was significantly lower when compared to PC (P = 0.0084), which is related to a decrease in intestinal permeability and therefore intestinal integrity. The effect of dietary treatments in reducing Salmonella was further supported by the analysis of 16S rRNA gene sequences using Linear discriminant analysis effect size (LEfSe) since Salmonella was significantly enriched in PC group (LDA score > 2.0 and P < 0.05) compared to other groups. In (Continued on next page) Background l ll Furthermore, reactive oxygen species produced by cop- per ions induce further damage to cellular structures and even DNA degradation [18]. Salmonella, a Gram-negative intracellular bacteria, is a food-borne pathogen that can cause gastroenteritis and severe systemic infections in humans [1–3], as well as significant economic losses in poultry production be- cause it can cause high mortality and affect growth per- formance parameters in broiler chickens [4, 5]. Recently, it has been reported that the global incidence of salmon- ellosis cases has increased, estimating that of the ap- proximately 94 million reported cases, 155,000 lead to death each year [6]. Furthermore, the estimated costs of medical expenses, sick leaves, and loss of productivity range from US$1.3 to US$4.0 billion a year in the United States of America (USA) [7]. Another alternative is curcumin (CR), a mixture of polyphenolic compounds obtained from the rhizome or root of the Curcuma longa plant, member of the Zingiberaceae or ginger family that is characterized by its excellent antioxidant, anti-inflammatory, and im- munomodulatory properties, as well as its antimicro- bial and growth-promoting effects [19–21]. However, an essential limitation of CR is its low solubility and permeability. Recent studies performed by our labora- tories have shown that the use of solid dispersions with polyvinylpyrrolidone can increase these bio- pharmaceutical properties [22, 23]. Therefore, the ob- jective of the present study was to evaluate the effect of dietary supplementation of CA, CR, and their com- bination (CA-CR) against S. Typhimurium colonization, intestinal permeability, and cecal micro- biota composition using a model of S. Typhimurium infection in broiler chickens. It is known that young chickens are more susceptible to colonization by Salmonella [8], with the cecum being the target site for establishing chronic infection [9]. Al- though the genus Salmonella consists of more than 2600 serovars, the most common serotypes isolated from chicken-associated outbreaks are Salmonella enterica serovar Enteritidis (20%) and Salmonella enterica sero- var Typhimurium (17%) [10, 11]. Nowadays, due to regulations on the use of antibiotics in poultry production derived from the problems of bac- terial resistance, and considering that S. Typhimurium is a bacterium capable of developing antimicrobial resist- ance more quickly than other Salmonella species [12, 13], several strategies have been proposed to treat and control Salmonella infections [14]. Background l ll Among the large number of alternatives that have been tested in recent years, it has been reported that the combination of polyphenolic compounds with metal ions, such as cop- per, have potentiated antioxidant, anti-inflammatory and antimicrobial effects, having the additional advantage of reducing toxicity of metal ions due to complex forma- tion [15, 16]. Conclusions: CR treatment was the most effective treatment to reduce S. Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota. Keywords: Broiler chickens, Copper acetate, Curcumin, Intestinal permeability, Microbiota composition, Salmonella Typhimurium Conclusions: CR treatment was the most effective treatment to reduce S. Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota. Keywords: Broiler chickens, Copper acetate, Curcumin, Intestinal permeability, Microbiota composition, Salmonella Typhimurium © 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. Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 2 of 12 (Continued from previous page) addition, Coprobacillus, Eubacterium, and Clostridium were significantly higher in the PC group compared to other treatment groups. On the contrary, Fecalibacterium and Enterococcus in CR, unknown genus of Erysipelotrichaceae at CA-CR, and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively. Conclusions: CR treatment was the most effective treatment to reduce S Typhimurium intestinal colonization and (Continued from previous page) addition, Coprobacillus, Eubacterium, and Clostridium were significantly higher in the PC group compared to other treatment groups. On the contrary, Fecalibacterium and Enterococcus in CR, unknown genus of Erysipelotrichaceae at CA-CR, and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively. Conclusions: CR treatment was the most effective treatment to reduce S. Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota. Keywords: Broiler chickens, Copper acetate, Curcumin, Intestinal permeability, Microbiota composition, Salmonella Typhimurium g addition, Coprobacillus, Eubacterium, and Clostridium were significantly higher in the PC group compared to other treatment groups. On the contrary, Fecalibacterium and Enterococcus in CR, unknown genus of Erysipelotrichaceae at CA-CR, and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively. treatment groups. On the contrary, Fecalibacterium and Enterococcus in CR, unknown genus of Erysipelotrichaceae at CA-CR, and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively. Preparation of experimental treatments and diets Preparation of experimental treatments and diets CR treatment consisted of a solid dispersion of curcumin with polyvinylpyrrolidone in a 1:9 ratio previously de- scribed [22, 23], CA treatment was copper(II) acetate hy- drate (98%, Catalog No. 341746, Sigma), and CA-CR treatment consisted of a mixture of the previous treat- ments. Solid dispersion of curcumin was prepared by dissolving 1 part of curcumin in 9 parts of a polyvinyl- pyrrolidone (PVP) K30 solution, followed by water evap- oration at 40 °C and sieving. Mash corn-soybean-based broiler starter basal diet was formulated to approximate the nutritional requirements of broiler chickens, as rec- ommended by the National Research Council [24] and then adjusted to breeder’s recommendations [25]. No antibiotics, coccidiostats or enzymes were added to the feed (Table 1). All animal handling procedures complied with the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (protocol #18029). Copper compounds such as copper acetate (CA) are believed to promote growth by regulating gastrointes- tinal microbiota through bactericidal and bacteriostatic effects [17]. The mechanisms that explain the antimicro- bial effect of the copper ion are related to direct damage to the bacteria’s membrane, which generates a loss of membrane potential and cytoplasmic content. Leyva-Diaz et al. Animal source and experimental design In the present study, two independent trials with 75 day- of-hatch male Cobb-Vantress broiler chickens (Fayette- ville, AR, USA) were conducted. Chicks were individually weighed and randomly assigned to one of five groups (n = 15 chickens/group): 1) Negative control (NC, basal diet); 2) Positive control (PC, basal diet + challenged with 104 CFU of S. Typhimurium per bird on hatching day); 3) CA (basal diet supplemented with 250 mg/kg of copper(II) acetate hydrate + challenged with 104 CFU of S. Typhi- murium per bird on hatching day); 4) CR (basal diet sup- plemented with 0.2% curcumin + challenged with 104 CFU of S. Typhimurium per bird on hatching day); and 5) CA-CR (basal diet supplemented with 250 mg/kg of cop- per (II) acetate hydrate and 0.2% curcumin + challenged with 104 CFU of S. Typhimurium per bird on hatching day). In both trials, chicks were raised in floor pens (118 in × 59 in), provided with their diet, water ad libitum, and maintained at an age-appropriate temperature during all experiments. Body weight (BW) and body weight gain (BWG) were evaluated at 10 days of age. On day ten post- S. Typhimurium challenge, chickens were given an appro- priate dose of fluorescein isothiocyanate dextran (FITC-d) by oral gavage 1 h before the chickens were euthanized by CO2 inhalation and only the right cecal tonsil (CT) from 12 broilers per group were aseptically collected for S. Typhimurium recovery. Furthermore, blood samples were also collected from the femoral vein for the determination of FITC-d. The concentration of FITC-d administered was calculated based on group body weight at day nine post-S. Typhimurium challenge. For microbiota analysis, the content of the left CT was collected aseptically and stored at −20 °C until analysis. The number of broilers chosen per group for each determination was based on re- producible results from experiments previously described and published by our laboratory [22, 23]. bMineral premix supplied per kg of diet: Mn, 120 mg; Zn, 100 mg; Fe, 120 mg; copper, 10 to 15 mg; iodine, 0.7 mg; selenium, 0.2 mg; and cobalt, 0.2 mg Salmonella strain and culture conditions The poultry strain of Salmonella Typhimurium (PHL- 2020) was obtained from the USDA National Veterinary Services Laboratory (Ames, IA, USA). This strain was se- lected for resistance to 25 μg/mL of novobiocin (NO, Catalog No. N-1628, Sigma) and 20 μg/mL of nalidixic acid (NA, Catalog No. N-4382, Sigma) in our laboratory. In the present study, 100 μL of S. Typhimurium from a frozen aliquot was added to 10 mL of tryptic soy broth (TSB, Catalog No. 22092, Sigma, St. Louis, MO, USA), incubated at 37 °C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase as Preparation of experimental treatments and diets Journal of Animal Science and Biotechnology (2021) 12:23 Page 3 of 12 Salmonella strain and culture conditions The poultry strain of Salmonella Typhimurium (PHL- 2020) was obtained from the USDA National Veterinary ( ) Table 1 Ingredient composition and nutrient content of a basal starter diet used in the experiment on as-fed basis Item Corn soybean-based diet Ingredients, % Corn 57.34 Soybean meal 34.66 Poultry fat 3.45 Dicalcium phosphate 1.86 Calcium carbonate 0.99 Salt 0.38 DL-Methionine 0.33 L-Lysine HCl 0.31 Threonine 0.16 Vitamin premixa 0.20 Mineral premixb 0.10 Choline chloride 60% 0.20 Calculated analysis Metabolizable energy, kcal/kg 3035 Crude protein, % 22.16 Ether extract, % 5.68 Lysine, % 1.35 Methionine, % 0.64 Methionine + cystine, % 0.99 Threonine, % 0.92 Tryptophan, % 0.28 Total calcium, % 0.90 Available phosphorus, % 0.45 Determined analysis Crude protein, % 21.15 Ether extract, % 6.05 Calcium, % 0.94 Phosphorus, % 0.73 aVitamin premix supplied per kg of diet: retinol, 6 mg; cholecalciferol, 150 μg; DL-α-tocopherol, 67.5 mg; menadione, 9 mg; thiamine, 3 mg; riboflavin, 12 mg; pantothenic acid, 18 mg; niacin, 60 mg; pyridoxine, 5 mg; folic acid, 2 mg; biotin, 0.3 mg; cyanocobalamin, 0.4 mg bMineral premix supplied per kg of diet: Mn, 120 mg; Zn, 100 mg; Fe, 120 mg; copper, 10 to 15 mg; iodine, 0.7 mg; selenium, 0.2 mg; and cobalt, 0.2 mg previously described [26]. Post-incubation, bacteria were washed three times with sterile 0.9% saline by centrifu- gation at 1864×g for 10 min, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20 DC, Spectronic Instruments Thermo Sci- entific, Rochester, NY, USA) and finally diluted to an ap- proximate concentration of 104 CFU/mL. Levels of S. Typhimurium were further verified by serial dilutions and plated on brilliant green agar (BGA, Catalog No. 70134, Sigma, St. Louis, MO, USA) with NO and NA for enumeration of actual CFU used in the experiment. Serum determination of FITC-d leakage Serum determination of FITC-d leakage FITC-d (MW 3–5 kDa; Sigma-Aldrich Co., St. Louis, MO, USA) was provided by oral gavage to 12 broiler chickens from each group at a dose of 8.32 mg/kg of body weight 1 h before the chicks were euthanized by CO2 inhalation with the purpose of evaluating the para- cellular transport and mucosal barrier dysfunction [27, 28]. Three remaining broiler chickens of each group were used as controls. The blood samples were centrifuged (1000×g for 15 min) to separate the serum. Then, serum samples obtained were diluted (1:5) and measured fluoro- metrically at an excitation wavelength of 485 nm and an emission wavelength of 528 nm (Synergy HT, Multi-mode microplate reader, BioTek Instruments, Inc., VT, USA) to determine the serum FITC-d levels [29]. Salmonella recovery In both independent trials, the right CT samples from 12 chickens per group were individually homogenized and diluted with saline (1:4; w/v), and 10-fold dilutions were plated on Xylose Lysine Tergitol-4 (XLT-4, Catalog Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 4 of 12 Leyva-Diaz et al. Journal of Animal Science and Biotechnology samples have shown reliable results since even omitting outliers the trend is the same [33, 34]. No. 223410, BD DifcoTM) with NO and NA for S. Typhimurium recovery. Plates were incubated at 37 °C for 24 h to enumerate total S. Typhimurium colony- forming units. Subsequently, the CT samples were enriched in 2× concentrated tetrathionate enrichment broth and further incubated at 37 °C for 24 h. Enrich- ment samples were streaked onto XLT-4 with NO and NA selective media for confirmation of Salmonella pres- ence. Samples that were negative in the plate dilution method but positive after enrichment with tetrathionate received an arbitrary value of 500 CFU/g (lower limit of detection). Amplicons sequence analysis Nebula cloud computing platform of the University of Arkansas was used to process raw sequencing reads in QIIME 2 version 2018.8 utilizing the pipelines developed for paired-end data types [35]. In sum, “demux emp- paired” method of q2-demux plugin was used to demul- tiplex sequencing reads, followed by quality filtering and denoising with “dada2 denoise-paired” method of q2- dada2 [36] plugin available at QIIME 2. The truncation length of forward and reverse reads were set at 220 and 200 bp, respectively, which was based on the quality score criteria (≥30). Taxonomic assignment was per- formed using a Naive Bayes classifier pre-trained with Greengenes (version 13.8) 99% OTUs [37] and q2- feature-classifier plugin, where the sequences have been trimmed to include only the V4 region of the 16S rRNA gene region which is defined by the 515F/806R primer pair. We detected the sequence reads assigned to Chloroplast and Mitochondria, which were subsequen- cingtly removed using taxonomy-based filtering option in QIIME2. The core-metrics-phylogenetic method at a sampling depth of 69,566 was used to analyze Alpha and Beta diversity. Observed OTUs were used to calculate alpha diversity, while weighted UniFrac distance and un- weighted UniFrac distance metrics were used for beta di- versity analysis. All figures were created using ggplot2 packages of R [38]. Data and statistical analysis y After demonstrating that data from S. Typhimurium counts (log10 CFU/g), serum determination of FITC-d leakage, BW and BWG presented a normal distribution and homogeneity of the variances using the Levene and Ryan-Joiner procedures, respectively, these data were subjected to analysis of variance (ANOVA) as a com- pletely randomized design using the General Linear Models procedure of Statistical Analysis System (SAS®) [39]. Significant differences among the means were de- termined by Duncan’s multiple range test at p < 0.05. En- richment data were expressed as positive/total chickens (%), and the percentage of S. Typhimurium positive sam- ples were compared by a Chi-square test of independ- ence [40], testing all possible combinations to determine the significance (P < 0.05). V4 region of 16S rRNA gene from the genomic DNA of each of the 25 samples of cecal content (5 samples per group × 5 groups) was amplified using the primers 515F [30] and 806R [31]. The library of amplicons for DNA sequencing was prepared according to the 16S Illumina PCR protocol described in the Earth Microbiome Project (http://www.earthmicrobiome.org) with slight modifica- tions [32]. In brief, Q5® High-Fidelity DNA Polymerase user guide protocol (New England Biolabs, Catalog No. M0491S) was used to conduct PCR in a 25-μL final reac- tion volume via 30 amplification cycles. The length of the amplified product was confirmed with 1% agarose gel electrophoresis, and equal amount (~ 300 ng) of the amplicons from each sample as measured by Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Catalog No. Q32850) were pooled together. The pooled ampli- cons were finally run on 1% agarose gel electrophoresis, purified using Zymoclean Gel DNA Recovery Kit (Zymo Research, Catalog No. D4007), and sequenced with Illu- mina MiSeq paired-end 300 cycle options at Admera Health, LLC (New Jersey, USA). Despite the small num- ber of samples analyzed, previous studies using 6 Statistical differences of bacterial taxa at different levels (family and genus) among treatment groups were determined using linear discriminant analysis effect size (LEfSe) using all against all comparison mode, where the level of significance was set at LDA score > 2.0 and P < 0.05 [41]. The significant differences in alpha diversity were calculated using an alpha-group-significance Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 5 of 12 Page 5 of 12 (2021) 12:23 Leyva-Diaz et al. Data and statistical analysis Journal of Animal Science and Biotechnology the samples of the experimental groups, with the excep- tion of NC. command of QIIME2, which is based on the Kruskal- Wallis test. Statistical differences in beta diversity among groups were calculated by PERMANOVA [41] test using a beta-group-significance command of QIIME2 with a pair- wise option. For both diversities analysis, the corrected P values for multiple comparisons (q) were used to report a significant difference between the two groups, where the level of significance was set at q < 0.05. Table 2 shows the results of the dietary administration of CA, CR, and CA-CR on serum FITC-d concentration in broiler chickens on day ten post-S. Typhimurium challenge. In both trials, there were no significant differ- ences in the serum FITC-d concentration when the CA and CA-CR groups were compared to groups PC and NC. However, the serum FITC-d concentration in chick- ens treated with CR was significantly lower when com- pared to PC (P = 0.008), but there were no significant differences when compared to NC. Results The results of the antimicrobial effect of CA, CR, and CA-CR on S. Typhimurium colonization in the CT of broiler chickens in trial one and trial two are summa- rized in Table 2. In both trials, all experimental treat- ments were able to significantly reduce the S. Typhimurium colonization in CT (P < 0.0001) when compared to the PC group. However, CR and CA-CR were the most effective treatments, since they reduced the colonization of S. Typhimurium more than 2.1 and 2.3 log10 (P = 0.002 and P = 0.008), respectively, com- pared to PC. Although the data are not presented in Table 2, the presence of Salmonella was confirmed in all The effect of the dietary inclusion of the treatment into the feed on the body weight at day 10, as well as the body weight gained of the broilers in each of the inde- pendent studies is summarized in Table 3. At the begin- ning of the experiment, no significant differences were shown in the weights of the broilers. However, at day 10, only the group treated with CR presented significant dif- ferences in BW compared to PC. Furthermore, at the end of the experiments, BWG increased significantly in the group treated with CR when compared to PC. Although the groups treated with CA and CA-CR did not show significant differences in BW on day 10 and BWG, a tendency to improve these parameters was Table 2 Evaluation of copper acetate (CA), curcumin (CR) and copper acetate – curcumin (CA-CR) on cecal tonsils (CT) colonization of Salmonella Typhimurium1 and serum concentration of fluorescein isothiocyanate-dextran (FITC-d) in broiler chickens on day ten post-S. Typhimurium challenge2 in trial 1 and trial 2 Table 3 Evaluation of copper acetate (CA), curcumin (CR) and copper acetate-curcumin (CA-CR) on body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) in broiler chickens on day ten post-S. Typhimurium challenge in trial 1 and trial 21 Table 3 Evaluation of copper acetate (CA), curcumin (CR) and copper acetate-curcumin (CA-CR) on body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) in broiler chickens on day ten post-S. Discussion Modern animal production has been changing in recent years due to the problems of bacterial resistance derived from the overuse of antimicrobials for prophylactic and growth promotion purposes [42, 43]. In this regard, many investigations have focused on probiotics, prebi- otics, enzymes, acidifiers, plant extracts, and some metals (copper and zinc) as feed additives, given their antimicrobial properties and effects in promoting growth, mainly [44]. In the present study, selection of CA was based on its advantages over inorganic sources since it has been described that inorganic sources tend to dissociate in the upper part of the gastrointestinal tract, causing a decrease in the availability of copper due to its interaction with other metals (chelation) and therefore a reduction in its activity [18, 45]. In contrast, the solubility of organic sources of copper is higher in weak acid enviroments, making their dissolution slower and increasing their availability and activity [45]. Fur- thermore, lower fecal copper excretion rates have been reported in broilers exposed to an organic source of cop- per compared to inorganic sources [46]. Although the copper ion is known to be more effective against Gram- positive bacteria [47], dietary supplementation with CA significantly reduced more than 18% the colonization of S. Typhimurium in both trials compared to PC group (Table 2). Copper ion has been reported to cause dam- age at the bacterial membrane level due to its adhesion to membranes and the generation of reactive oxygen species [48]. Additionally, it can be associated with the functional groups of proteins and enzymes, leading to the inactivation or inhibition of some cellular processes, as well as having a direct negative effect on the genetic material of bacteria [47, 48]. In addition, this reduction in the colonization of S. Typhimurium presented a posi- tive effect on BW and BWG since they tend to improve in both experiments (P = 0.081 and P = 0.085, respect- ively), when compared to PC. Alpha diversity analysis among the groups, as mea- sured by observed OTUs is shown in Fig. 4. Although there were no significant differences among the groups (Kruskal-Wallis test; P > 0.05), it was observed that PC Fig. 1 Taxonomic composition of the cecal microbiota in the different treatments at family level. NC: negative control, PC: positive control, CA: cooper acetate, CR: curcumin, and CA-CR: copper acetate-curcumin. Results Typhimurium challenge in trial 1 and trial 21 Treatments BW, g/broiler (D 0) BW, g/broiler (D 10) BWG, g/broiler (D 0–10) Trial 1 CTRL (−) 40.60 ± 0.57 232.33 ± 9.46 ab 191.73 ± 9.39 ab CTRL (+) 40.13 ± 0.86 204.97 ± 10.06 b 164.83 ± 10.05 b CA 40.67 ± 0.70 227.50 ± 9.32 ab 186.83 ± 9.40 ab CR 40.87 ± 1.01 237.93 ± 7.80 a 197.07 ± 7.60 a CA-CR 40.47 ± 0.62 226.8 ± 8.75 ab 186.33 ± 8.83 ab SEM2 0.34 4.17 4.15 P-value 0.973 0.122 0.134 Trial 2 CTRL (−) 40.93 ± 0.64 231.83 ± 7.54 ab 190.90 ± 7.28 ab CTRL (+) 40.07 ± 0.95 205.6 ± 7.75 b 165.53 ± 7.90 b CA 40.40 ± 0.79 226.83 ± 8.82 ab 186.43 ± 8.66 ab CR 41.27 ± 0.71 236.63 ± 8.00 a 195.37 ± 8.04 a CA-CR 40.40 ± 0.84 226.6 ± 10.51 ab 186.2 ± 10.68 ab SEM2 0.35 3.94 3.92 P-value 0.833 0.119 0.143 1 Data expressed as mean ± SE from 15 chickens. a -b Values within columns with different superscripts differ significantly (P < 0.05) 2 Standard error of the means trial 1 and trial 2 Treatments CT, log10 CFU/g FITC-d, ng/mL Trial 1 CTRL (−) 0.00 ± 0.00 d 17.03 ± 5.44 b CTRL (+) 6.18 ± 0.33 a 54.99 ± 10.51 a CA 4.99 ± 0.32 b 35.19 ± 8.80 ab CR 3.92 ± 0.55 bc 17.60 ± 7.50 b CA-CR 3.76 ± 0.54 c 32.99 ± 10.34 ab SEM3 0.33 4.18 P-value 0.000 0.020 Trial 2 CTRL (−) 0.00 ± 0.00 d 19.80 ± 9.26 b CTRL (+) 6.09 ± 0.276 a 59.38 ± 9.81 a CA 4.94 ± 0.32 b 32.99 ± 11.31 ab CR 3.91 ± 0.19 c 15.40 ± 7.60 b CA-CR 3.78 ± 0.31 c 39.59 ± 15.06 ab SEM3 0.30 5.13 P-value 0.000 0.048 1 Data expressed in log10 CFU/g of tissue. Mean ± SE from 12 chickens. a–d Values within treatments columns for each treatment with different superscripts differ significantly (P < 0 .05) 2 Chickens were orally gavaged with 104 CFU of S. Typhimurium per chicken at 1 day old, samples were collected at day 10 post-challenge 3 Standard error of the means Page 6 of 12 Leyva-Diaz et al. Results Journal of Animal Science and Biotechnology (2021) 12:23 observed in comparison with PC (P = 0.085 and P = 0.119, respectively). group presented a numerically lower diversity compared to the other groups. Cecal microbiota was analyzed in samples collected from day ten post-S. Typhimurium challenged birds. The relative abundance of different bacterial families re- covered across different groups is shown in Fig. 1. In all five groups, either Ruminococcaceae or Lachnospiraceae were the most predominant families. Lachnospiraceae was the most dominant bacterial family in NC (44.10%) and CA (46.37%) followed by Ruminococcaceae (NC, 33.23%; CA, 32.86%). However, Ruminococcaceae was found the highest in PC (36.50%), CR (44.17%), and CA- CR (63.12%) followed by Lachnospiraceae (PC, 31.37%; CR, 31.78%; CA-CR, 17.69%). LEfSe analysis (LDA score > 2.0 and P < 0.05) revealed some important differ- entially abundant taxa at both bacterial family and genus level. As shown in Fig. 2, Enterococcaceae was signifi- cantly higher in the PC group while Clostridiaceae was significantly enriched in CR group. Furthermore, at genus level, Salmonella, Coprobacillus, Eubacterium, and Clostridium were significantly abundant in PC, while the genera Fecalibacterium and Enterococcus were significantly enriched in CR group and the unknown genera that belong to Erysipelotrichaceae and Lachnos- piraceae were significantly enriched in CA-CR and CA groups, respectively (Fig. 3). To assess the accuracy of the taxonomic assignment of the reads matching to genus Salmonella, we did BLAST analysis using the cor- responding amplicon sequence variant (ASV) sequence as shown in Supplementary Materials, which strongly support correct identification of genus Salmonella. Beta diversities among different groups as measured by weighted and unweighted UniFrac distance metrics are illustrated in PCoA plots (Fig. 5a and b, respectively). Results of the Permutational multivariate analysis of variance (PERMANOVA) showed that there were no significant differences in the structure of the microbial community among the groups at q < 0.05. Discussion “NA” refers to the bacterial taxa that were not assigned to the family level but were assigned at the higher taxonomic level. “Others” represent the minor bacterial families whose relative abundance were < 0.1% Fig. 1 Taxonomic composition of the cecal microbiota in the different treatments at family level. NC: negative control, PC: positive control, CA: cooper acetate, CR: curcumin, and CA-CR: copper acetate-curcumin. “NA” refers to the bacterial taxa that were not assigned to the family level but were assigned at the higher taxonomic level. “Others” represent the minor bacterial families whose relative abundance were < 0.1% Fig. 1 Taxonomic composition of the cecal microbiota in the different treatments at family level. NC: negative control, PC: positive control, CA: cooper acetate, CR: curcumin, and CA-CR: copper acetate-curcumin. “NA” refers to the bacterial taxa that were not assigned to the family level but were assigned at the higher taxonomic level. “Others” represent the minor bacterial families whose relative abundance were < 0.1% In the case of the group treated with the solid disper- sion of curcumin (CR), which was previously described by our research group and is characterized by being more soluble and permeable [22], the colonization of S. Typhimurium significantly decreased by more than 35% (more than 2 log10) with respect to the PC group after Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 7 of 12 Leyva-Diaz et al. Journal of Animal Science and Biotechnology Fig. 2 Taxonomic difference of the main families in the microbiota between the curcumin-treated group (CR) and the positive control (PC) After oral infection with Salmonella, this pathogen must overcome the conditions of the gastrointestinal tract to interact with the intestinal epithelium [55]. Inva- sion of epithelial layers by S. Typhimurium is known to increase intestinal permeability in both in vivo and in vitro models since the expression of some markers such as claudin-1, occludin, and mucin-2, mRNA levels of zonula occludens-1 and E-cadherin was reduced [55, 56]. In the present study, FITC-d, a large molecule (3–5 kDa) that, under normal intestinal health conditions, does not leak through the epithelium, was used to assess intestinal permeability. However, when there is damage to the epithelium, the permeability of FITC-d increases so that it can be quantified in serum [57]. In the present study, all treated groups showed lower serum FITC-d concentrations compared to the PC group (Table 2). Discussion However, only the group treated with CR had signifi- cantly lower concentrations when compared to PC and turned out to have serum FITC-d concentrations com- parable to the NC group. Perhaps, this result is due to the ability of CR to restore the intestinal barrier function 10 days of treatment (Table 2). These results are due to the antimicrobial action of curcumin, which in general, is associated with damage to the bacterial membrane and inhibition of bacterial cell proliferation [49, 50]. Furthermore, it has been published that curcumin can induce some physical and mechanical changes of the S. Typhimurium flagellar filament, causing a de- crease in motility, adherence, and invasion of the host cells, which results in a reduction or elimin- ation of its virulence [51]. Likewise, curcumin has been reported to decrease bacterial cell division pro- cesses since it interacts with the FtsZ protein, a cytoskeleton protein essential for this process [52]. The treatment containing the mixture of CA and CR (CA-CR) reduced 2% and 37% the S. Typhimurium colonization compared to the group treated with CR and the PC group, respectively. These results con- trast with those obtained in other articles where the combination of curcumin with heavy metals, includ- ing copper showed better effects and even decrease the toxicity of metals [16, 53, 54]. 10 days of treatment (Table 2). These results are due to the antimicrobial action of curcumin, which in general, is associated with damage to the bacterial membrane and inhibition of bacterial cell proliferation [49, 50]. Furthermore, it has been published that curcumin can induce some physical and mechanical changes of the S. Typhimurium flagellar filament, causing a de- crease in motility, adherence, and invasion of the host cells, which results in a reduction or elimin- ation of its virulence [51]. Likewise, curcumin has been reported to decrease bacterial cell division pro- cesses since it interacts with the FtsZ protein, a cytoskeleton protein essential for this process [52]. The treatment containing the mixture of CA and CR (CA-CR) reduced 2% and 37% the S. Typhimurium colonization compared to the group treated with CR and the PC group, respectively. These results con- trast with those obtained in other articles where the combination of curcumin with heavy metals, includ- ing copper showed better effects and even decrease the toxicity of metals [16, 53, 54]. Fig. Discussion 3 Taxonomic composition of the microbiota in the groups treated at the genus level. PC: positive control, CA: cooper acetate, CR: curcumin, and CA-CR: copper acetate – curcumin Fig. 3 Taxonomic composition of the microbiota in the groups treated at the genus level. PC: positive control, CA: cooper acetate, CR: curcumin, and CA-CR: copper acetate – curcumin Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 8 of 12 Page 8 of 12 Leyva-Diaz et al. Journal of Animal Science and Biotechnology Fig. 4 Comparison of the alpha diversities among the groups as measured by observed OTUs FITC-d concentration only decreased numerically since it has been described that the production of reactive oxygen species by copper affects not only bacteria but also epithelial cells [60]. The chicken gut microbiota are densely populated with complex microbial communities that are involved in digestion and metabolism, regulation of enterocytes, vitamin synthesis, and development and regulation of the host immune system [61]. Cecum is by far the most densely colonized microbial habitat in chickens [62]. Despite the absence of any clinical signs of Salmonella infection, it has been reported that the composition of the microbiota is affected, but the changes are quite weak at the level of the caecal tonsils [63, 64], which supports our results since no significant differences in alpha (measured by the observed OTUs) and beta diver- sity were observed in the cecal samples at day ten post- S. Typhimurium challenge. This means that there were no changes in the relationship of the number of different species per sample (richness) and in the diversity of the microbial community between different samples, re- spectively [65]. Notwithstanding the above, the taxo- nomic composition showed some significant differences at the family and genus levels when the groups were compared. At the family level, abundance of Enterococcaceae was lower in all groups supplemented with CA when compared to the PC group. Enterococcaceae, one of the six families of the order Lactobacillales [66], is com- prised of the genera Enterococcus, Bavariicoccus, Catelli- coccus, Melissococcus, Pilibacter, Tetragenococcus, and Vagococcus [67]. However, it has been described that the dietary copper supplementation alters the intestinal and the expression of proteins associated with the tight junctions, the proliferation-regeneration of the intestinal epithelium, and its antimicrobial action, resulting in de- creased paracellular permeability as has been previously reported [58, 59]. Discussion Regarding the treatments with CA and CA-CR, although the S. Typhimurium counts decreased significantly compared to the PC group, the serum Fig. 5 PCoA plots showing beta diversity among the groups according to a weighted UniFrac distance metric and b unweighted UniFrac distance metric Fig. 5 PCoA plots showing beta diversity among the groups according to a weighted UniFrac distance metric and b unweighted UniFrac distance metric Leyva-Diaz et al. Journal of Animal Science and Biotechnology (2021) 12:23 Page 9 of 12 (2021) 12:23 microbiota, decreasing the abundance of Enterococca- ceae due to the total reduction of lactic acid bacteria [68]. In contrast, Salmonella infection is known to in- crease the relative abundance of Enterococcaceae, Lactobacillaceae, Clostridiaceae, Lachnospiraceae, Erysi- pelotrichaceae, Peptostreptococcaceae, and Ruminococ- caceae, but decrease that of Enterobacteriaceae [69]. Furthermore, the family of Clostridiaceae was signifi- cantly lower in chickens whose diet contained CR in common compared to PC. Clostridiaceae is one of the responsible families for converting polysaccharides into short-chain fatty acids (SCFAs) [70]. It has been described that SCFAs such as acetate, propionate, and butyrate, are important in maintaining intestinal homeo- stasis due to their immunomodulatory capacity, main- tenance of metabolism, proliferation, differentiation and promotion at low pH, favoring beneficial bacteria, and reducing the growth and viability of pathogenic bacteria [71]. Therefore, these results support the lower Salmon- ella counts in CT and the improvement in BW and BGW in the CR treated group. However, in the case of the group treated with CR, the in- crease in Faecalibacterium, a genus of bacteria responsible for the production of butyrate and related to health benefits in poultry, could be mainly due to the prebiotic effect of cur- cumin, like other substances with the same activity [76]. It has been described that CR could act as a factor of promo- tion, proliferation, growth, and survival for the beneficial bac- teria of the intestinal microbiota from its biotransformation [77]. Finally, the bacterial genera that belong to Erysipelotri- chaceae and Lachnospiraceae were significantly enriched in the CA-CR and CA groups, respectively. It has been pub- lished that in chickens infected with Salmonella this genus of bacteria decreases markedly, which could negatively affect the diversity and development of intestinal bacteria [69]. Supplementary Information Th li i i l Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40104-021-00545-7. pp y The online version contains supplementary material available at https://doi. org/10.1186/s40104-021-00545-7. Additional file 1. Identification of genus Salmonella in microbiota analysis Additional file 1. Identification of genus Salmonella in microbiota analysis Furthermore, the genus Faecalibacterium and Entero- coccus were significantly enriched in the group treated with CR. After infection with Salmonella, this pathogenic bacteria alters the intestinal microbiota, causing a decrease in bacteria of the genus Blautia, Enorma, Faecalibacterium, Shuttle- worthia, Sellimonas, Intestinimonas, and Subdoligranulum, as well as an increase in the abundance of Butyricicoccus, Erysipelatoclostridium, Oscillibacter and Flavonifractor [61]. Discussion In the specific case of CA and CA-CR, copper is known to in- crease the relative abundance of these bacterial genera, which are the most active microbial components in the healthy gut and are responsible for preventing the production of inflam- matory cytokines and induce intestinal production of SCFAs by fermenting carbohydrates [78, 79]. Although the sample size for microbiome analysis is small, the results are promis- ing and suggestive since there is a close relationship with what was observed in the other determinations. At the genus level, Salmonella, Coprobacillus, Eubacter- ium, and Clostridium were significantly enriched in the PC group, which is closely related to the severity of the Sal- monella infection process. Coprobacillus, Clostridium, and Eubacterium have an important role in the production of SCFAs essential amino acids and the digestion of non- starch polysaccharides, which stimulate the production of SCFAs for metabolic balance [70, 72]. Likewise, it has been reported that the reduction of Clostridium and the main- tenance of Eubacterium and Coprobacillus levels could be related to the effectiveness of the treatments since they rep- resent a positive effect in the maintenance of intestinal homeostasis [72–74]. Regarding the high abundance of Sal- monella, it has been reported that it is related to its colonization in CT [75]. Although sequencing of the V4 re- gion of 16S rRNA gene is not able to distinguish between Enterobacteriaceae, BLAST analysis using an amplicon se- quence variant (ASV) that matched to genus Salmonella strongly supports that the taxonomic assignment of this ASV to genus Salmonella in this study was accurate (see Supplementary Materials). Hence, these results confirm again the effect of the treatments, especially CR, on the de- crease in Salmonella counts, the maintenance of intestinal integrity as indirectly measured by the serum FITC-d con- centration, and the improvement in BW and BWG. Conclusion d According to the previous results, it can be concluded that the treatment with CR was the most effective in re- ducing S. Typhimurium counts. Furthermore, it was de- termined that the antimicrobial activity of CR, when administered at 0.2% into the feed using an S. Typhi- murium infection laboratory model, is based on a com- bined mechanism in which direct activity on pathogenic bacteria and the prebiotic effect is mainly involved. Fi- nally, it is clear that physical mixtures of CR with a metal such as copper (CA) are not effective, increasing the antimicrobial effect. Studies to confirm and expand these results with a larger number of animals and sam- ples, and considering the analysis of inflammatory and antioxidant biomarkers to get a complete description of CR required further investigation. References 1. Uzzau S, Brown DJ, Wallis T, Rubino S, Leori G, Bernard S, et al. Host adapted serotypes of Salmonella enterica. Epidemiol Infect. 2000;125:229–55. 1. Uzzau S, Brown DJ, Wallis T, Rubino S, Leori G, Bernard S, et al. Host adapted serotypes of Salmonella enterica. Epidemiol Infect. 2000;125:229–55. 2. Ochman H, Groisman EA. The origin and evolution of species differences in Escherichia coli and Salmonella Typhimurium. 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https://openalex.org/W3102483034	https://bmcneurol.biomedcentral.com/track/pdf/10.1186/s12883-020-01985-w	English	null	Elevated blood urea nitrogen is associated with recurrence of post-operative chronic subdural hematoma	BMC neurology	2,020	cc-by	6,372	Open Access Elevated blood urea nitrogen is associated with recurrence of post-operative chronic subdural hematoma Ning Wang1,2, Jiangnan Hu3, Anthony Oppong-Gyebi3, Xuanhao Zhu1,2, Yihao Li1,2, Jianjing Yang1,2, Linhui Ruan1,2, Qichuan Zhuge1,2 and Sheng Ye1,2* Abstract Background: Chronic subdural hematoma (CSDH) is fundamentally treatable with about a 2–31% recurrence rate. Recently, there has been renewed interest in the association between Blood Urea Nitrogen (BUN) and intracranial lesion. Therefore, this paper attempts to show the relationship between BUN and CSDH recurrence. Methods: A total of 653 CSDH cases with Burr-hole Irrigation (BHI) were enrolled from December 2014 to April 2019. The analyzed parameters included age, gender, comorbidities, laboratory investigations, medication use and hematoma location. The cases were divided into recurrence and non-recurrence groups while postoperative BUN concentration was further separated into quartiles (Q1 ≤4.0 mmol/L, 4.0 < Q2 ≤4.9 mmol/L, 4.9 < Q3 ≤6.4 mmol/L, Q4 > 6.4 mmol/L). Restricted cubic spline regressions and logistic regression models were performed to estimate the effect of BUN on CSDH recurrence. Results: CSDH recurrence was observed in 96 (14.7%) cases. Significant distinctions were found between recurrence and non-recurrence groups in postoperative BUN quartiles of cases (P = 0.003). After adjusting for the potential confounders, the odds ratio of recurrence was 3.069 (95%CI =1.488–6.330, p = 0.002) for the highest quartile of BUN compared with the lowest quartile. In multiple-adjusted spline regression, a high BUN level visually showed a significantly high OR value of recurrence risk. Conclusions: Elevated BUN at post-operation is significantly associated with the recurrence of CSDH, and it is indicated that high levels of serum BUN after evacuation may serve as a risk factor for CSDH recurrence. Keywords: Chronic subdural hematoma, Blood urea nitrogen (BUN), Recurrence rate * Correspondence: yesheng342@163.com p y g @ 1Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 2Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China Full list of author information is available at the end of the article Wang et al. BMC Neurology (2020) 20:411 https://doi.org/10.1186/s12883-020-01985-w Wang et al. BMC Neurology (2020) 20:411 https://doi.org/10.1186/s12883-020-01985-w Correspondence: yesheng342@163.com 1Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 2Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Neurology (2020) 20:411 Page 2 of 10 Page 2 of 10 Wang et al. BMC Neurology (2020) 20:411 Background associated with intracranial disorders. But very little attention has been paid to the relationship between BUN and CSDH recurrence so far. Chronic subdural hematoma (CSDH) is a frequently encountered neurosurgical disorder that is common among the aged. The incidence of CSDH is estimated to be 8.2–17.6 per 100,000 persons per year which in- creases along with increasing age [1–3]. Burr-hole irriga- tion (BHI) is most widely used to treat CSDH with a relatively good outcome, but about 2–31% of patients re- lapse after the initial operation [1]. In this paper, variables associated with CSDH recur- rence were investigated, focusing on the postoperative BUN concentrations of patients with CSDH who under- went evacuation at a single facility from 2014 to 2019. This research aimed to explore the relationship between serum BUN levels and CSDH recurrence to provide a new orientation to study the pathophysiology of CSDH. Factors found to influence CSDH recurrence have been reported in several studies, including age, Glasgow coma score (GCS), antiplatelet or anticoagulant agents, bilateral hematomas, postoperative pneumocephalus, and other certain related computed tomography (CT) findings [4–7]. However, the findings of the connection between laboratory investigation and CSDH recurrence are still scarce. Patients and parameters This study enrolled admitted patients with the diagnosis of CSDH at the First Affiliated Hospital of Wenzhou Medical University, Zhejiang province, China, between December 2014 and April 2019. Patients with CSDH were diagnosed by head magnetic resonance imaging (MRI) and CT. The following exclusive criteria were used:(1) without surgical treatment;(2) younger than 18 years old;(3) severe epilepsy;(4) severe renal or blood dis- eases;(5) hematoma organized or bad operation result;(6) craniotomy or evacuated by other departments;(7) lack of laboratory investigation;(8) hospital mortality. Patient with two operations was defined as two cases. Blood urea nitrogen (BUN) concentration is an eas- ily overlooked investigation. Interestingly, there is a growing amount of literature that recognizes high BUN/creatinine(Cr) as an independent risk factor of poor outcomes in acute ischemic stroke (AIS) patients [8–10]. Furthermore, elevated BUN concentration was revealed to increase in-hospital mortality in AIS pa- tients [11]. These studies indicated that BUN was Fig. 1 CT imagings of two typical patients without and with recurrence of CSDH. a, the preoperative presentation of patient A without recurrence; b the imaging of the same patient within 48 h postoperatively; c the CT scan at 3-month follow-up of the patient A; d the presurgical imaging of patient B with recurrence of CSDH; e the presentation of patient B within 48 h postoperatively; f the CT scan of patient B at 2 months after operation, a significant increase volume of subdural collection could be seen in left side Fig. 1 CT imagings of two typical patients without and with recurrence of CSDH. a, the preoperative presentation of patient A without recurrence; b the imaging of the same patient within 48 h postoperatively; c the CT scan at 3-month follow-up of the patient A; d the presurgical imaging of patient B with recurrence of CSDH; e the presentation of patient B within 48 h postoperatively; f the CT scan of patient B at 2 months after operation, a significant increase volume of subdural collection could be seen in left side Wang et al. BMC Neurology (2020) 20:411 Page 3 of 10 Page 3 of 10 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology (2020) 20:411 The following clinical characteristics were analyzed: age, gender, demographic parameters, comorbidities, postoperative laboratory investigations, postoperative medication use, location of hematoma, and preoperative BUN level. Glasgow outcome scale (GOS) at discharge was performed to evaluate the neurologic function of patients. Surgery procedure and follow-up All patients underwent BHI with general anesthesia. After burr-hole exposure, dura incision and irrigation, we used closed system drainage with a subdural catheter connected to a vacuum plastic bag. Most catheters were withdrawn within 72 h after the operation. Head CT was performed within the first 48 h and on day 6 or 7 post- operatively. For good measure, patients were followed up with head CT at 3 months at the outpatient service. Patients and parameters surgery through CT scans, and clinical criteria in which preoperative symptoms and signs abided or recurred (Fig. 1). The classification of recurrence was determined by two experienced neurosurgeons who were blinded to the study. The cases were first divided into 2 groups according to recurrence and non-recurrence to investigate the risk factors for CSDH recurrence. To identify the specific ef- fect of postoperative BUN, it was further divided into quartiles to explore the different influences on the CSDH recurrence in each category. The local ethics committee ruled that no formal ethics approval was re- quired in this particular study. Statistical analysis Data for continuous variables were shown as mean ± standard deviation or medians and interquartile range. To verify whether the data follows a normal distribution or not, Kolmogorov-Smirnov test was performed. Cat- egorical variables were shown as relative frequencies and percentages. Normally distributed variables were com- pared by Student’s t-test, whereas the Mann–Whitney U test was used for the asymmetrically distributed Definition of recurrence and grouping CSDH recurrence was evaluated by radiological criteria, which included an increased volume of the subdural col- lection and brain compression on either side within 3 months compared with those first measured after Fig. 2 Study flow diagram. CSDH, chronic subdural hematoma Wang et al. Definition of recurrence and grouping BMC Neurology (2020) 20:411 Page 4 of 10 Table 1 Clinical and Demographic Characteristics of Patients with CSDH Variables All patients (N = 653) Recurrence(n = 96) Non-Recurrence(n = 557) P-value Postoperative BUN (mmol/L) 4.9 (4,6.4) 5.9 (4.4,6.9) 4.9 (3.9,6.3) < 0.001 Demographic parameters Age (years) 72 (64,80) 75 (69,82) 71 (63,80) 0.012 Gender 0.263 Male, n (%) 561 (85.9%) 86 (89.6%) 475 (85.3%) Female, n (%) 92 (14.1%) 10 (10.4%) 82 (14.7%) Current drinking, n (%) 234 (35.8%) 34 (35.4%) 200 (35.9%) 0.926 Current smoking, n (%) 237 (36.3%) 33 (34.4%) 204 (36.6%) 0.672 Baseline SBP (mmHg) 138 (126,153) 135 (124,151) 140 (127,153) 0.122 Baseline DBP (mmHg) 79 (71,87) 77 (71,84) 79 (71,87) 0.091 Laboratory investigation Blood glucose (mmol/L) 5.5 (4.7,6.6) 5.7 (5,6.7) 5.4 (4.7,6.6) 0.265 Cr (μmol/L) 67 (58,77) 68 (60,80) 66 (57,77) 0.097 Leukocyte (X10^9/L) 6.94 (5.81,8.43) 6.51 (5.52,7.55) 7.01 (5.85,8.62) 0.010 Neutrophil (X10^9/L) 4.62 (3.68,6.15) 4.30 (3.51,5.31) 4.70 (3.71,6.26) 0.012 Lymphocyte (X10^9/L) 1.46 (1.10,1.81) 1.46 (1.17,1.83) 1.47 (1.1,1.81) 0.650 Erythrocyte (X10^12/L) 4.23 ± 0.51 4.19 ± 0.52 4.24 ± 0.51 0.379 Hemoglobin (g/L) 132 (120,142) 132 (119,142) 132 (119,140) 0.442 Platelet (X10^9/L) 211 (172,249) 194 (164,239) 213 (175,251) 0.010 Prothrombin Time(s) 13.3 (12.8,13.9) 13.4 (12.8,14.0) 13.3 (12.8,13.9) 0.642 INR 1.02 (0.97,1.08) 1.02 (0.98,1.10) 1.02 (0.97,1.07) 0.719 Fibrinogen (g/L) 3.61 (3.09,4.28) 3.50 (2.89,4.15) 3.64 (3.15,4.30) 0.046 Comorbidities Hypertension, n (%) 252 (38.6%) 40 (41.7%) 212 (38.1%) 0.503 Diabetes mellitus, n (%) 77 (11.8%) 11 (11.5%) 66 (11.8%) 0.913 Cardiovascular disease, n (%) 42 (6.4%) 6 (6.3%) 36 (6.5%) 0.937 Medication use Atorvastatin therapy, n (%) 478 (73.2%) 74 (77.1%) 404 (72.5%) 0.352 PAMBA therapy, n (%) 189 (28.9%) 26 (27.1%) 163 (29.3%) 0.663 Hematoma location 0.957 Unilateral left, n (%) 288 (44.1%) 42 (43.8%) 246 (44.2%) Unilateral right, n (%) 216 (33.1%) 31 (32.3%) 185 (33.2%) Bilateral, n (%) 149 (22.8%) 23 (24.0%) 126 (22.6%) Postoperative bleeding 136 (20.8%) 22 (22.9%) 114 (20.5%) 0.585 GOS at discharge 0.549 5, n (%) 593 (90.8%) 90 (93.8%) 503 (90.3%) 4, n (%) 48 (7.4%) 5 (5.2%) 43 (7.7%) 3, n (%) 12 (1.8%) 1 (1.0%) 11 (2.0%) Preoperative investigation N = 632 n = 95 n = 537 BUN (mmol/L) 5.3 (4.4,6.5) 5.9 (4.8,6.9) 5.2 (4.3,6.4) 0.001 NOTE. NOTE. SBP, systolic blood pressure; DBP, diastolic blood pressure; Cr, creatinine; INR, international normalized ratio; PAMBA, para-aminomethylbenzoic acid; BUN, blood urea nitrogen; GOS, Glasgow Outcome Scale Baseline characteristics of all cases in BUN quartiles Baseline characteristics of all cases in BUN quartiles For further exploration, the cases were divided into 4 groups based on quartiles of the postoperative BUN con- centration. The cut-off points for this stratification of the BUN concentration into quartiles were: Q1 ≤4.0 mmol/L, 4.0 < Q2 ≤4.9 mmol/L, 4.9 < Q3 ≤6.4 mmol/L and Q4 > 6.4 mmol/L. Table 3 summarizes the character- istics of the CSDH cases by the quartiles of BUN. Cases with different postoperative BUN concentration appeared to be similar in most features except for age, Cr, erythrocyte and hemoglobin. These factors would be adjusted for multivariate-adjusted binary logistic regres- sion for good measure. 22 (14%) of 157 patients in the highest quartile of BUN suffered moderate disability at discharge which was statistically higher than in other quartiles. Definition of recurrence and grouping SBP, systolic blood pressure; DBP, diastolic blood pressure; Cr, creatinine; INR, international normalized ratio; PAMBA, para-aminomethylbenzoic acid; BUN, blood urea nitrogen; GOS, Glasgow Outcome Scale Table 1 Clinical and Demographic Characteristics of Patients with CSDH Wang et al. BMC Neurology (2020) 20:411 Page 5 of 10 continuous variables. Chi-squared (χ2) test was used to compare categorical variables. Statistical comparisons among BUN concentrations stratification were estimated by one-way analysis of variance (ANOVA) or Kruskal– Wallis test for continuous variables, and Pearson’s chi- square test for categorical variables where appropriate. Wilcoxon signed-rank test was performed to compare the difference between pre and post-operative BUN levels. Odds ratio (OR) and 95% confidence interval (CI) for recurrence risk were calculated by performing multivariate-adjusted binary logistic regression. Re- stricted cubic spline regressions model was built to examine the linear relation between serum BUN concen- tration and the risk of recurrence. In this study, a two- tailed p-value less than 0.05 (P < 0.05) was considered as statistical significance. All statistical analyses were done using SPSS (version 23.0, IBM Corp.) and STATA soft- ware (version 12, StataCorp LP) was employed to analyze the restricted cubic spline regressions. continuous variables. Chi-squared (χ2) test was used to compare categorical variables. Statistical comparisons among BUN concentrations stratification were estimated by one-way analysis of variance (ANOVA) or Kruskal– Wallis test for continuous variables, and Pearson’s chi- square test for categorical variables where appropriate. Wilcoxon signed-rank test was performed to compare the difference between pre and post-operative BUN levels. Odds ratio (OR) and 95% confidence interval (CI) for recurrence risk were calculated by performing multivariate-adjusted binary logistic regression. Re- stricted cubic spline regressions model was built to examine the linear relation between serum BUN concen- tration and the risk of recurrence. In this study, a two- tailed p-value less than 0.05 (P < 0.05) was considered as statistical significance. All statistical analyses were done using SPSS (version 23.0, IBM Corp.) and STATA soft- ware (version 12, StataCorp LP) was employed to analyze the restricted cubic spline regressions. post-operative BUN level in the non-recurrence group (P < 0.001) while it showed no obvious difference in the recurrence group. Baseline characteristics of all cases in the recurrence group and non-recurrence group A total of 653 CSDH cases were enrolled in this study (Fig. 2). This study group included 561 male cases (85.9%) and 92 female cases (14.1%). Patient’s age ranged from 21 to 100 years with a median of 72 years (inter- quartile range 64 to 80 years). (Table 1). 8 patients with hospitalized mortality were excluded and there was no mortality upon follow-up in this study. Significant differences were observed between the recur- rence and non-recurrence groups in BUN concentration quartiles of cases (P = 0.003). The proportion of cases in the lowest quartile (≤4.0 mmol/L) was dramatically low in the recurrence group (P = 0.027), whilst the propor- tion of cases in the highest quartile (> 6.4 mmol/L) was significantly high in the recurrence group (P = 0.012) (Table 4). The descriptive characteristics between groups with and without recurrence are shown in Table 1, including the demographics, laboratory, imaging, medication and comorbidity characteristics. In this study, 96 (14.7%) cases were diagnosed as CSDH recurrence, including 16 patients who needed a second operation. Compared with non-recurrence, the cases in the group of recurrence were older and have lower levels of serum leukocyte, neutrophil and platelet count. However, lower serum fi- brinogen concentration was examined in recurrence cases (P < 0.05). Moreover, there was a statistical difference between pre and post-operative groups on serum BUN concentration (P = 0.001 and P < 0.001, re- spectively). Table 2 revealed that the BUN level of the pre-operative group was significantly higher than the In Table 5, with all cases taken as a whole, the condi- tion that CSDH recurrence was interpreted as a dependent variable and the lowest quartile was inter- preted as the reference was used for postoperative BUN level in the binary logistic regression models. The high- est quartile of BUN concentration (> 6.4 mmol/L) was independently estimated as a risk factor of CSDH recurrence with an unadjusted OR of 3.315 (95%CI: 1.711–6.423, P < 0.001). Baseline characteristics of all cases in the recurrence group and non-recurrence group After adjusting for the con- founders including sex, age, current alcohol drinking, current smoking, comorbidities (hypertension, diabetes mellitus, coronary heart disease), medicine (Atorvastatin and PAMBA), and laboratory investigation (platelet, fibrinogen, leukocyte, erythrocyte, hemoglobin, Cr), the Table 2 The comparison between pre- and post-operative BUN levels Variable Preoperative BUN (mmol/L) Postoperative BUN (mmol/L) P-value Total (n = 632) 5.3 (4.4,6.5) 4.9 (4.0,6.4) < 0.001 Recurrence (n = 95) 5.9 (4.8,6.9) 5.85 (4.4,6.9) 0.177 Non-Recurrence (n = 537) 5.2 (44.3,6.4) 4.9 (3.9,6.25) < 0.001 BUN, blood urea nitrogen; Wang et al. Baseline characteristics of all cases in the recurrence group and non-recurrence group BMC Neurology (2020) 20:411 Page 6 of 10 Table 3 Baseline Characteristics of Patients with Chronic Subdural Hematoma according to postoperative BUN quartile Variables BUN quartiles quartile 1 n = 170(≤4.0) quartile 2 n = 162(> 4.0,≤4.9) quartile 3 n = 164(> 4.9,≤6.4) quartile 4 n = 157(> 6.4) P-value BUN (mmol/L) 3.4 (3.0,3.7) 4.5 (4.3,4.8) 5.7 (5.3,6.0) 7.4 (6.9,8.3) < 0.001 Demographic parameters Age (years) 68 (61,76) 72 (63,79) 73 (66,82) 76 (69,83) < 0.001 Gender, male, n (%) 140 (82.4%) 148 (91.4%) 141 (86.0%) 132 (84.1%) 0.103 Current drinking, n (%) 61 (35.9%) 62 (38.3%) 58 (35.4%) 53 (33.8%) 0.866 Current smoking, n (%) 68 (39.4%) 56 (34.6%) 63 (38.4%) 51 (32.5%) 0.526 Baseline SBP (mmHg) 138.4 ± 19.4 139.3 ± 19.7 141.0 ± 20.6 142.5 ± 21.0 0.274 Baseline DBP (mmHg) 79 (71,87) 79 (73,86) 77 (72,88) 78 (71,87) 0.984 Laboratory investigation Blood glucose (mmol/L) 5.5 (4.8,6.5) 5.6 (4.7,6.6) 5.6 (4.7,6.8) 5.4 (4.5,6.7) 0.818 Cr (μmol/L) 60 (54,68) 66 (57,75) 69 (60,80) 74 (63,87) < 0.001 Leukocyte (X10^9/L) 7.00 (6.14,8.45) 6.97 (5.81,8.62) 6.98 (5.73,8.33) 6.77 (5.55,8.39) 0.677 Neutrophil (X10^9/L) 4.78 (3.84,6.31) 4.68 (3.76,6.30) 4.52 (3.59,5.92) 4.52 (3.60,5.95) 0.369 Lymphocyte (X10^9/L) 1.50 (1.10,1.80) 1.40 (1.10,1.71) 1.50 (1.11,1.87) 1.45 (1.10,1.81) 0.518 Erythrocyte (X10^12/L) 4.30 ± 0.51 4.27 ± 0.50 4.24 ± 0.52 4.12 ± 0.52 0.008 Hemoglobin (g/L) 133 (120,144) 133 (123,142) 132 (120,144) 130 (116,139) 0.047 Platelet (X10^9/L) 219 (174,259) 206 (175,250) 203 (175,243) 201 (162,249) 0.183 Prothrombin Time (s) 13.3 (12.7,13.8) 13.2 (12.9,13.9) 13.4 (12.9,13.9) 13.4 (12.9,14.1) 0.523 INR 1.02 (0.97,1.07) 1.02 (0.97,1.08) 1.03 (0.97,1.08) 1.03 (0.97,1.10) 0.484 Fibrinogen (g/L) 3.55 (3.11,4.38) 3.71 (3.10,4.35) 3.61 (2.97,4.18) 3.67 (3.12,4.26) 0.740 Comorbidities Hypertension, n (%) 61 (35.9%) 61 (37.7%) 60 (36.6%) 70 (44.6%) 0.355 Diabetes mellitus, n (%) 18 (10.6%) 18 (11.1%) 17 (10.4%) 24 (15.3%) 0.480 Cardiovascular disease, n (%) 9 (5.3%) 10 (6.2%) 11 (6.7%) 12 (7.6%) 0.853 Medication use Atorvastatin therapy, n (%) 124 (72.9%) 118 (72.8%) 114 (69.5%) 122 (77.7%) 0.427 PAMBA therapy, n (%) 40 (23.5%) 45 (27.8%) 53 (32.3%) 51 (32.5%) 0.222 Hematoma location 0.367 Unilateral left, n (%) 68 (40.0%) 82 (50.6%) 75 (45.7%) 63 (40.1%) Unilateral right, n (%) 62 (36.5%) 48 (29.6%) 55 (33.5%) 51 (32.5%) Bilateral, n (%) 40 (23.5%) 32 (19.8%) 34 (20.7%) 43 (27.4%) GOS at discharge 0.042 5, n (%) 160 (94.1%) 149 (92.0%) 152 (92.7%) 132 (84.1%) 4, n (%) 8 (4.7%) 10 (6.2%) 8 (4.9%) 22 (14%) 3, n (%) 2 (1.2%) 3 (1.9%) 4 (2.4%) 3 (1.9%) NOTE. Discussion To the best of our knowledge, this is the first study to investigate the association between BUN concentration and the recurrence of CSDH after evacuation. Our results indicated that high postoperative BUN concen- tration was related to the prevalence of CSDH recur- rence within 3 months after the operation. Our findings hence suggest that the postoperative BUN concentration could be an available risk factor for CSDH recurrence. BUN is a waste product of protein catabolism which is linked with poor prognosis and mortality in acute or chronic heart failure [12]. In addition, a study of 3355 AIS patients by You et al. observed that higher BUN had a 3.75-fold higher risk of in-hospital mortality [11]. In the present study, we found that patients with a rela- tively higher level of BUN were more likely to be moder- ately disabled at discharge. This was consistent with the previous studies that elevated BUN levels during hospitalization was related to a poor outcome. However, Table 5 Multivariate adjusted odds ratios for the association between BUN levels and Recurrence Quartile ORa 95%CI P-value Unadjusted Middle 1.751 0.863–3.554 0.121 Higher 1.910 0.951–3.837 0.069 Highest 3.315 1.711–6.423 < 0.001 Model 1b Middle 1.590 0.778–3.252 0.204 Higher 1.713 0.842–3.483 0.137 Highest 2.892 1.463–5.717 0.002 Model 2c Middle 1.612 0.788–3.299 0.191 Higher 1.768 0.866–3.611 0.118 Highest 2.939 1.480–5.836 0.002 Model 3d Middle 1.612 0.780–3.333 0.197 Higher 1.733 0.837–3.590 0.139 Highest 3.069 1.488–6.330 0.002 OR, odds radio; CI confidence level; Cr, creatinine; Recurrence of postoperative CSDH a Reference OR (1.000) is the lowest quartile of BUN for Recurrence of CSDH b Model 1: adjusted for age, sex, current smoking, current alcohol drinking c Modal 2: adjusted for covariates from Model 1 and further adjusted for medical history (coronary heart disease, diabetes mellitus, hypertension) and medicine (Atorvastatin and PAMBA) d Modal 3: adjusted for covariates from Model 2 and further adjusted for Platelet, Fibrinogen, Leukocyte, Erythrocyte, Hemoglobin, Cr Table 5 Multivariate adjusted odds ratios for the association between BUN levels and Recurrence The precise mechanisms underlying the association between elevated BUN and CSDH recurrence remained unclear. A potential explanation for this might be that a systemic increase in serum BUN induces intracranial multiple responses, which in turn can produce hematoma expansion and bring out CSDH recurrence. Baseline characteristics of all cases in the recurrence group and non-recurrence group SBP, systolic blood pressure; DBP, diastolic blood pressure; Cr, creatinine; INR, international normalized ratio; PAMBA, para-aminomethylbenzoic acid; BUN, blood urea nitrogen; GOS, Glasgow Outcome Scale Table 3 Baseline Characteristics of Patients with Chronic Subdural Hematoma according to postoperative BUN quartile highest quartile of BUN remained significantly and inde- pendently associated with CSDH recurrence (model 1: OR = 2.892, 95% CI:1.463–5.717, P = 0.002; model 2: OR = 2.939, 95% CI:1.480–5.836, P = 0.002; model 3: OR = 3.069, 95%CI:1.488–6.330, P = 0.002). There was no multicollinearity between the independent variables in model 3. Furthermore, restricted cubic spline regres- sions were used to explore the linear relationship be- tween BUN concentration and the risk of CSDH recurrence (Fig. 3). Most importantly, it could be ob- served visually that the highest quartile had a signifi- cantly high OR value. Page 7 of 10 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology Table 4 BUN quartiles of patients Variable Recurrence (n = 96) Non-Recurrence (n = 557) χ2 P-value BUN (mmol/L) 14.308 0.003 Quartile 1(≤4.0) 14 (14.6%) 156 (28.0%) 4.870 0.027 Quartile 2(> 4.0,≤4.9) 22 (22.9%) 140 (25.1%) 0.132 0.717 Quartile 3(> 4.9,≤6.4) 24 (25.0%) 140 (25.1%) 0.000 0.983 Quartile 4(> 6.4) 36 (37.5%) 121 (21.7%) 6.283 0.012 BUN, blood urea nitrogen; little data was found on the relationship between BUN and recurrence of CSDH. In this study, we observed that cases with a higher BUN level presented a trend toward a higher recurrence rate of CSDH. Furthermore, elevated BUN was associated with an enhanced risk of CSDH re- currence and cases with the highest postoperative BUN (> 6.4 mmol/L) seemed to have a 3.069-fold increase in danger of CSDH recurrence after adjusting for the po- tential confounders. The normal range of BUN level in the research hospital is 2.8–7.2 mmol/L which means the majority of patients presented with BUN level in the normal range. The BUN levels reflected not only the renal function but also other reactions that were men- tioned below. In this study, the Cr level did not have an effect on the recurrence of CSDH which means there was no direct association between renal function and CSDH recurrence. The cutoff value of the postoperative BUN level in this study is 6.4 mmol/L and it indicated that the normal range of BUN level for intracranial le- sion might be different from the systematic level. Baseline characteristics of all cases in the recurrence group and non-recurrence group Also, elevated preoperative BUN levels showed an association with the recurrence of CSDH after logistic regression (See Supplemental Table, Additional file 1). Comparison between pre and post-operative BUN levels revealed that the BUN in the non-recurrence group significantly de- creased after evacuation while it did not occur in the re- currence group, further confirming the relationship between BUN and CSDH recurrence. OR, odds radio; CI confidence level; Cr, creatinine; Recurrence of postoperative CSDH a Reference OR (1.000) is the lowest quartile of BUN for Recurrence of CSDH b Model 1: adjusted for age, sex, current smoking, current alcohol drinking c Modal 2: adjusted for covariates from Model 1 and further adjusted for medical history (coronary heart disease, diabetes mellitus, hypertension) and medicine (Atorvastatin and PAMBA) d Modal 3: adjusted for covariates from Model 2 and further adjusted for Platelet, Fibrinogen, Leukocyte, Erythrocyte, Hemoglobin, Cr Supplementary Information Th li i i l y The online version contains supplementary material available at https://doi. org/10.1186/s12883-020-01985-w. Additional file 1. Supplemental table of multivariate analyzed association between preoperative BUN and Recurrence. Additional file 2. The complete data used and analysed during the current study. Discussion Some studies have revealed that the predictive value of BUN may be induced by its connection with other vari- ables, such as protein intake, protein catabolism, nitro- gen production and neurohormonal activation [12, 13]. On the other hand, CSDH was recently suggested to be formed through complex processes including angiogen- esis, fibrinolysis and inflammation [14], and the recur- rence of CSDH follows the same process as formation. There is strong evidence to show that vascular endothe- lial growth factor (VEGF) was related to the generation Wang et al. BMC Neurology (2020) 20:411 Page 8 of 10 Fig. 3 Association of BUN levels with risk of Recurrence. Dashed lines are 95% confidence intervals. Dotted line is where BUN concentration is 6.4 mmol/L. Odds ratios and 95% confidence intervals derived from restricted cubic spline regression. Odds ratios were estimated using logistic regression modeling, adjusting for the same variables as model 3 in Table 5 Fig. 3 Association of BUN levels with risk of Recurrence. Dashed lines are 95% confidence intervals. Dotted line is where BUN concentration is 6.4 mmol/L. Odds ratios and 95% confidence intervals derived from restricted cubic spline regression. Odds ratios were estimated using logistic regression modeling, adjusting for the same variables as model 3 in Table 5 and steady increase of CSDH fluid volume [15–17] as well as the risk of CSDH recurrence [18]. Interestingly, Lin et al. observed that VEGF expression was positively correlated with BUN [19]. Hence, these discoveries might explain the relatively good correlation between el- evated BUN and CSDH recurrence. single-center clinical finding, which may impact the generalization of the results, thus further prospective re- search with plenty of patients might be required. Finally, considering that the role BUN plays in the CSDH recur- rence is still confusing, more studies need to be pursued to further and better delineate CSDH formation in the future. Compared with CSDH cases without recurrence, our re- sult also showed that CSDH cases with recurrence had sig- nificantly higher age, which is consistent with the reports of previous studies [7, 20]. However, we found that the older cases were associated with higher BUN and the difference of patient age was not statistically significant after multivari- ate logistic regression analysis though age affects the recur- rence of CSDH through the change of BUN. Conclusion The most obvious finding from this study was that ele- vated BUN at post-operation was independently associ- ated with the recurrence of CSDH. The result suggested that a high level of postoperative BUN might serve as a risk factor of CSDH recurrence, so the determination of serum BUN after evacuation is important for patients with CSDH. Further prospective studies need to be undertaken to validate the causal relationship between BUN and CSDH recurrence. Additionally, decreased fibrinogen level was found to be associated with the CSDH recurrence. In fact, fibrinogen has been proven to be a potentially available risk factor for postoperative intracranial bleeding [21, 22] considering that elevating serum fibrinogen was targeted as a valid therapy for intracranial hemorrhage [23]. Moreover, high levels of fibrinogen degradation products (FDPs) have been examined in CSDH fluid [24, 25] and fresh red cells were identified within CSDH fluid [24], suggesting that bleeding is an essential part of CSDH formation. The current study did not reveal a significant difference between postoperative atorvastatin use and CSDH recur- rence. This finding runs contrary to existing studies [26, 27], hence, making the relationship between atorvastatin administration and CSDH recurrence puzzling. Funding This work was supported by grants from the National Natural Science Foundation of China (No. 81771262), Zhejiang Health Science and Technology Project (2016RCA022), Zhejiang Provincial Natural Science Foundation of China (LQ20H090005) and Wenzhou Municipal Science and Technology Bureau Project of China (Y20190568). 10. Deng L, Wang C, Qiu S, Bian H, Wang L, Li Y, et al. Association between blood urea nitrogen-to-creatinine ratio and three-month outcome in patients with acute ischemic stroke. Curr Neurovasc Res. 2019;16(2):166–72. https://doi.org/10.2174/1567202616666190412123705. Acknowledgments Not applicable. 7. Leroy HA, Aboukais R, Reyns N, Bourgeois P, Labreuche J, Duhamel A, et al. Predictors of functional outcomes and recurrence of chronic subdural hematomas. J Clin Neurosci. 2015;22(12):1895–900. https://doi.org/10.1016/j. jocn.2015.03.064. Received: 10 March 2020 Accepted: 29 October 2020 20. Motoie R, Karashima S, Otsuji R, Ren N, Nagaoka S, Maeda K, et al. Recurrence in 787 patients with chronic subdural hematoma: retrospective cohort investigation of associated factors including direct Oral anticoagulant use. World Neurosurg. 2018;118:e87–91. https://doi.org/10. 1016/j.wneu.2018.06.124. Competing interests 15. Weigel R, Hohenstein A, Schilling L. Vascular endothelial growth factor concentration in chronic subdural hematoma fluid is related to computed tomography appearance and exudation rate. J Neurotrauma. 2014;31(7): 670–3. https://doi.org/10.1089/neu.2013.2884. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or be- liefs) in the subject matter or materials discussed in this manuscript. 16. Shono T, Inamura T, Morioka T, Matsumoto K, Suzuki SO, Ikezaki K, et al. Vascular endothelial growth factor in chronic subdural haematomas. J Clin Neurosci. 2001;8(5):411–5. https://doi.org/10.1054/jocn.2000.0951. 17. Hua C, Zhao G, Feng Y, Yuan H, Song H, Bie L. Role of matrix Metalloproteinase-2, matrix Metalloproteinase-9, and vascular endothelial growth factor in the development of chronic subdural hematoma. J Neurotrauma. 2016;33(1):65–70. https://doi.org/10.1089/neu.2014.3724. Authors’ contributions NW, JH and AO wrote the manuscript. NW performed the statistical analyses. NW, XZ and YL gathered the data and are responsible for the integrity of registered data. LR, JY and QZ designed and coordinated the study. SY contributed to the analysis and interpretation of data. The authors read and approved the submitted version. 8. Schrock JW, Glasenapp M, Drogell K. Elevated blood urea nitrogen/ creatinine ratio is associated with poor outcome in patients with ischemic stroke. Clin Neurol Neurosurg. 2012;114(7):881–4. https://doi.org/10.1016/j. clineuro.2012.01.031. 9. Lin CJ, Yang JT, Huang YC, Tsai YH, Lee MH, Lee M, et al. Favorable outcome of blood urea nitrogen/creatinine-based hydration therapy 3 months after acute ischemic stroke. Am J Emerg Med. 2016;34(12):2414–8. https://doi.org/10.1016/j.ajem.2016.09.033. Abbreviations CSDH: Chronic subdural hematoma; BUN: Blood Urea Nitrogen; BHI: Burr-hole irrigation; GCS: Glasgow coma score; CT: Computed tomography; MRI: Magnetic resonance imaging; AIS: Acute ischemic stroke; GOS: Glasgow Outcome Scale; ANOVA: Analysis of variance; OR: Odds ratio; CI: Confidence interval; Cr: Creatinine; PAMBA: Para-aminomethylbenzoic acid; SBP: Systolic There were a few limitations recognized in the present study. First, this study is a retrospective study and the selection bias is inevitable. Secondly, this is only a Page 9 of 10 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology (2020) 20:411 blood pressure; DBP: Diastolic blood pressure; INR: International normalized ratio; VEGF: Vascular endothelial growth factor; FDPs: Fibrinogen degradation products meta-analysis. J Clin Neurosci. 2017;38:79–83. https://doi.org/10.1016/j.jocn. 2016.12.001. meta-analysis. J Clin Neurosci. 2017;38:79–83. https://doi.org/10.1016/j.jocn. 2016.12.001. 6. Rust T, Kiemer N, Erasmus A. Chronic subdural haematomas and anticoagulation or anti-thrombotic therapy. J Clin Neurosci. 2006;13(8):823– 7. https://doi.org/10.1016/j.jocn.2004.12.013. Availability of data and materials 11. You S, Zheng D, Zhong C, Wang X, Tang W, Sheng L, et al. Prognostic significance of blood urea nitrogen in acute ischemic stroke. Circ J. 2018; 82(2):572–8. https://doi.org/10.1253/circj.CJ-17-0485. The datasets used and analyzed during the current study are available in supported Additional file 2 and the confidential patient data was erased. 12. Damman K, Voors AA, Navis G, van Veldhuisen DJ, Hillege HL. Current and novel renal biomarkers in heart failure. Heart Fail Rev. 2012;17(2):241–50. https://doi.org/10.1007/s10741-011-9254-2. Author details 1 1Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China. 2Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China. 3Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. 18. Hong HJ, Kim YJ, Yi HJ, Ko Y, Oh SJ, Kim JM. Role of angiogenic growth factors and inflammatory cytokine on recurrence of chronic subdural hematoma. Surg Neurol. 2009;71(2):161–5discussion 5-6. https://doi.org/10. 1016/j.surneu.2008.01.023. 19. Lin S, Teng J, Li J, Sun F, Yuan D, Chang J. Association of Chemerin and Vascular Endothelial Growth Factor (VEGF) with diabetic nephropathy. Med Sci Monit. 2016;22:3209–14. https://doi.org/10.12659/msm.896781. 19. Lin S, Teng J, Li J, Sun F, Yuan D, Chang J. Association of Chemerin and Vascular Endothelial Growth Factor (VEGF) with diabetic nephropathy. Med Sci Monit. 2016;22:3209–14. https://doi.org/10.12659/msm.896781. Received: 10 March 2020 Accepted: 29 October 2020 Received: 10 March 2020 Accepted: 29 October 2020 Ethics approval and consent to participate pp p p The ethics committee of Wenzhou Medical University First Affiliated Hospital ruled that no formal ethics approval was required in this retrospective study and determined that informed consent was not required. 13. Schrier RW. Blood urea nitrogen and serum creatinine: not married in heart failure. Circ Heart Fail. 2008;1(1):2–5. https://doi.org/10.1161/ CIRCHEARTFAILURE.108.770834. 13. Schrier RW. Blood urea nitrogen and serum creatinine: not married in heart failure. Circ Heart Fail. 2008;1(1):2–5. https://doi.org/10.1161/ CIRCHEARTFAILURE.108.770834. 14. Edlmann E, Giorgi-Coll S, Whitfield PC, Carpenter KLH, Hutchinson PJ. Pathophysiology of chronic subdural haematoma: inflammation, angiogenesis and implications for pharmacotherapy. J Neuroinflammation. 2017;14(1):108. https://doi.org/10.1186/s12974-017-0881-y. 14. Edlmann E, Giorgi-Coll S, Whitfield PC, Carpenter KLH, Hutchinson PJ. Pathophysiology of chronic subdural haematoma: inflammation, angiogenesis and implications for pharmacotherapy. J Neuroinflammation. 2017;14(1):108. https://doi.org/10.1186/s12974-017-0881-y. References Characterization of chronic subdural hematoma fluid proteome. Neurosurgery. 2013;73(2):317–31. https://doi.org/10.1227/01.neu.0000430323.24623.de. 26. Tang R, Shi J, Li X, Zou Y, Wang L, Chen Y, et al. Effects of atorvastatin on surgical treatments of chronic subdural hematoma. World Neurosurg. 2018; 117:e425–e9. https://doi.org/10.1016/j.wneu.2018.06.047. 26. Tang R, Shi J, Li X, Zou Y, Wang L, Chen Y, et al. Effects of atorvastatin on surgical treatments of chronic subdural hematoma. World Neurosurg. 2018; 117:e425–e9. https://doi.org/10.1016/j.wneu.2018.06.047. 27. Jiang R, Zhao S, Wang R, Feng H, Zhang J, Li X, et al. Safety and efficacy of atorvastatin for chronic subdural hematoma in Chinese patients: a randomized ClinicalTrial. JAMA Neurol. 2018;75(11):1338–46. https://doi.org/ 10.1001/jamaneurol.2018.2030. 27. Jiang R, Zhao S, Wang R, Feng H, Zhang J, Li X, et al. Safety and efficacy of atorvastatin for chronic subdural hematoma in Chinese patients: a randomized ClinicalTrial. JAMA Neurol. 2018;75(11):1338–46. https://doi.org/ 10.1001/jamaneurol.2018.2030. References 1. Ducruet AF, Grobelny BT, Zacharia BE, Hickman ZL, DeRosa PL, Andersen KN, et al. The surgical management of chronic subdural hematoma. Neurosurg Rev. 2012;35(2):155–69discussion 69. https://doi.org/10.1007/ s10143-011-0349-y. 1. Ducruet AF, Grobelny BT, Zacharia BE, Hickman ZL, DeRosa PL, Andersen KN, et al. The surgical management of chronic subdural hematoma. Neurosurg Rev. 2012;35(2):155–69discussion 69. https://doi.org/10.1007/ s10143-011-0349-y. 21. Zhang XH, Wang QM, Chen H, Chen YH, Han W, Wang FR, et al. Clinical characteristics and risk factors of intracranial hemorrhage in patients following allogeneic hematopoietic stem cell transplantation. Ann Hematol. 2016;95(10):1637–43. https://doi.org/10.1007/s00277-016-2767-y. 2. Rauhala M, Luoto TM, Huhtala H, Iverson GL, Niskakangas T, Ohman J, et al. The incidence of chronic subdural hematomas from 1990 to 2015 in a defined Finnish population. J Neurosurg. 2019:1–11. https://doi.org/10.3171/ 2018.12.JNS183035. 2. Rauhala M, Luoto TM, Huhtala H, Iverson GL, Niskakangas T, Ohman J, et al. The incidence of chronic subdural hematomas from 1990 to 2015 in a defined Finnish population. J Neurosurg. 2019:1–11. https://doi.org/10.3171/ 2018.12.JNS183035. 22. Adelmann D, Klaus DA, Illievich UM, Krenn CG, Krall C, Kozek-Langenecker S, et al. 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Ito H, Yamamoto S, Komai T, Mizukoshi H. Role of local hyperfibrinolysis in the etiology of chronic subdural hematoma. J Neurosurg. 1976;45(1):26–31. https://doi.org/10.3171/jns.1976.45.1.0026. 5. Wang Y, Zhou J, Fan C, Wang D, Jiao F, Liu B, et al. Influence of antithrombotic agents on the recurrence of chronic subdural hematomas and the quest about the recommencement of antithrombotic agents: a Page 10 of 10 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology (2020) 20:411 Wang et al. BMC Neurology (2020) 20:411 25. Heula AL, Ohlmeier S, Sajanti J, Majamaa K. Characterization of chronic subdural hematoma fluid proteome. Neurosurgery. 2013;73(2):317–31. https://doi.org/10.1227/01.neu.0000430323.24623.de. 25. Heula AL, Ohlmeier S, Sajanti J, Majamaa K. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4387238415	https://journals.asmarya.edu.ly/jau/index.php/jau/article/download/306/226	Japanese	null	حجية أحكام التلاوة	Mağallaẗ al-ğāmiʿaẗ al-Asmariyyaẗ	2,008	cc-by	5,888	 @ @ @@@@@ @  @@ @ ¯pBÏƒªA ”_Úº‰ß Â ÚC‰j_Ù¥‰NÍª Â B‰ƒÙ≥‰j_ÚØ ıB√EÊj_Û≥‰À ı›Õ¯l_Êƒ‰M Â B‰ƒÙªÏl__‰√‰À ÎS_Ù∏Âø ”_Úº‰ß @  @ 106 @ @@@   @ @ @@@@ @ ı›ŒÍMÊj_‰M ‰∆EÊj_Û¥ÙªA ¯Ω_ëM‰i‰À  @  @ @@ 4 @@@@ @@@@@@@@@@@@ @@ @@@@@@ @  @   @@  @ @@  @ @ @ @ Í…ŒÍØ BÁIB‰NÍ∑ Ê¡Û∏ÊŒÚª¯G B‰ƒÙª‰l√ÚC ÊfÚ¥Úª ‰∆Ã_ÛºÍ¥Ê®‰M Ú›_ÚØÚC Ê¡Û∑Âj_Ù∑Íg  @ @ @ 10 @ @@@@@ @ ‰j_Ù∑ëhªA B‰ƒÙªÏl_‰√ Â≈_ÊZ‰√ B_Ï√¯G ‰∆Ã_Û§ÍØB‰ZÚª Â…_Úª B_Ï√¯G‰À  @ @ @9@ @@@@@@@@@@ @ @@@@@@@@@@@@ @@@@@ @@@@@@  @@@@ @@   א … @N sîË@†àª@ku‰ ∗ Journal of Alasmarya University Volume 9, June 2008 ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ اﻟﻣﺟﻠد اﻟﺗﺎﺳﻊ، ﯾوﻧﯾو 2008  א … @N sîË@†àª@ku‰ ∗ Journal of Alasmarya University Volume 9, June 2008 ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ اﻟﻣﺟﻠد اﻟﺗﺎﺳﻊ، ﯾوﻧﯾو 2008 אא @@ @ @@@@@@@@@  @@@@@@@@@@@@ @@@@@ @@@@@@@  @@  @@@@  @  ∗ @  41 א  @@@  @@ ) 1 (@ @@@@@@@@ @@ @@@@@@@ @@@ @ @@@@@@@@@@@@@ @@@@@@@@ @@  @@@@@@@@@@ @ @@@@@@ @@@@@  @@@ @@  @@@@ @  @@  @  @ @@  @@@@  @@  @@@@@  @@@  @@ ) 1 (@ @@@@@@@@ @@ @@@@@@@ @@@ @ @@@@@@@@@@@@@ @@@@@@@@ @@  @@@@@@@@@@ @ @@@@@@ @@@@@  @@@ @@  @@@@ @  @@  @  @ @@  @@@@  @@  @@@@@  @@@@@  @@@@ ) 2 ( @@ @@ @ @@@@@@@  @@@@@  @  @@  @@@  @@@@@@@@@@@@@@ @@ @@ @ @@ @ @@@@@@@@@@@@  @ @ @@@@@  @@@  @@  @@@ @ @ @ @  @@  @ @  @ @@ @ @@@@  @@@@@ @ @@@@  @ @ @  @@@@  @@@  @@@@@@@  @ @ @@@@@@@@  @ @  @  @ @ @@@  @@@@@ @ @@@@ @@@@@@@@@@@ @@@@@@  @@@@@@@@@@ @ @@@@ @ @@@@@@@@@@ @@@@@@@@@@@@ @@@@@@@  @@@@@  @@@@ ) 2 ( @@ @@ @ @@@@@@@  @@@@@  @  @@  @@@  @@@@@@@@@@@@@@ @@ @@ @ @@ @ @@@@@@@@@@@@  @ @ @@@@@  @@@  @@  @@@ @ @ @ @  @@  @ @  @ @@ @ @@@@  @@@@@ @ @@@@  @ @ @  @@@@  @@@  @@@@@@@  @ @ @@@@@@@@  @ @  @  @ @ @@@  @@@@@ @ @@@@ @@@@@@@@@@@ @@@@@@  @@@@@@@@@@ @ @@@@ @ @@@@@@@@@@ @@@@@@@@@@@@ @@@@@@@  @ @@ @ @@@@@ @@@@@ @ @ 324 @@  @@ @ @@  @ @ 665 @@ @ @@@@@@@@  @ 1 @ @@@@ 1 @ 210  42 K  @@@@@@@ @ @@@ @ @@@@@ @@@@@  @@@@  @ @@ @@@@@@@@@  @ @ @ @@  @@@ ) 3 (@ @@@@@@@@@ @  @   @ 32 @@  @@@  @ @@@  @@@  @@@ @@ @  @   ) 4 ( @@@@@ @    1  @@@ k6 j @@  @ @@@@@@@ @@@@@  ) 5 (@ @@@  @@ @ @@@@ @@@  @@@@@@ @@@@ @@  @@  @@ @@@@@@@@@  @@@    @@ @@ @ @@@@@  @@@@@ @  @@  @ @ @@@@@  @@@@@  3 @ @@@  @@ @ 45 @ @@@@@ @  3 4 @ @@@@@ 203 @@@@@ 1@ 1975 . אא 5 @ @@@@@ 2 @ 192  6 @ @@@@@@@@@@ @  @@ @ @@@ @ 15 @  @@ 3 @ 603  7 @  @ @@  8 @ @ @ @@@@@@@  †‡ÈÛa@ 9 @òäÛa 5 †‡ÈÛa@ 9 @òäÛa 5 43 א  òí‹þa@òÈßb§a@òÜª@ @ 44 @@ @ @@@@@@@ @ @@@  @@@ @ @ @@  @ @ @@  @@@@@ @ @@@  @ @@ @@@@@@@  @@@  ) 9 ( @@ @ @ @@  @ 20 @@  @@ @  @@@@@ @@@@@@@@@@@@@@ @@ @@@@  @ @ @@@ @ @  ) 10 ( @@@@@@  @ 32 @@  @@@ @  @@@ @  @ @@ ) 11 (@ @@@@@@@@@@@ @@@@@ @@ @ @   ) 12 ( @  @@@@@@  @@ @  @ @@@@ @@@@@  @@@@@ @@@@ @@@@@@@@@@@  @@@ @ @@@@@@@@@@@@@@ @ @@@ @@ @@@@ @ @@@  @@@@  @@ @@@  ) 13 (@ 3 @ @@@  @@  @ @@@  @@@@  ëL‰i Í…_˙ºÍª Âf_Ê¿‰ZÙªA ‰îÍ¿ÚªB_‰®ÙªA  @ @@@@   ¯¡ŒÍYÏj_ªA ¯≈‰¿ÊYÏj_ªA  @ @@@@@@@ @@ @  ) 14 ( @   @@@@@  @@@@@  @@@ @@@@@@@@@@@@ @@@@@@ @ @@@@@@@  9 @ @@ 3 @ 226  @@ 2 @ 202 @@@@ 2 @ 101  10 @ @@@@ 2 @ 203 @ @@ @ 5 @ 127  11 @ @@@@  12 @ @@ 2 @@ 343 @@@ 2885  13 @ @@  @@ 203 @ @ . 14 @ @@ 5 @ 185 @ @@@ @ 2 @ 522 @ @@ 2910 . 15 @ @@ 1 @ 137  אא @@ @ @@@@@@@ @ @@@  @@@ @ @ @@  @ @ @@  @@@@@ @ @@@  @ @@ @@@@@@@  @@@  ) 9 ( @@ @ @ @@  @ 20 @@  @@ @  @@@@@ @@@@@@@@@@@@@@ @@ @@@@  @ @ @@@ @ @  ) 10 ( @@@@@@  @ 32 @@  @@@ @  @@@ @  @ @@ ) 11 (@ @@@@@@@@@@@ @@@@@ @@ @ @   ) 12 ( @  @@@@@@  @@ @  @ @@@@ @@@@@  @@@@@ @@@@ @@@@@@@@@@@  @@@ @ @@@@@@@@@@@@@@ @ @@@ @@ @@@@ @ @@@  @@@@  @@ @@@  ) 13 (@ 3 @ @@@  @@  @ @@@  @@@@  ëL‰i Í…_˙ºÍª Âf_Ê¿‰ZÙªA ‰îÍ¿ÚªB_‰®ÙªA  @ @@@@   ¯¡ŒÍYÏj_ªA ¯≈‰¿ÊYÏj_ªA  @ @@@@@@@ @@ @  ) 14 ( 3 @ @@@  @@  @ @@@  @@@@  ëL‰i Í…_˙ºÍª Âf_Ê¿‰ZÙªA ‰îÍ¿ÚªB_‰®ÙªA  @ @@@@   ¯¡ŒÍYÏj_ªA ¯≈‰¿ÊYÏj_ªA  @ @@@@@@@ @@ @  ) 14 ( @   @@@@@  @@@@@  @@@ @@@@@@@@@@@@ @@@@@@ @ @@@@@@@ òí‹þa@òÈßb§a@òÜª@ @ 44 9 @ @@ 3 @ 226  @@ 2 @ 202 @@@@ 2 @ 101  10 @ @@@@ 2 @ 203 @ @@ @ 5 @ 127  11 @ @@@@  12 @ @@ 2 @@ 343 @@@ 2885  13 @ @@  @@ 203 @ @ . 14 @ @@ 5 @ 185 @ @@@ @ 2 @ 522 @ @@ 2910 . 9 @ @@ 3 @ 226  @@ 2 @ 202 @@@@ 2 @ 101  10 @ @@@@ 2 @ 203 @ @@ @ 5 @ 127  12 @ @@ 2 @@ 343 @@@ 2885  12 @ @@ 2 @@ 343 @@@ 2885  14 @ @@ 5 @ 185 @ @@@ @ 2 @ 522 @ @@ 2910 . 14 @ @@ 5 @ 185 @ @@@ @ 2 @ 522 @ @@ 2910 . 14 @ @@ 5 @ 185 @ @@@ @ 2 @ 522 @ @@ 2910 . òí‹þa@òÈßb§a@òÜª@ @ 19 @  @@  @@  @@  @@ 6 @@  20 @ @  @@@@ 177  21 @ @  @  @@ 1 @ 225  22 @ @@  @  @@ @ @@  @@@ 152 @ @   23 @ @ @@@@ @  @@@@@ 378  24 @ @  @  @@ 1 @ 230  26 @ @@@@@@@@@@@@@  @@    . @ 2 @ 298 .@ @@@@@ 2 @ 166  27 @ @@ @ @@@@ 2 @ 25  28 @ @@@@@@@@ @@@@ @@@  @@@@  @@@ 2 @ 166  29 @ @@@ @ 2 @ 25  30 @ @@@ 2 @ 298  אא 44 K  @@@@@@ @@@ @ @@@@ @@@@@@@@@@@@ @@ @ @@@@@  @@ @  @@@ @@  @@ @ @@@@@@@@@@ @   @@  @@ @@@@@@@@@@@@@    @@@@@@ @@@ @ @@@@ @@@@@@@@@@@@ @@ @ @@@@@  @@ @  @@@ @@  @@ @ @@@@@@@@@@ @   @@  @@ @@@@@@@@@@@@@    @@@@ @@ @@@@@@@ @@@@@@@@@@@@@@@ @@@@@@@@ @@  @@@@@@@@@ @@@  @@@@ @ @@@@@@ @ @@ @  @@@@@@@@@@@@ @@@@@@ @ @@ @@@@@@ @ @@@@@@@@@@@@@ @ @  @@@@@@@@@@@  @@@ @ @ @@@@@  @ @@ @@@@ @@ @@@@@@@ @@@@@@@@@@@@@@@ @@@@@@@@ @@  @@@@@@@@@ @@@  @@@@ @ @@@@@@ @ @@ @  @@@@@@@@@@@@ @@@@@@ @ @@ @@@@@@ @ @@@@@@@@@@@@@ @ @  @@@@@@@@@@@  @@@ @ @ @@@@@  @ @@ @@ @  @   @@@@ @   @@@  @@@  @@  @  @  @@@@  @@   @ @@ @ @@ @  @ @  @  @@@@@   @ @ ) 15 (@ @@@@@  @ @@@  @@@@@@ @ @@  @@@@  @@ @@  @@ @@@  @@@ @@@  @@  @@@  @@@@@  @@  @ @ @@@@@@@ @@@  @@@@@@@@@@ @ @ @  @ @@@@  @@@@@@@@@@@@@@@ @@@ @@@@@ @@@@@@@@@@  @@ @ @@@@ @  @@@@@@ @ @  @@@@@@@@@ 15 @ @@ 1 @ 137  15 @ @@ 1 @ 137  †‡ÈÛa@ 9 @òäÛa 5 45 א  @@@ @ @@ @  @@  @ @@@ @ @ @@@@ @ @@  @@@  @@@@ @@@@@  @@@@@@  @@@@ @@  @ @@@  @@@ @@ @@@@@@@@@ @@@@@@@@@@@@@@ @@@@@@@@@@  @@@@@ @ @@@@@ @@  @@@@@@@@@@@@@ @ @@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@ @  @@@@@ @@@@  @@@@@ 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@@@@@@@@@    @@@@@@@@@  @@@@@ @@@@@@@@  @@@@@@@@ @@  @@@@@@ @@@@@@@ @@@ @ @@@@  @ 852 @@ @ @@@@@ @@@@ ) 17 (@ @@@@@@@@@  @@@     @@@@@@@@@  @@@@ @@@@@ @ @@@@@@@@@@ @ @@@   @ 405 @@  ) 18 (@ @@@@  @@  @@@ @  ¯≈‰¿ÊYÏj_ªA Í…_˙ºªA ¯¡_ÊnÍI ¯¡ŒÍYÏj_______________ªA  1 ‰îÍ¿ÚªB_______________‰®ÙªA ëL‰i Í…_______________˙ºÍª Âf_______________Ê¿‰ZÙªA  2 ¯¡ŒÍYÏj_______________ªA ¯≈‰¿ÊYÏj_______________ªA  3 Íπ_______________ÍªB‰ø ¯≈Õëf_______________ªA ¯¬ÊÃ_______________‰Õ  4 ÂfÂJ_______________Ê®‰√ 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@@@   @@@@@@  17 @ @  18 @ @@@@ 2 @ 2 52 @@@ 2910  †‡ÈÛa@ 9 @òäÛa 5 47 א   @ @@@@  @@@ @@@  @ @@@@@ @ @@  @@ @@@@@ @  @@@@ @  ‰îÍ¿ÚªB_‰®ÙªA ëL‰i Í…_˙ºÍª Âf_Ê¿‰ZÙªA  @@ @   @@ @ @@@@@@@@@@ @@@@@@@@@@@@ @ @@@ @  @@@@@@@@@@ @@@@@@@@  ) 19 (@ @@@@ @ @@ @@@@@  ) 20 ( @@@@@@ @ @@@  @@@@ @@ @ @@@@@@@@@@@@@   @@ ) 21 (@ @ @ @@@@@@@@@ @@@@@@@@@@@@@@@  @ @@  @@ @@@@@@@@@@@@@ @@@@@@@  @@@@@@@@@@ @@@ @   @ @@@@@@@@@@ @  Ï∆¯G ¯œÊZ‰N_Ên‰Õ Ú‹ ‰…_˙ºªA   @ @ 26  @ @ “‰QÚ›‰Q ÂSÍªB‰Q ‰…˙ºªA Ï∆¯G    @ 73  @ @ Â…˙ºªA ‰h‰bÏMA AÁf_Úª‰À   @ @ 68 @ @@@@@@ @ @@ @ @@@@ @@@@@@@@@@@@@@ @@ @ @@@@ @@@@@@@@ @ AÃ_ÛªBÚ≥ ‰≈ÕÍh_˙ªA ‰j_Ú∞Ú∑ Êf_Ú¥Úª    @ 73 @ @ @  AÃÛªBÚ≥ ‰≈ÕÍh˙ªA ‰æÊÃÚ≥ Â…˙ºªA ‰©Í¿‰m ÊfÚ¥Úª   @ @ 181 @ @ @ @ @@@@@@@@@@ ) 22 (@ @@@  @@ @  @@@@@   @  @ @@@ ) 23 ( 48 K  Ûa Ûa @@@@  @ @@@ @ @@@@@@ @@@@@@@@@@@ @@@@@ @ @ @ @@@@@@@@@@@ @@    @@  @@@@ @ @@@@@@@@@@@ @@@@@@  @@  @@  @@@@@@ @  @@@@   @@@@@@@@@  @@@ @ @@@ @ @@@@@@@@@@@@@@ @@@@@@@@@ @ @@  @@@@@@@@ @@@@@@@@@@ @   @@  @@ @@@@ @@@  @@  ) 24 ( @@@@@@@@@ @@@@ @@@  @@@@@@@@@@@@  @@ @@@@@@@@@@@@ @@ @@  @@@@@@@@ @@@@@@@@  @@  @ @  @@@@@@  @@@@   ) 25 (  @ @@@@ ﺃ- ﺟﻮﺍﺯ ﺍﻟﻮﻗﻒ @@@ @@@@@@  1  @@@@@@@@@@@@@@@ @  Ú‹ ∆ÃÂƒ_ÍøÊ¤ÂÕ ‰ @@@@@@@@@ @ Â…_˙ºªA ‰¡‰N_‰a @ @ @@@ @  ‰ß Ë’A‰Ã_‰m AÀÂj_Ú∞Ú∑ ‰≈ÕÍh_˙ªA Ï∆¯G ‰∆ÃÂƒ_ÍøÊ¤ÂÕ B_Úª Ê¡Â«ÊiÍhƒ_ÂM Ê¡_Úª Ê¬ÚC Ê¡Â»‰MÊi‰h_√ÚCÚC Ê¡¯»ÊŒ_Úº ‰¡‰N_‰a Â…__˙ºªA Ê¡¯»Í®Ê¿___‰m ”__Úº‰ß‰À Ê¡¯»ÍIÃ___ÛºÛ≥ ”__Úº‰ß    @6  7@ @ @ Ú‹ÚC Ê¡___Â»Ï√ÚC ÚπÍ◊__ÚªÀÛC Ì≈___Û§‰Õ ‰∆ÃÂQÃÂ®ÊJ_‰ø   @ @4 @ @@@@@@@@@@@@@@@ @@@@ @  24 @ @  @  @@ 1 @ 230  25 @ @  @  @@ 5 @@@@  @ @@@ @ @@@@@@ @@@@@@@@@@@ @@@@@ @ @ @ @@@@@@@@@@@ @@    @@  @@@@ @ @@@@@@@@@@@ @@@@@@  @@  @@  @@@@@@ @  @@@@   @@@@@@@@@  @@@ @ @@@ @ @@@@@@@@@@@@@@ @@@@@@@@@ @ @@  @@@@@@@@ @@@@@@@@@@ @   @@  @@ @@@@ @@@  @@  ) 24 ( @@@@@@@@@ @@@@ @@@  @@@@@@@@@@@@  @@ @@@@@@@@@@@@ @@ @@  @@@@@@@@ @@@@@@@@  @@  @ @  @@@@@@  @@@@   ) 25 ( @@@@  @ @@@ @ @@@@@@ @@@@@@@@@@@ @@@@@ @ @ @ @@@@@@@@@@@ @@    @@  @@@@ @ @@@@@@@@@@@ @@@@@@  @@  @@  @@@@@@ @  @@@@   @@@@@@@@@  @@@ @ @@@ @ @@@@@@@@@@@@@@ @@@@@@@@@ @ @@  @@@@@@@@ @@@@@@@@@@ @   @@  @@ @@@@ @@@  @@  ) 24 (      @@@@@@@@@ @@@@ @@@  @@@@@@@@@@@@  @@ @@@@@@@@@@@@ @@ @@  @@@@@@@@ @@@@@@@@  @@  @ @  @@@@@@  @@@@   ) 25 (  @ @@@@  @ @@@@ @@@ @@@@@@  1  @@@@@@@@@@@@@@@ @  Ú‹ ∆ÃÂƒ_ÍøÊ¤ÂÕ ‰ @@@@@@@@@ @ Â…_˙ºªA ‰¡‰N_‰a @ @ @@@ @  ‰ß Ë’A‰Ã_‰m AÀÂj_Ú∞Ú∑ ‰≈ÕÍh_˙ªA Ï∆¯G ‰∆ÃÂƒ_ÍøÊ¤ÂÕ B_Úª Ê¡Â«ÊiÍhƒ_ÂM Ê¡_Úª Ê¬ÚC Ê¡Â»‰MÊi‰h_√ÚCÚC Ê¡¯»ÊŒ_Úº ‰¡‰N_‰a Â…__˙ºªA Ê¡¯»Í®Ê¿___‰m ”__Úº‰ß‰À Ê¡¯»ÍIÃ___ÛºÛ≥ ”__Úº‰ß    @6  7@ @ @ Ú‹ÚC Ê¡___Â»Ï√ÚC ÚπÍ◊__ÚªÀÛC Ì≈___Û§‰Õ ‰∆ÃÂQÃÂ®ÊJ_‰ø   @ @4 @ @@@@@@@@@@@@@@@ @@@@ @ †‡ÈÛa@ 9 @òäÛa 5 49 א  @@@@@@@@@@@@@@ @@@@@@@@@@@@ @@@@  2  @@@@@@@@@@@@@@ @@@@@ @ @@@ @ @@@ @@@@@ @  Ú‹ÚC ∆ÃÛªÃÛ¥‰Œ_Úª Ê¡_¯»Í∏ÙØ¯G Ê≈_Íø Ê¡_Â»Ï√¯G    @ 151 @ @@@ @ Â…_˙ºªA ‰f_Úª‰À @ @@@@@@@@@@@@@@@@@@ @ @@@@@@@@@@@@@@@@@@@@@ @@@ @@@@   2  @@@@@@@@@@@@@@ @@@@@ @ @@@ @ @@@ @@@@@ @  Ú‹ÚC ∆ÃÛªÃÛ¥‰Œ_Úª Ê¡_¯»Í∏ÙØ¯G Ê≈_Íø Ê¡_Â»Ï√¯G    @ 151 @ @@@ @ Â…_˙ºªA ‰f_Úª‰À @ @@@@@@@@@@@@@@@@@@ @ @@@@@@@@@@@@@@@@@@@@@ @@@ @@@@  @@@@@@@@@  1  @@@@@@@@@@@@@@ @  ËΩÊÕ‰Ã_ÚØ ‰î˛º_‰vÂ¿ÙºÍª   @ @4 @ @@@@@@@@ @@@  @@ @ @@@@@  2  @@@@@@@@@@@  Í¢A‰j_Íu ”_Úª¯G Ê¡_¯»ëI‰i ¯∆Êg¯H_ÍI fŒ_Í¿‰ZÙªA ¯l_Õ¯l‰®ÙªA Í  @ @1 @ @@@@@@@@@ @@  @ –Íh_˙ªA Í…_˙ºªA @ @@@@@ ) 26 ( @@ @ @@@ @@@@@@@@@ @@@@ ) 27 (@ @@ @@@  –h_ªA ÛA  @@@@@@ ) 28 ( @@@@@ @@  @ @  @@@@  @@ ) 29 (@ @ @ @@@  @@  @ @ @  æB‰uFÙªA‰À ëÀÂfÂ¨ÙªBÍI B‰»ŒÍØ Â…Úª Â\ëJ_‰nÂÕ Í  @ @ 36 @ @@@@    @@@@ ) 30 (@ @@@ @@@@@@@@@  1  @@@@@@@@@@@@@@ @  ËΩÊÕ‰Ã_ÚØ ‰î˛º_‰vÂ¿ÙºÍª   @ @4 @ @@@@@@@@ @@@  @@ @ @@@@@  2  @@@@@@@@@@@  Í¢A‰j_Íu ”_Úª¯G Ê¡_¯»ëI‰i ¯∆Êg¯H_ÍI fŒ_Í¿‰ZÙªA ¯l_Õ¯l‰®ÙªA Í  @ @1 @ @@@@@@@@@ @@  @ –Íh_˙ªA Í…_˙ºªA @ @@@@@ ) 26 ( @@ @ @@@ @@@@@@@@@ @@@@ ) 27 (@ @@ @@@  –h_ªA ÛA  @@@@@@ ) 28 ( @@@@@ @@  @ @  @@@@  @@ ) 29 (@ @ @ @@@  @@  @ @ @  æB‰uFÙªA‰À ëÀÂfÂ¨ÙªBÍI B‰»ŒÍØ Â…Úª Â\ëJ_‰nÂÕ Í  @ @ 36 @ @@@@    @@@@ ) 30 (@ @@@ 26 @ @@@@@@@@@@@@@  @@    . אא @ 2 @ 298 .@ @@@@@ 2 @ 166  27 @ @@ @ @@@@ 2 @ 25  28 @ @@@@@@@@ @@@@ @@@  @@@@  @@@ 2 @ 166  29 @ @@@ @ 2 @ 25  30 @ @@@ 2 @ 298  òí‹þa@òÈßb§a@òÜª@ @ 50 K  @@@  @@ @@@@@@    @@  @ @@    @@@  @ @@@@@ @ @@@ @ @ @ @ @@@@  @@ @  @ @@@@ ) 31 (@ @  @@@@ @ @    @@@    @@ @@ ) 32 (@ @  æB_‰uFÙªA Í @@@  @@@@ @ @@@@@ @@  Â\ëJ_‰nÂÕ  @@@ ) 33 ( @@ @@@  @@@    @@ @ @  Ú‹ ËæB‰U¯i ˆ—‰iB‰VÍM Ê¡¯»Œ¯»ÙºÂM  @@ @  @ @@@@ @ @  Â\ëJ_‰nÂÕ  @@@ ) 34 ( @@@@@  ËæB_‰U¯i  @@@@ @  @@@  @@  @@@@@@@  @@@@@@@ @  @@  @@@@@@@  @@@@ @@@@@@@@@@@ @@@ @@@@@@@@@@@@@@@@@@@@@@@@@ @@@@ @@@@@@@@@  @@@@   @@ ) 35 ( @@ @@@@ @ @@@@@@@ @@ @@@@@@@@@@@@@@@ @@@@ @ @ @@@@@@@@  31 @  @@ @ 2 @ 229  32 @ @@@@@@@@ 452  33 @  @@ @ 2 @ 229  34 @ @@@@@@  @@  @@@ 2 @ 298  35 @ @ @@@@ @@ 175  32 @ @@@@@@@@ 452  32 @ @@@@@@@@ 452  34 @ @@@@@@  @@  @@@ 2 @ 298  35 @ @ @@@@ @@ 175  †‡ÈÛa@ 9 @òäÛa 5 51 א  @@@@@@@@@@@@ @@@@@@@ @@  @ @@ @ @@@@@@ @@@@@@@@@@@@@ @  @@@@@@@@ @@    ) 36 (@   @  @@@  @ @@  @ @ @    @  @  @   @@@@@@@@@@  @@@@@@@@ @@    ) 36 (@   @  @@@  @ @@  @ @ @    @  @  @   @@@@@@@@@@  òí‹þa@òÈßb§a@òÜª@ @ 52  W אא 1  @@@@  ı›ŒÍMÊj__‰M ‰∆EÊj_Û¥ÙªA ÊΩ__ëM‰i‰À    @4@ @@@@@ @ @@@@@@@@@@@@@ @@@@@@@@@@@@ @ @@@ @@@@@@@@@@@ @ @  @@ @@@@  @@@  @ 40 @  @ @@@@@@@ @ @  @@@@@  ) 37 (@ @@@  @ 68 @@ @ @  ) 38 (@ @@@@ @@  2  @@@ @ Ú›ÍM Ïµ_‰Y Â…‰√ÃÛºÊN_‰Õ ‰LB‰N_Í∏ÙªA Ê¡Â«B‰ƒ_ÊŒ‰ME ‰≈ÕÍh_˙ªA Í…_ÍM‰À    @ 121 @ @@@ @@@@@@@@   W  א @@@@@@@@  @@  @   @@@@ @@@@  @@@@@@@@@ @@ @@@@@  1  @ @@  @@@ @@ @@@@  @@ @ @@ @@@  @  36 @ @ @ @@ 19  37 @ @@@@ 1 @ 209 @@@@ 1 . אא 38 @ @@ 198 @  @ @ @ .     1  @@@@  ı›ŒÍMÊj__‰M ‰∆EÊj_Û¥ÙªA ÊΩ__ëM‰i‰À    @4@ @@@@@ @ @@@@@@@@@@@@@ @@@@@@@@@@@@ @ @@@ @@@@@@@@@@@ @ @  @@ @@@@  @@@  @ 40 @  @ @@@@@@@ @ @  @@@@@  ) 37 (@ @@@  @ 68 @@ @ @  ) 38 (@ @@@@ @@  2  @@@ @ Ú›M Ïµ_‰Y Â…‰√ÃÛºÊN_‰Õ ‰LB‰N_∏ÙªA Ê¡Â«B‰ƒ_ÊŒ‰ME ‰≈Õh_˙ªA …_M‰À    @ 121 @ @@@ @@@@@@@@  @@  @   @@@@ @@@@  @@@@@@@@@ @@ @@@@@  1  @ @@  @@@ @@ @@@@  @@ @ @@ @@@  @  òí‹þa@òÈßb§a@òÜª@ @ 52 36 @ @ @ @@ 19  37 @ @@@@ 1 @ 209 @@@@ 1 . 38 @ @@ 198 @  @ @ @ . òí‹þa@òÈßb§a@òÜª@ @ 52 36 @ @ @ @@ 19  37 @ @@@@ 1 @ 209 @@@@ 1 . 38 @ @@ 198 @  @ @ @ . 43 @ @@@@ 1 @ 551  44 @ @@@@@ 3 @ 384  45 @ @@@ 798  46 @ @ @ 13 @ 519  47 @   @@@ 4937  48 @ @  @ 6 @ 84  49 @  @ 1 @ 7 50 @ @  @ 6 @@ 84  51 @  @ 1 @ 7 52 @  @ @@  53 @ @ @ @ @@@  @  @ @@ 7 @ 55 @@@@@   @@ @ @ 1 @ 316  54 @ @@@@ 2 @ 31  52 @  @ @@  אא 52 K  @@ @@ @@@@  @@  @ @@@  @@@@@ @@@@ @@@@@@@  ) 40 (@ @@@@ @@@@@@@@@@@@@@ @@@@@@@@ @  @@@@@@  @@ @@@@@ @@@  @@@@@@@@@  @@@@@  @ @ @  @@@@@@@@@@@@@@ @@  @@@@@@@@@@@@  @@@@@@@@@@ ) 41 (@ @@@ @@@@@@@  @@@@@@@@@ @@@@@@@@@@@@@@@@@@ @ @@ ) 42 ( 2  @@@@@@@@@@@@@ @ @ @@@@@@@@@@@@@@  @  Â B‰ƒÙªÏl‰√‰À ÎSÙ∏Âø ”Úº‰ß ¯pBÏƒªA ”Úº‰ß Â ÚC‰jÙ¥‰NÍª Â B‰ƒÙ≥‰jÚØ ıB√EÊjÛ≥‰À ı›Õ¯lÊƒ‰M  @  @ 106  2  @@@@@@@@@@@@@ @ @ @@@@@@@@@@@@@@  @  Â B‰ƒÙªÏl‰√‰À ÎSÙ∏Âø ”Úº‰ß ¯pBÏƒªA ”Úº‰ß Â ÚC‰jÙ¥‰NÍª Â B‰ƒÙ≥‰jÚØ ıB√EÊjÛ≥‰À ı›Õ¯lÊƒ‰M  @  @ 106  3  @@@  @@@@@  @ @@@@@@@@ @@@@@@@@@@@@@@ @@@@@@@@@ @ @@@@ @  @ 261 @  @ @@@@@ @  @ @ 93 @@ @ @@@  @@@  @ 20 @@ @  @@ 3  @@@  @@@@@  @ @@@@@@@@ @@@@@@@@@@@@@@ @@@@@@@@@ @ @@@@ @  @ 261 @  @ @@@@@ @  @ @ 93 @@ @ @@@  @@@  @ 20 @@ @  @@ 40 @ @@ 5 @ 22  41 @ @@@ 1 @ 541  41 @ @@@ 1 @ 541  42 @ @@ 1 @ 541  †‡ÈÛa@ 9 @òäÛa 5 53 א  @@@@@@  @ @@  @@  @@@ @@@@@ @@@@@@@ ) 46 (@ @@@@@@@@@  @@@@@  @@  @@@ @ @  @@@ @ @@@  ) 47 ( @@ @@@@@  @  @ @@ @@@@@ @@@@@@@@@@@@@@ @ ‰LB‰N_Í∏ÙªA B‰ƒ_ÊQ‰iÊÀÚC Ï¡_ÂQ B‰√ÍeB‰J_Íß Ê≈_Íø B‰ƒÊŒÚ∞ÚÒ_ÊuA ‰≈ÕÍh_˙ªA   @ @ 32 @ @@  @@@@@@@@ @@@@@@@ @  @@@ @ @@@ @@@@@@@@@@@@ @@@ @@@@@@@@@@@@ ) 48 ( 48 @ @  @ 6 @ 84  54 K  @@@@  @@@@ ) 49 (@  @@ @@  @ @  @@@@@@@@@@ @ @@ @ @@@@@@@@ @ @@@@@@@@ @@@@@@@@@@@@@@@@ @ ) 50 (@ @@ @  @@@@@  @@@@@@@@@@ @ @@@@@@@@ ) 51 ( 5  @@@  @ 360 @@  @@ @@@@@@@@@@@@ @@ @@@@ @ Í’A‰j__Ú¥Û∞ÙºÍª ÂPBÚ≥‰f__ÏvªA B__‰¿Ï√¯G B‰»ÊŒ__Úº‰ß ‰îÍºÍøB_‰®ÙªA‰À ¯îÍ∑B__‰n‰¿ÙªA‰À  @  @ 60 @  ) 52 (@ @@ @ @@@@@  @ @ @ @@ @@@@ @ @ @ @  Í’A‰j_Ú¥Û∞ÙºÍª ÂPBÚ≥‰f_ÏvªA B_‰¿Ï√¯G  @@@ ) 53 (@ @ @ @@@  @@ @@@@@@@@@@ @@@@@@@@@@@@@@ @ @@@@@@  @@  @@@  @@@@@@@@@@@@  @@@@@ @@@    @@@  ) 54 (@ @   @@@@    @@@@@@@ @@@ @ @ @ @@@  @@@  @  @@@ @@@@ @ @@@@@@@@@@@@  @  6  @ @@ @@@ @ @@@  @ 405 @@ @ @@@@  @ 71 @@ @ @@@  @@ @ @@@@@@@ @@@@  @@@@ ) 49 (@  @@ @@  @ @  @@@@@@@@@@ @ @@ @ @@@@@@@@ @ @@@@@@@@ @@@@@@@@@@@@@@@@ @ ) 50 (@ @@ @  @@@@@  @@@@@@@@@@ @ @@@@@@@@ ) 51 ( 5  @@@  @ 360 @@  @@ @@@@@@@@@@@@ @@ @@@@ @ Í’A‰j__Ú¥Û∞ÙºÍª ÂPBÚ≥‰f__ÏvªA B__‰¿Ï√¯G B‰»ÊŒ__Úº‰ß ‰îÍºÍøB_‰®ÙªA‰À ¯îÍ∑B__‰n‰¿ÙªA‰À  @  @ 60 @  ) 52 (@ @@ @ @@@@@  @ @ @ @@ @@@@ @ @ @ @  Í’A‰j_Ú¥Û∞ÙºÍª ÂPBÚ≥‰f_ÏvªA B_‰¿Ï√¯G  @@@ ) 53 (@ @ @ @@@  @@ @@@@@@@@@@ @@@@@@@@@@@@@@ @ @@@@@@  @@  @@@  @@@@@@@@@@@@  @@@@@ @@@    @@@  ) 54 (@ @   @@@@    @@@@@@@ @@@ @ @ @ @@@  @@@  @  @@@ @@@@ @ @@@@@@@@@@@@  @  6  @ @@ @@@ @ @@@  @ 405 @@ @ @@@@  @ 71 @@ @ @@@  @@ @ @@@@@@@ †‡ÈÛa@ 9 @òäÛa 5 55 א  @@@@  ) 55 (@ @@@@@@@@ @ @@ @@ @@@ @ @@ @@@@ @@@@@@@@@@@@@@   òí‹þa@òÈßb§a@òÜª@ @ 74 @ @ 3 @ 994  75 @ @@@@ 1924  76 @ @@ 1924  77 @ @@@@ 2 @ 179  78 @ @@ @ @ @@ 182   79 @ @@ 9 @ 91  78 @ @@ @ @ @@ 182  79 @ @@ 9 @ 91  80 @ @  81 @ @@@@ 1 @ 319  81 @ @@@@ 1 @ 319  †‡ÈÛa@ 9 @òäÛa 5 80 @ @   79 @ @@ 9 @ 91  87 @ @@@ 149  אאא  אאא  @@@@@@@@@@@ @ @ @@ @@@@@@@@@@ ) 56 (@ @   @ @@ @ @@@@@@ ) 57 (@ @@@@@ @@@@@@ @  @@@@@@   ) 58 ( @@@@@  @@@@@@@@ @  @@@@@@@  ) 59 (@ @@@ @@@ @ @ @@@@@@@@@@@@@ @@@ ) 60 (@ @@ @  @ @@@@  ) 61 (@ @@@ @@@@ @ @@@@ ) 62 ( @@@@@ @ @@ @@@@@@@ @ @@@@@@@ @@@ @  @ @@  @@@@@@  ) 63 (@ @@@@@ @ @@@@@@ @@@@@@@@@ @ @  55 @ @@@ 1 @ 768 @@@@@ @ 55 @ @@@ 1 @ 768 @@@@@ @ 56 @ @ @ @  57 @ @  @ @@@  58 @ @@ 5 @ 244  63 @ @@@ 9 @ 47  56 K    -   @@@ @ -   @@  @  @@@@@  @@@ @ @@@  @@  @@@@@@@@@@@@  @@ @ @ @@@@@@@@ @ @@@@@@ @  @  @@ @  -   @@@ @ -   @@@ @  -   @@  @  @@@@@  @@@ @ @@@  @@  @@@@@@@@@@@@  @@ @ @ @@@@@@@@ @ @@@@@@ @  @  @@ @ אא @@@@@@@@@@@ @@@@@@@@ @@@@@@ @@@@ @@@@@@@@@@@@@@ @@@@@@@@@@@@@@ @@@@@@@@@@@@ @@@@  @@@@@@@@@@ @ @@@ @  @ 833 @  @@@ @@ @@@@@@@ @@ ) 64 ( @@@@@@@@@@  @@@@@@@ @@@@@ @@@@ @@ @ @@@@@@@@@@@ @@@ @ @@@@@@@@@@ @@@@@@@@@@@    64 @ @@@@ 76 @@@@@@@ 4@ 1997  †‡ÈÛa@ 9 @òäÛa 5 57 א  @ ) 65 (@ @@@@@@@   @@@@@ @@@@@@@@@@@@@ @ @ @  @@ @@@@@@ @@@@@@@@@ @ @ @@@@ @ @@@@@@@@@  @@@ @@@@@@@ @  @@@@@@@@@@@ ) 66 (@ @@  ÂOÙ∑‰j_‰M  @@@@ @  ÂOÙ∑‰j_‰M B‰¿Œ_ÍØ BÁZÍªB_‰u ÂΩ_‰__¿ÊßÚC œ_˛º‰_®Úª    @ 100 @ @ @@@@@@@   BÁ¿Œ_ÍºÙ∏‰M ”_‰mÃÂø Â…_˙ºªA ‰¡_˙ºÚ∑‰À    @ 164 @ @ @@@@@@  @@@@@ @@@ @ ”_Úº‰ß ‰…_˙ºªA Ï∆¯G Ëj_ÕÍfÚ≥ Î’Êœ_‰q ëΩ_Û∑   @ @ 77 @ @@@@@@@@@ @@@@@@@@@@@@ @@@@@@  @@ @@@ @@@@@  òí‹þa@òÈßb§a@òÜª@ @ 58 @ @@  @@@@@@@@@@@@@ @@ @ @@@@@@@  @@@@@ @@@@@@@  @@ @ @@@@@@@@ @@@ @@@@ @ @@@@ @ @@@@@@ @@@@@@ @ @@@@@  @@ @@@  @  @  @@  @@@  @  @ א  @@@@@@@ @@@ @ @@@@@ @@@@@@@@@@ @@ @@@@@@@@@@@ @ @ @@@@ @ @@@@@ @@  65 @ @@  @ @@@@@@@@@@ 11 @@ 1971  66 @ @  @ @@  @@@@@@@@@@@@@ @@ @ @@@@@@@  @@@@@ @@@@@@@  @@ @ @@@@@@@@ @@@ @@@@ @ @@@@ @ @@@@@@ @@@@@@ @ @@@@@  @@ @@@  @  @  @@  @@@  @  @ 58 K  †‡ÈÛa@ 9 @òäÛa 5 @@@@@@@ @ @@@@@ ) 67 ( @@  @ @@@@@@@@@@  אא @@@  @ 646 @@  ) 68 (@ @@  @@  @@ @ @ @@@ @@@@@@@@@@@ @  ) 69 ( א @@  @@@  @ 241 @  ) 70 ( @@@@@ @ @@ @@@@ ) 71 (@  @ @@@@@@@@@      @ @@  @@  @@@  @@ ) 72 ( @@@@ @ @ @@ @ @@@@@@@@@@@@@  ) 73 ( א @@@  @ 808 @@  @@@ @  @@@@@  67 @ @@@@@@@@@@@@ @ @ 1 @  @@@ @@@@@@ 239 @@ @@  68 @ @@ @@@@@@@@  @@@@ 323  69 @  @ @ 1 @ 319 @@@@@ 3@ 1980 @@ 1 @ 30 @ @ 1 @ 223 @  @ @@ 1 @ 306  70 @ @@@@@@@@@@@@   @@@@ 164 @@@ @@ @ @  @ 4 @ 415 @@@ 2 @ 96 @ @ 229 @@@ @ 1  4@ @@@ 7 @ 92  71 @  @ @ 1 @ 320 @@ 72 @ @ @ @  @@@@@  @@@@@@ 80 @@@ @ @@ @ @  @ 3 @ 313 @ @@@  @@ 1 @ 111  73 @  @ @@@@ 1 @ 79 @@@@@@ 1@ 1972  K  @@@@@@@ @ @@@@@ ) 67 ( @@  @ @@@@@@@@@@  אא @@@  @ 646 @@  ) 68 (@ @@  @@  @@ @ @ @@@ @@@@@@@@@@@ @  ) 69 ( א @@  @@@  @ 241 @  ) 70 ( @@@@@ @ @@ @@@@ ) 71 (@  @ @@@@@@@@@      @ @@  @@  @@@  @@ ) 72 ( @@@@ @ @ @@ @ @@@@@@@@@@@@@  ) 73 ( א @@@  @ 808 @@  @@@ @  @@@@@ 67 @ @@@@@@@@@@@@ @ @ 1 @  @@@ @@@@@@ 239 @@ @@   68 @ @@ @@@@@@@@  @@@@ 323  69 @  @ @ 1 @ 319 @@@@@ 3@ 1980 @@ 1 @ 30 @ @ 1 @ 223 @  @ @@ 1 @ 306  70 @ @@@@@@@@@@@@   @@@@ 164 @@@ @@ @ @  @ 4 @ 415 @@@ 2 @ 96 @ @ 229 @@@ @ 1  4@ @@@ 7 @ 92  71 @  @ @ 1 @ 320 @@ 72 @ @ @ @  @@@@@  @@@@@@ 80 @@@ @ @@ @ @  @ 3 @ 313 @ @@@  @@ 1 @ 111   73 @  @ @@@@ 1 @ 79 @@@@@@ 1@ 1972  †‡ÈÛa@ 9 @òäÛa 5 59 א  @@@@@ @@  @@@@@@@ @@@@@  @@@@@  @ @ @ @@@@@@@@@@@   ) 74 ( @@@@@@@ @@@@@@@@  @@@@@@@@@@ @ @@ @ @@@@@@@@@@@  @@@@ @@@@@@@@@@@ @@@ @@@@@@@@@@@@ ) 75 (@ @@ @@@@@@@@@@@  1  @@@@@@@  @ @@@@@ @ @@@@ @@  @@ @ @@@ ) 76 (@ @@@@@@@ @@@@@@@@ ) 77 (@ @@@@@ @@  @@@@ @ @@ @ @@@@@@ @ @ @@@@@@@ @@@@@ @ @ @@@@@@@@@@@@@@ @@@@@@@@@@@@@@@ @@@@@@@@@@@@ @@ @@@@@@@@@@@@@ @ @@@@@@@@@@@@@@@@   @ 444 @ @ @@@ @ @@@@ @ @@@@ @@@@@@@ @@@@@@ @@@@@@@@@@@@ @@@@@@@@@@@@ 77 @ @@@@ 2 @ 179  òí‹þa@òÈßb§a@òÜª@ @ 60 K  @@@ ) 78 ( 2 @ @@@@@@@@@@ @ @ @@  @@@@ @   @ @@@ @  @@@@  @ 10 @ @    ) 79 ( @@@  @ @ @@@@ @ @@@@  @@@@@ @ @ @@@  @@@@@@     3  @@@ @  @@@@@@ @ @@@@ @@ @  @@ @@   B‰»ÊŒÚº‰ß ‰îÍºÍøB‰®ÙªA‰À ¯îÍ∑B‰n‰¿ÙªA‰À Í’A‰j_Ú¥Û∞ÙºÍª ÂPBÚ≥‰f_ÏvªA B_‰¿Ï√¯G    @ 60 @  @ @@ @ @@@@@  @ @ @ @@ @@@ @@ @ @@ @  Í’A‰j_Ú¥Û∞ÙºÍª ÂPBÚ≥‰f_ÏvªA B_‰¿Ï√¯G  @@@ ) 80 (@  @ @@@@@@@   Í’A‰j_Ú¥Û∞ÙºÍª  @@@@@@@ @@@@@@@@ @ @@@@@  @@ @@@ @@@@@@@@@@ @@@@   4  @@@@@  @ 794 @@  @@@@@@@@ @  @@@@@@@@@  @@@@@ @ @@@@@ @@@@@@@@@@@ @@@@@@@@@@@@@@@ @ @ @@@@@@ @ @@ @  @ @@ @ @ @   @ @   @@  ) 81 ( 5  @@ @@@  @ 923 @@  @@@@@@@@@ @  @@@@    @@@@  @@@@@@ @@@@@@@@ @@@@@@@@ @ @ @@@@@@@ @ @@ @@@@@@@ 4  @@@@@  @ 794 @@  @@@@@@@@ @  @@@@@@@@@  @@@@@ @ @@@@@ @@@@@@@@@@@ @@@@@@@@@@@@@@@ @ @ @@@@@@ @ @@ @  @ @@ @ @ @   @ @   @@  ) 81 (     5  @@ @@@  @ 923 @@  @@@@@@@@@ @  @@@@    @@@@  @@@@@@ @@@@@@@@ @@@@@@@@ @ @ @@@@@@@ @ @@ @@@@@@@ 81 @ @@@@ 1 @ 319  †‡ÈÛa@ 9 @òäÛa 5 61 א  @@  ) 82 ( 6  @@@@@@  @ 833 @@  @@@@@@@@@@ @@@@@ @@@@@@@@@@@@  @ @ @@@@@@@@ @  @@@  @@ @@@@@@@@@@@@@@@@@ ) 83 ( @@@@@@@@@@@@@    @@@@@@    @@@    @@@    @ @@ @@  @@@ @@   –@ @@@@@@@@@@@@@ @@@@@  @@@@  @@@  @@ @@@@  @@@@ @  – @@ @@@@@@@@ @@  @@@ @@  ) 84 (  @@@@@@@@ @ @@@@@@ @@@@@@ @@@@@@@@@@@@@  @@@@@@@@@@@@ ) 85 (   @@@@@@@@@@@@@ @ @ @@@@@@@@@@@ @@@ ) 86 (  @ @ @@@@  @@@@@@@@ @@@  @ @@@@@@@  @@@ א  @@  ) 82 ( 6  @@@@@@  @ 833 @@  @@@@@@@@@@ @@@@@ @@@@@@@@@@@@  @ @ @@@@@@@@ @  @@@  @@ @@@@@@@@@@@@@@@@@ ) 83 ( @@@@@@@@@@@@@   @@@@@@@@@@@@@    @@@@@@    @@@    @@@    @ @@ @@  @@@ @@     –@ @@@@@@@@@@@@@ @@@@@  @@@@  @@@  @@ @@@@  @@@@ @   – @@ @@@@@@@@ @@  @@@ @@  ) 84 (   – @@ @@@@@@@@ @@  @@@ @@  ) 84 (  @@@@@@@@ @ @@@@@@ @@@@@@ @@@@@@@@@@@@@  @@@@@@@@@@@@ ) 85 (     @@@@@@@@@@@@@ @ @ @@@@@@@@@@@ @@@ ) 86 ( òí‹þa@òÈßb§a@òÜª@ @ 62 K  @ @ @@   ) 87 (@ @@@   @@@@@@  @@ @@@@  @ @@@@@@  @@  @@@@@@  @@@  @@@@@ @ @@@  @@@@@@@@@@@@ @@@@@@@@@@ @@@@ @@@ @ @@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@  †‡ÈÛa@ 9 @òäÛa 5 63
https://openalex.org/W4234271673	https://www.qeios.com/read/H2JOO6/pdf	English	null	Glucose Urine Excretion Rate	Definitions	2,020	cc-by	66	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Glucose Urine Excretion Rate National Cancer Institute National Cancer Institute Qeios ID: H2JOO6 · https://doi.org/10.32388/H2JOO6 Source National Cancer Institute. Glucose Urine Excretion Rate. NCI Thesaurus. Code C117834. A determination of the amount of glucose being excreted in urine over a defined period of time. Qeios ID: H2JOO6 · https://doi.org/10.32388/H2JOO6 1/1
https://openalex.org/W3213524185	https://zenodo.org/records/8371918/files/auditory_visual_interactions_in_egocentric_distance_perception_Ventriloquism_effect_and_aftereffect.pdf	English	null	Auditory-visual interactions in egocentric distance perception: Ventriloquism effect and aftereffect	The Journal of the Acoustical Society of America/The journal of the Acoustical Society of America	2,021	cc-by	13,878	a)Portions of this work were presented at the 165th meeting of the Acoustical Society of America. b)Current address: Audio Information Processing, Technical University of Munich, Munich, 80333, Germany. Electronic mail: lubos.hladek@ tum.de, ORCID: 0000-0002-0870-7612. c)ORCID: 0000-0003-4936-9303. Auditory-visual interactions in egocentric distance perception: Ventriloquism effect and aftereffecta) L’ubos Hladek,1,b) Aaron R. Seitz,2,c) and Norbert Kopcˇo1 1Institute of Computer Science, P. J. Safarik University, Jesenna 5, Kosice, 040 01, Slovakia 2Department of Psychology, University of California Riverside, 900 University Avenue, Riverside, California 92521, USA Auditory-visual interactions in egocentric distance perception: Ventriloquism effect and aftereffecta) L’ubos Hladek,1,b) Aaron R. Seitz,2,c) and Norbert Kopcˇo1 1Institute of Computer Science, P. J. Safarik University, Jesenna 5, Kosice, 040 01, Slovakia 2Department of Psychology, University of California Riverside, 900 University Avenue, Riverside, California 92521, USA ABSTRACT: This study describes data on auditory-visual integration and visually-guided adaptation of auditory distance percep- tion using the ventriloquism effect (VE) and ventriloquism aftereffect (VAE). In an experiment, participants judged egocentric distance of interleaved auditory or auditory-visual stimuli with the auditory component located from 0.7 to 2.04 m in front of listeners in a real reverberant environment. The visual component of auditory-visual stimuli was displaced 30% closer (V-closer), 30% farther (V-farther), or aligned (V-aligned) with respect to the auditory compo- nent. The VE and VAE were measured in auditory and auditory-visual trials, respectively. Both effects were approx- imately independent of target distance when expressed in logarithmic units. The VE strength, deﬁned as a difference of V-misaligned and V-aligned response bias, was approximately 72% of the auditory-visual disparity regardless of the visual-displacement direction, while the VAE was stronger in the V-farther (44%) than the V-closer (31%) con- dition. The VAE persisted to post-adaptation auditory-only blocks of trials, although it was diminished. The rates of build-up/break-down of the VAE were asymmetrical, with slower adaptation in the V-closer condition. These results suggest that auditory-visual distance integration is independent of the direction of induced shift, while the re- calibration is stronger and faster when evoked by more distant visual stimuli. V C 2021 Acoustical Society of America. https://doi.org/10.1121/10.0007066 (Received 26 August 2020; revised 15 October 2021; accepted 19 October 2021; published online 15 November 2021) [Editor: Laurie M. Heller] Pages: 3593–3607 Calcagno et al., 2012; Cubick et al., 2015; Gardner, 1968; Mendonc¸a et al., 2016; Mershon et al., 1980; Mershon et al., 1981; Rebillat et al., 2012; Voss, 2016; Zahorik, 2001). The primary cues for auditory distance perception are sound level and direct-to-reverberant ratio. Visual dis- tance perception relies upon binocular disparity and ver- gence cues, which also underlie auditory-visual interactions (Tresilian et al., 1999; Zahorik et al., 2005). The main goal of the current study is to systematically examine the basic properties of the ventriloquism effect and aftereffect in distance. V C 2021 Acoustical Society of America 3593 0001-4966/2021/150(5)/3593/15/$30.00 ARTICLE ................................... ARTICLE ................................... https://doi.org/10.1121/10.0007066 Finally, the dynamics of the ventriloquism buildup and decay are addressed, as VE and VAE occur on different time scales and the rates of adap- tation might also be different for the V-closer vs V-farther conditions. y g p Many previous auditory distance and ventriloquism studies used virtual acoustics to present stimuli. Here, the main goal is to understand the relative effects of visual stim- uli on auditory percepts, for which it is critical that the visual and auditory location percepts are veridical. Therefore, the study was performed in a real environment using loudspeakers and LEDs, to ascertain that the physical locations of the stimuli are unambiguous, and the percepts are as close to natural as possible. Of note, this naturalistic listening approach resulted in some artifacts, such as acous- tic shadowing of speakers by each other. While this could have been avoided, e.g., by staggering the loudspeakers in azimuth or elevation, this design choice was made as stag- gering would result in additional azimuth or elevation cues that could help identify individual loudspeakers. Broadband noise stimuli were employed to provide strong localization cues such as the rich spectral cues which aid sound localiza- tion in distance and onset cues which are important for sound localization in rooms (Hartmann, 1983), while at the same time assuring that participants were unable to ﬁxate on a speciﬁc spectral characteristic of the stimulus, as the noise was random and independent from trial to trial. LEDs were used as visual stimuli, as is commonly in ventriloquism lit- erature (Bertelson et al., 2006; Recanzone, 1998). They are small enough so that all of them were clearly visible in the dense array used here, and they have good temporal charac- teristics (quick onset and offset), which was important to keep the synchrony of the auditory-visual stimuli. To assess the possible effect of these experimental choices on baseline distance perception, Appendix A provides a comparison of the performance with current setup to available data from the literature. To address these questions, a behavioral experiment was performed that investigated VE and VAE at interme- diate egocentric distances from 0.7 to 2.04 m. The experi- ment was conducted in a small semi-reverberant room in which targets were located in a line in front of partici- pants. https://doi.org/10.1121/10.0007066 distance cues become available (Kopcˇo and Shinn- Cunningham, 2011). However, even in reverberation, audi- tory distance judgments are more accurate if the speakers are visible or if there are visual references in the room (Calcagno et al., 2012; Zahorik, 2001). The current study examined whether there is an asymmetry in the strength of VE and VAE for the visual (V)-closer vs V-farther stimuli in a reverberant room. ventriloquism adaptation on the timescale of minutes. Each session took approximately one hour and consisted of several experimental runs in which the direction of the V-stimulus displacement was ﬁxed, combined with several pre-adaptation and post-adaptation control runs. While VE was expected to occur immediately upon the presentation of AV stimuli, VAE was expected to grow gradually as more and more AV stimuli were presented, and to decay gradually when the AV stimulus presentation stopped. The ﬁnal analysis examined the VAE dynamics of adaptation across runs within a session, focusing on whether VAE strength differs for the V-closer and V-farther conditions, which would be another indication that the adaptation pro- cess underlying VAE is direction-dependent. Most previous ventriloquism studies examining ven- triloquism have utilized one or only a few ﬁxed visual stimulus locations combined with several auditory loca- tions to examine the spatial dependence of the distance VE and/or VAE (Gardner, 1968; Mershon et al., 1980; Zahorik, 2003). An important question that can elucidate the adaptive nature of the neural structures underlying ventriloquism is whether the auditory distance representa- tion adapted is linear or non-linear (Bedford, 1993; Shinn- Cunningham et al., 1998a). Typically, auditory distance studies analyze data on a logarithmic scale, as variance in distance judgments is approximately constant on this scale (Anderson and Zahorik, 2014; Kopcˇo et al., 2012). The current study used a range of auditory-visual stimulus pairs with a ﬁxed auditory-visual (AV) distance ratio to test whether adaptation by a constant amount in logarith- mic units over a range of distances will induce constant VE and VAE shifts in logarithmic units. The relative strength of the induced VAE vs VE in both V-closer and V-farther conditions is examined to determine whether the size of aftereffects correlate with the size of VE and whether adaptation caused by ventriloquism is equally large for the two directions. https://doi.org/10.1121/10.0007066 The auditory (A) and V stimuli were presented on an array of light-emitting diodes (LEDs) densely covering the distances of 0.45 to 2.68 m, respectively. The ventrilo- quism effect was induced by presenting AV stimuli in which the A component (one of the 8 speakers) was paired with a V component (LED) positioned either 30% closer (V-closer) or 30% farther (V-farther). The size of V- component shift was ﬁxed throughout the study so that the effects of displacement direction (V-closer vs V-farther) and stimulus distance could be examined. The ventrilo- quism aftereffect was examined in A-only trials inter- leaved with the AV trials, and by separate A-only runs performed before and after adaptation. The interleaved A-only trials were expected to show the immediate after- effect caused by preceding AV stimulation on the time- scale of seconds, while the shifts observed in the separate A-only runs were expected to reﬂect a more persistent I. INTRODUCTION The human ability to localize sounds requires a lot of ﬂexibility. For example, whenever we enter a new room, we need to adapt our mapping of acoustic localization cues to perceived locations to reﬂect the reverberation-related changes in those acoustic cues (Shinn-Cunningham et al., 2005). Vision plays an important role in these calibration processes, causing both an immediate re-alignment of audi- tory spatial percepts to match the corresponding visual sig- nals, and a long-lasting adaptation of the acoustic localization cue mapping based on the visual inputs. The immediate alignment of auditory percepts with visual sig- nals is known as visual capture or the ventriloquism effect (VE) (Jack and Thurlow, 1973; Bertelson et al., 2000; Alais and Burr, 2004; Recanzone, 2009). Persisting visually induced adaptation has been referred to as the ventriloquism aftereffect (VAE) (Kopcˇo et al., 2009; Recanzone, 1998; Wozny and Shams, 2011). VE and VAE have been exten- sively studied for horizontal sound localization; however, fewer studies have researched how vision inﬂuences audi- tory distance perception (Anderson and Zahorik, 2014; One previously reported property of ventriloquism in distance is that the strength of the effect depends on whether it is induced by visual stimuli displaced closer than the audi- tory signal (a condition referred to here as V-closer) vs visual stimuli displaced farther away (a condition called here V-farther). For example, in anechoic space, VE creates a strong illusion of the sound coming from the nearest plau- sible visual target located closer than the sound source (“proximity image effect”; Gardner, 1968; Min and Mershon, 2005). Later, Mershon et al. (1980) found an effect similar to the proximity image effect that applied to visual targets located farther than the sound source. These effects are strong in anechoic rooms in part because very few intensity-independent distance cues are available for these sources (Brungart and Rabinowitz, 1999). In reverber- ant rooms, the visual dominance in distance dimension can be expected to be weakened, as intensity-independent V C 2021 Acoustical Society of America 3593 0001-4966/2021/150(5)/3593/15/$30.00 J. Acoust. Soc. Am. 150 (5), November 2021 3593 B. Setup, stimuli, and procedures corrected for the distance-dependent level increase and after subtracting the across-distance mean at each frequency to assess speaker-to-speaker spectral differences and the effect of acoustic shadowing of the speakers. Responses vary mostly by up to 2 dB for frequencies up to 1 kHz, with no systematic effect of distance. At frequencies 2 kHz and higher, there is a systematic variation of up to 3 dB such that more distant speakers are attenuated relatively to closer ones (for the closest speaker at the highest frequency of 8 kHz, this deviation is more than 5 dB). Figure 1(E) also shows the spectra of the individual random-noise stimuli (gray traces). This ﬁgure shows that the stimulus-to-stimulus spectral variation for frequencies up to 1 kHz was on the order comparable with the speaker-to-speaker variation, which means that these two sources of variation were not separable for the listener. For frequencies of 2 kHz and higher, the across-speaker differences were systematic and larger than the stimulus-to-stimulus variation. However, for broadband stimulus distance perception, these frequencies are weighted much less than for the low frequencies of 500 Hz and less (Kopcˇo and Shinn-Cunningham, 2011). Although the differences could have provided some cues for distance perception, the study focuses on the effects of the visual stimulus and any such effects would be minimized by considering the performance in the baseline condition. For further reference on the distance perception in the base- line condition and comparison with the literature see Appendix A. Finally, Fig. 1(F) shows the A-weighed sound pressure level of the direct and reverberant portion of the stimuli, showing that the direct sound level decreased sys- tematically with distance, while the reverberant portion was approximately constant at all distances. The experiment was conducted in a small semi- reverberant acoustically treated room (T20 ¼ [405, 444, 577, 552, 505, 411] ms in octave frequencies from 250 Hz to 8 kHz [Berzborn et al., 2017; background noise 35 dB sound pressure level (SPL)] with internal dimensions of 2.6 m 3.33 m 3.05 m (H). Participants were seated on a barber chair with adjustable height and a headrest. The chair back faced the middle of one of the shorter walls in the room. The seated participant faced an array of nine uni- formly spaced loudspeakers arranged along a straight path in front of the participant [Fig. 1(A)] at ear level. B. Setup, stimuli, and procedures An acous- tically transparent fabric covered the loudspeaker array, so the participant was unaware of the number and exact loca- tions of the loudspeakers. Sound stimuli were delivered from 8 loudspeakers (Peerless 830984 Tymphany) mounted on stands. An additional loudspeaker placed closest to the listener was used to provide acoustic shadow so that every speaker was obstructed by at least one other speaker. Visual stimuli were delivered from an array of forty- eight uniformly spaced LEDs (4.76 cm spacing) mounted on a wooden frame raised 7 cm (at the far end) to 10 cm (at the near end) above the loudspeakers. The array height slightly decreased with distance so that all LEDs were clearly visible to the participants. The same LED array was used to collect responses. The loudspeakers and the LED array were coupled with a multichannel digital signal pro- cessor controller (RX-8, Tucker Davis Technologies, Alachua, FL) and an 8-channel power ampliﬁer (CROWN, Elkhart, IN). A trackball was used to control which light was lit and to collect the listener’s responses. An experi- mental computer outside the experimental room controlled the experiment, stimulus presentation, and response collec- tion. The experimental procedure, stimulus generation and data analysis were implemented in MATLAB (Mathworks, Natick, MA). The visual stimuli consisted of a 300-ms ﬂash of light from one of the LEDs. The AV stimuli consisted of the auditory component (A-component, identical to the audi- tory stimulus) and the visual component (V-component, identical to visual stimulus) presented synchronously. The stimulus timing was controlled by a separate program run- ning directly on the digital signal processor. The V-component of the AV stimuli was either aligned with the A-component (V-aligned), placed approximately 30% closer than the auditory component (V-closer), or approxi- mately 30% farther than the auditory component (V-farther). The exact relative placement of the V-component vs A- component deviated slightly from 30% because of the discrete positions of the LEDs [Fig. 1(B)]. Also note that, while the 30% shift results in a constant shift as a function of target distance in log units when the V-closer and V-farther positions are considered separately, it corresponds to a larger absolute logarithmic value in the V-closer direction than in the V-farther direction. Speciﬁcally, 70% of target distance is –0.36 log(cm) while 130% distance is 0.26 [compare the left- hand vs right-hand axis labels in Fig. A. Participants A group of 183 young adults participated in this study. All participants had normal or corrected-to-normal vision and normal hearing (by self-report). The experimental proto- col was approved by the University of California, Riverside Human Research Review Board. All participants provided written informed consent. 3594 J. Acoust. Soc. Am. 150 (5), November 2021 B. Setup, stimuli, and procedures 1(B)], which makes it more complicated to directly compare the effects of V-closer and V-farther adaptors in logarithmic units. A correction was applied to address this disparity, as described in the results section. The auditory stimuli were 300-ms broadband noise bursts. On each trial, one noise token was randomly selected from a set of 50 pre-generated tokens, and presented from one of eight loudspeakers. The presentation sound level was held constant, which led to a natural decrease in sound level from 56 to 53 dBA with increasing target distance (mea- sured, with reverberation, by a sound level meter Extech HD600, Flir Comercial System Inc., Elkhart, IN). A cali- brated microphone positioned at the location of the listener’s head and pointing towards the loudspeakers, connected to the recording and playback system, was used to record the stimuli for acoustic analysis. Frequency characteristics of the audio system and loudspeakers were estimated from the ﬁrst 5.8 ms of impulse responses measured for each loud- speaker measured using the maximum length sequence tech- nique (Rife and Vanderkooy, 1989; Vanderkooy, 1994). Figure 1(C) shows the octave-band spectrum of the direct portion of the responses. Responses are shifted systemati- cally upwards as the distance of the speakers decreases. Colored traces in Fig. 1(E) show the same responses Hladek et al. 3595 3595 J. Acoust. Soc. Am. 150 (5), November 2021 olor online) Setup, stimuli, and groups. (A) Setup and locations of the subject, loudspeakers, and LEDs in the room. Loudspeakers were spa nd the nearest loudspeaker (50 cm) was not used. An example AV stimulus pair is also shown, with the loudspeaker at D ¼ 127 cm (shown as dspeaker) and the corresponding V-farther LED (green), V-aligned LED (red), and V-closer LED (blue). (B) Actual physical locations of the ts of AV stimuli used to approximate the 30% AV separation in the V-farther and V-closer conditions. The V-component distance is shown re nent (on a log scale) as a function of the target location. Right-hand side axis shows the ratio of distances in per cents. The x axis was log tr d this transformation is used throughout the paper. (C) Frequency response of the audio delivery system measured for each loudspeaker in nds at the location of the listener head (without reverberation). B. Setup, stimuli, and procedures (D) Schematic of the experimental session structure for different participant gro cipant was assigned into one of three groups and participated in two sessions of eleven runs. Each row represents a series of runs performed by t from a given group from a given session. Session types F, A, and C differed by the type of AV stimulus (V-farther, V-aligned, and V-clo e adaptation runs 4-8. Session ordering was counterbalanced across participants within each group. (E) Thick colored lines show, for each lo he deviation of frequency response of that loudspeaker (from panel C) with respect to the across-loudspeaker mean, corrected for the distan overall level change. Thin gray lines show frequency response of each stimulus with respect to the across-stimulus mean computed in 1-oc A-weighted sound pressure levels of the direct and reverberant portions of the experimental signals. Critical distance is 103 cm. FIG. 1. (Color online) Setup, stimuli, and groups. (A) Setup and locations of the subject, loudspeakers, and LEDs in the room. Loudspeakers were spaced linearly, and the nearest loudspeaker (50 cm) was not used. An example AV stimulus pair is also shown, with the loudspeaker at D ¼ 127 cm (shown as the active loudspeaker) and the corresponding V-farther LED (green), V-aligned LED (red), and V-closer LED (blue). (B) Actual physical locations of the V- components of AV stimuli used to approximate the 30% AV separation in the V-farther and V-closer conditions. The V-component distance is shown re. the A-component (on a log scale) as a function of the target location. Right-hand side axis shows the ratio of distances in per cents. The x axis was log trans- formed and this transformation is used throughout the paper. (C) Frequency response of the audio delivery system measured for each loudspeaker in 1- octave bands at the location of the listener head (without reverberation). (D) Schematic of the experimental session structure for different participant groups. Each participant was assigned into one of three groups and participated in two sessions of eleven runs. Each row represents a series of runs performed by the participant from a given group from a given session. Session types F, A, and C differed by the type of AV stimulus (V-farther, V-aligned, and V-closer) used in the adaptation runs 4-8. Session ordering was counterbalanced across participants within each group. https://doi.org/10.1121/10.0007066 condition data were obtained from the C sessions of the par- ticipants from Group CF and Group CA. All responses were converted to the log scale (Anderson and Zahorik, 2014; Kopcˇo et al., 2012) before any manipulations. When possi- ble, percent scale is also plotted in graphs to provide a more intuitive description of the data. However, note that percent scales cannot be used to describe the error bars as the scale is non-linear. Unless speciﬁed otherwise, all results graphs show the across-subject mean and standard errors of the mean (SEM). Analysis of variance (ANOVA) with within- subject factor of distance (1–8) and between-subject factor of session type (F, C, A) assessed statistical signiﬁcance of effects. The statistical software CLEAVE (Herron, 2005) was used for the ANOVAs and the reported p values were corrected by using the Greenhouse-Geisser epsilon. response with a button-click on the trackball (Wozny and Shams, 2011). No immediate feedback was provided, and after 500 ms inter-trial interval, the next trial was initiated. There were two types of runs. The A-only runs con- sisted of auditory-only trials. The AV runs consisted of A- only and AV trials pseudorandomly interleaved with the ratio of 3:1 (75% AV trials, 25% A trials). In each run, each of the eight target loudspeakers was randomly chosen to pre- sent stimulus on two auditory trials and six AV trials. The relative displacement of the visual component was ﬁxed within AV runs. The AV runs were of three types, V-closer, V-farther, or V-aligned. In each A-only run, each target loudspeaker was randomly chosen eight times. There were three types of sessions, F, C, and A, differing by what type of shift was being induced [V-farther, V-closer, or V-aligned, respectively; Fig. 1(D)]. Each session, indepen- dent of its type, started with a pre-adaptation baseline period consisting of one V-aligned AV run followed by two A-only runs. The AV trials in run 1 were included both to minimize possible pre-adaptation biases that participants might have in their auditory-only distance responses and to establish a pre- adaptation reference. The sessions differed only in the adapta- tion period consisting of ﬁve AV runs (runs 4–8) with the AV disparity ﬁxed within the session, which determined the type of session (C, F, or A). III. RESULTS The main goal of this study was to examine the effect of visual stimulation on auditory distance perception. To separate this effect from possible biases due to the experi- mental methods used here (like sensitivity to the exact placement of the speakers) and/or inherent biases in auditory distance perception, data from adaptation runs were ana- lyzed relative to the pre-adaptation baseline runs 1–3. This section ﬁrst presents the baseline data and a detailed com- parison with other auditory distance studies is provided in Appendix A. Then, effects of congruent and incongruent visual stimuli on VE and VAE are presented as a function of target distance for the adaptation runs 4–8 and the post- adaptation runs 9–10. Finally, dynamics of the VE and VAE build-up and breakdown are analyzed as a function individ- ual runs within a session for data collapsed across distance. Participants performed two sessions that were conducted on separate days. Each participant was randomly assigned to one of three experimental groups [Fig. 1(D)] differing by the conditions they performed in the sessions. Group CF (34 par- ticipants) performed the F and C sessions, Group FA (40 par- ticipants) the F and A sessions, and Group CA (40 participants) the C and A sessions. The order of sessions was counterbalanced across participants within a group. A. Baseline Figure 2 shows baseline reported distance (upper row) and localization bias (lower row) for the Visual-only stimuli [Figs. 2(A) and 2(D)], V-aligned AV stimuli [Figs. 2(B) and 2(E)], and Auditory-only stimuli [Figs. 2(C) and 2(F)]. The visual baseline [Figs. 2(A) and 2(D)] was measured to vali- date the response method used here. The Visual-only responses were very accurate in the range in which the audi- tory targets were presented (70–203 cm), with only slight undershooting (by less than 5%) at the largest distances. However, this undershooting became more pronounced at larger distances, and reached 10% at the distance of 250 cm. It is not clear whether this bias is caused by actual biases of the visual distance perception or by some artifact of the response method. However, it is likely that this bias only inﬂuenced measurements for the most distant targets in the V-farther condition, for which the visual components were presented at distances larger than 2 m. To minimize the pos- sible effect of this response method bias on the VA and VAE data, the data were corrected by adding the inverse of the response method bias to the baseline-corrected perceived location of auditory and AV targets (see below). Since this correction only affected responses larger than 2 m, A separate preliminary calibration measurement was per- formed to establish if any biases in responses might be due to the experimental setup and, in particular, the response method based on a manually controlled visual pointer and/or due to biases in visual distance perception. In the calibration measure- ment, a group of new 69 participants judged the egocentric dis- tance of visual stimuli, using procedures identical to those described above, except for the following two differences. First, the calibration session had only one run of 80 trials. Second, on each trial, a visual stimulus was presented from one randomly chosen LED in the array of 48 LEDs such that each LED was chosen at least once and not more than twice. https://doi.org/10.1121/10.0007066 The post-adaptation period consisted of two A-only runs to assess the persistence of the induced adaptation and one V-aligned AV run to minimize any carry- over effects of adaptation on participant. A whole session took approximately 1 h to complete. There were 30-s breaks between the runs within a session. The participant received feedback about their progress during these breaks via voice commands played by one of the loudspeakers in the array. B. Setup, stimuli, and procedures (E) Thick colored lines show, for each loud- speaker, the deviation of frequency response of that loudspeaker (from panel C) with respect to the across-loudspeaker mean, corrected for the distance- dependent overall level change. Thin gray lines show frequency response of each stimulus with respect to the across-stimulus mean computed in 1-octave bands. (F) A-weighted sound pressure levels of the direct and reverberant portions of the experimental signals. Critical distance is 103 cm. Each participant participated in two experimental ses- sions, each consisting of 11 runs of 64 trials. Each trial started with the presentation of an A-only or AV-stimulus, followed by the participant’s response. The participant’s task was to indicate the perceived egocentric distance of the stimulus. When the AV stimulus was presented, participants were instructed to respond to the A component and ignore the V component. To collect participant responses, a random LED was activated after the presentation of the stimulus, and the participant then moved the active LED towards a perceived position using the trackball. Only one LED was active at any given time. The participant submitted their 3596 J. Acoust. Soc. Am. 150 (5), November 2021 https://doi.org/10.1121/10.0007066 C. Data analysis The data from the sessions of the same type were pooled across the subject groups; e.g., the V-closer Hladek et al. 3597 Hladek et al. 3597 Hladek et al. J. Acoust. Soc. Am. 150 (5), November 2021 2. (Color online) Baseline performance as a function of target distance. Upper panels: Across-subject geometric mean of the response distance as a tion of actual target distance. Lower panels: across-subject bias in log of response re. log of actual target distance. (A) and (D), Visual-only stimulus line measured in a calibration experiment. (B) and (E), Auditory-visual baseline measured in run 1 of each session. (C) and (F), Auditory baseline from 1–3 in each session. Different symbols in (B), (C), (E), and (F) represent different types of sessions. The right-hand y axis of panels (D)–(F) show the of the response and target distance in percent. https://doi.org/10.1121/10.0007066 https://doi.org/10.1121/10.0007066 https://doi.org/10.1121/10.0007066 https://doi.org/10.1121/10.0007066 FIG. 2. (Color online) Baseline performance as a function of target distance. Upper panels: Across-subject geometric mean of the response distance a function of actual target distance. Lower panels: across-subject bias in log of response re. log of actual target distance. (A) and (D), Visual-only stimu baseline measured in a calibration experiment. (B) and (E), Auditory-visual baseline measured in run 1 of each session. (C) and (F), Auditory baseline fro runs 1–3 in each session. Different symbols in (B), (C), (E), and (F) represent different types of sessions. The right-hand y axis of panels (D)–(F) show t ratio of the response and target distance in percent. FIG. 2. (Color online) Baseline performance as a function of target distance. Upper panels: Across-subject geometric mean of the response distance as a function of actual target distance. Lower panels: across-subject bias in log of response re. log of actual target distance. (A) and (D), Visual-only stimulus baseline measured in a calibration experiment. (B) and (E), Auditory-visual baseline measured in run 1 of each session. (C) and (F), Auditory baseline from runs 1–3 in each session. Different symbols in (B), (C), (E), and (F) represent different types of sessions. The right-hand y axis of panels (D)–(F) show the ratio of the response and target distance in percent. by the bias observed in Fig. 2(A)]. This result is consistent with the auditory horizon observed in previous studies reporting that distance estimates are overestimated for dis- tances less than approximately 1–3 m and underestimated for larger distances (Zahorik, 2001). B. Ventriloquism effect and aftereffect The upper panels of Fig. 3 show the raw reported dis- tances as a function of target distance, separately for the FIG. 3. (Color online) Performance in adaptation and post-adaptation runs. (A)–(C), Across-subject geometric mean of response distance as a function o target distance. (D)–(F), Across-subject mean of the response distance (from respective A–C) re. Baseline-run response distance (from Fig. 2) plotted as function of target distance. (G)–(I), Across-subject mean of the V-closer (V-farther) data from the respective middle-row panel (D)–(F), plotted relative to h di V li d d (f h iddl l) d li d b h h i l di i b h i l d h G. 3. (Color online) Performance in adaptation and post-adaptation runs. (A)–(C), Across-subject geometric mean of response distance as a function of get distance. (D)–(F), Across-subject mean of the response distance (from respective A–C) re. Baseline-run response distance (from Fig. 2) plotted as a ction of target distance. (G)–(I), Across-subject mean of the V-closer (V-farther) data from the respective middle-row panel (D)–(F), plotted relative to corresponding V-aligned data (from the same middle-row panel) and normalized by the physical disparity between the visual component and the target om Fig. 1(D)]. The three types of sessions are shown by different symbols. The left-hand and middle columns show data averaged across adaptation runs 8 for AV targets (left-hand column) and A-only targets (middle column). The right-hand column (C, F, and I) show the A-only data averaged across post- ptation runs 9 and 10. The right-hand y axis of (D)–(F) show the ratio of the response distance and the baseline-run response distance (in per cent). Data FIG. 3. (Color online) Performance in adaptation and post-adaptation runs. (A)–(C), Across-subject geometric mean of response distance as a function of target distance. (D)–(F), Across-subject mean of the response distance (from respective A–C) re. Baseline-run response distance (from Fig. 2) plotted as a function of target distance. (G)–(I), Across-subject mean of the V-closer (V-farther) data from the respective middle-row panel (D)–(F), plotted relative to the corresponding V-aligned data (from the same middle-row panel) and normalized by the physical disparity between the visual component and the target [from Fig. 1(D)]. The three types of sessions are shown by different symbols. The left-hand and middle columns show data averaged across adaptation runs 4–8 for AV targets (left-hand column) and A-only targets (middle column). https://doi.org/10.1121/10.0007066 three types of sessions (represented by different symbols and colors). Figure 3(A) shows responses to AV stimuli dur- ing the adaptation runs 4–8, Fig. 3(B) shows responses to the A-only stimuli during the adaptation runs 4–8, and Fig. 3(C) shows responses to the A-only stimuli in the A- only post-adaptation runs 9–10. The main trend shown in these panels is that estimates grow with the target distance. To focus on the effect of the visual stimuli instead of the overall effect of distance, each panel in the middle row [Figs. 3(D)–3(F)] shows data from the respective upper panel, plotted relative to the response distance baselines [from Fig. 2(B) and 2(C)], and corrected for the response method biases by adding the inverse of the response method bias [computed from data in Fig. 2(A)]. (up to 20%) biases in the A-only responses are observed, even though in run 1 the A-only stimuli were interleaved with V- aligned AV stimuli for which no bias was observed (panels B and E). To account for this, the analysis of the VE and the VAE in the following sections (IIIB–IIIE) plots data relative to this A-only pre-adaptation baseline. And, since this study focuses on relative effects of the V-farther and V-closer stimuli referenced to the V-aligned condition, it is assumed that the baseline biases are not differentially affecting these relative comparisons. C. Data analysis In the current study, the location of the horizon appears to be at around 200 cm. Inconsistent with previous studies is the fact that the bias was reduced at the shortest distances. Even though a slight similar trend is observed in (Kopcˇo and Shinn- Cunningham, 2011), most previous studies performed in virtual environments show that overestimation grows as the sources approach the listener (see Appendix A, which compares the current baseline to an additional Auditory- only baseline measurement and to Anderson and Zahorik, 2014; Kopcˇo and Shinn-Cunningham, 2011; Rebillat et al., 2012; Zahorik et al., 2005; Zahorik and Wightman, 2001). Overall, this reduced bias suggests that, in the real environment and with the AV-aligned stimuli interleaved in the initial run, as used here, participants are more accu- rate at judging the distance of nearby sources than in vir- tual environments, even if the loudspeakers shadow each other. additional statistical analysis is also provided, where rele- vant, with the three most distant speakers excluded (consid- ering speakers below 1.5 m), to assure that the reported results are not affected by the correction (and Appendix B describes the results of this analysis without applying the V- response correction). The AV baseline [Figs. 2(B) and 2(E)] was measured in run 1 which used the V-aligned AV stimuli. Very little bias was observed (less than þ/–5%), with an undershooting trend at the largest distances, consistent with the bias observed in the Visual-only calibration measurement [Fig. 2(A)]. Importantly, there were no large differences between the three conditions (shown by different symbols), indicat- ing that the groups were similar in their AV baseline performance. Figures 2(C) and 2(F) show the auditory baseline obtained from auditory responses in runs 1–3. Again, there were no systematic differences between subject groups (shown by different symbols). However, participants tended to overestimate the auditory targets at most distan- ces. The overestimation was largest (10%–20%) at distan- ces around 100 cm, and gradually decreased at larger distances, ﬂipping to underestimation by approximately 5% at 200 cm [this underestimation, again, is likely caused Given the large overestimation observed in the previous auditory distance studies, it is not surprising that these small 3598 J. Acoust. Soc. Am. 150 (5), November 2021 C. Ventriloquism effect Two trends in Fig. 3(D) suggest that additional correc- tions need to be applied before the effects of interest can be properly evaluated. First, the AV response biases in Fig. 3(D) are overall larger for the V-closer stimuli than the V-farther stimuli (relative to the V-aligned stimuli; compare the blue triangles and green diamonds to the red squares). However, this asymmetry is likely caused by the fact that a 30% shift of the visual component corresponds to a larger disparity in logarithmic units for the V-closer than V-farther condition. Second, while the V-farther data (green dia- monds) are relatively smooth, the V-closer graph (blue triangles) has a peak at the 89-cm target and a trough at the 165-cm target. This non-uniformity might be due to the slight variability in the size of the physical disparity between the V and A components, which was not always exactly 30% [Fig. 1(B)]. To account for these artifacts, as well as for the compressive bias observed even in the V-aligned condition, the V-closer and V-farther data from Fig. 3(A) were re-plotted in Fig. 3(G) relative to the across-subject average of the V-aligned data and scaled by the size of the physical AV dis- parity. Thus, to show 7, 1022 strength of the ventriloquism effect, Fig. 3(G) plots the size of the shift in the perceived loca- tion as a proportion of the shift in the physical displacement of the V-component which induced it. AV responses in the V-aligned condition [red squares in Fig. 3(D)] are approximately at 0 log(cm), slightly over- shooting near targets and undershooting distant targets. The V-farther (green diamonds) and V-closer (blue triangles) data are both shifted in the direction of the visual compo- nent, conﬁrming the existence of the VE in the distance dimension. Also, their dependence on target distance is sim- ilar to that for the V-aligned data (slight downward slope). On average V-farther responses are shifted up by 0.2 log(cm) and V-closer responses are shifted down by 0.25 log(cm). g Similar to the AV-responses in panel D, the auditory- only responses during adaptation runs [Fig. 3(E)] show a slight compression (downward slope) in all conditions. There is also a clear effect of the interleaved AV stimuli, such that both V-closer (blue triangles) and V-farther (green diamonds) stimuli shift the auditory-only responses in the direction of the visual component. C. Ventriloquism effect The effect is approxi- mately linear on the logarithmic scale (the blue and green lines are parallel with the red line); however, it is weaker than in the AV responses [Fig. 3(D)], in particular for the V- closer stimuli (compare the ﬁlled blue triangles of Fig. 3(E) to the open blue triangles of Fig. 3(E)]. VE data from Fig. 3(G) were subjected to a two-way mixed ANOVA with factors of target location and condi- tion, which showed that the VE is approximately equally strong in the V-closer and V-farther conditions [main effect of condition: F(1,146) ¼ 0.29, n.s.; for targets<1.5 m, i.e., for targets unaffected by the V-response correction: F(1,146) ¼ 0.63, n.s.]. While there was considerable varia- tion in the size of the effect as a function of target distance in both the V-closer and V-farther graphs [interaction of condition and target distance: F(7, 1022) ¼ 5.97; p < 0.0001; for targets < 1.5 m: F(4,584) ¼ 4.72, p ¼ 0.0016], there was no clear systematic trend of increasing or decreasing effect size with target distance. Moreover, this variation is rela- tively small compared to the overall VE and it is likely driven by noise, e.g., due to factors like the limited accuracy in the estimation of the V-aligned responses. Speciﬁcally, the observed pattern of maxima and minima in the V-closer data is exactly mirroring that in the V-farther data (i.e., the maxima in the green line are aligned with the minima in the blue line, and vice versa), which would be expected if the effect is mostly driven by noise in the V-aligned data estimation, as that estimate gets subtracted from the V-farther data and added to the V-closer data. Therefore, the dependence of results on target distance, while signiﬁcant, was not further examined here. In post-adaptation runs [Fig. 3(F)], the observed trends in the A responses are similar to those during adaptation [Fig. 3(E)], but the magnitude of the effect of V-closer and V-farther adaptation is smaller than that in Fig. 3(E), and approximately equal for all targets independent of their loca- tion or the direction of the visual component shift. The downward sloping red lines (as well as the green and blue lines) in Figs. 3(D)–3(F) indicate that responses tended to be more compressed during the adaptation and post-adaptation relative to the baseline. https://doi.org/10.1121/10.0007066 perception. The effect appears approximately constant as a function of target distance and depends in strength on the direction of the induced shift (closer vs farther). Sections III B through III D provide a detailed analysis of these results. Finally, the lower panels of Fig. 3 show the magnitudes of the ventriloquism effect [VE; Fig. 3(G)], immediate after- effect [VAE; Fig. 3(H)], and persistent aftereffect [persistent VAE; Fig. 3(I)] derived from the data in the corresponding middle panels of the ﬁgure. Speciﬁcally, to compute the effect of a visual component placed closer (farther) than the auditory component in the lower panels, the differences between the corresponding V-closer (V-farther) and V- aligned data are taken from the middle panel and scaled by the physical disparities between the visual and auditory components [from Fig. 1(B)]. 3600 J. Acoust. Soc. Am. 150 (5), November 2021 B. Ventriloquism effect and aftereffect The right-hand column (C, F, and I) show the A-only data averaged across post- adaptation runs 9 and 10. The right-hand y axis of (D)–(F) show the ratio of the response distance and the baseline-run response distance (in per cent). Data in (D)–(I) were corrected for the response method bias shown in Fig. 2(D). Hladek et al. 3599 Hladek et al. 3599 Hladek et al. 3599 3599 J. Acoust. Soc. Am. 150 (5), November 2021 https://doi.org/10.1121/10.0007066 In summary, the effect of spatially displaced visual stimulus on the perceived distance of a simultaneously pre- sented auditory stimulus is approximately constant on a log- arithmic scale at 72% of the visual stimulus displacement when the visual component is shifted by a constant 30% re. auditory component over a range of distances from 0.7 to 2 m. This effect is independent of whether the visual compo- nent is located closer or farther than the auditory component (V-closer vs V-farther) and it does not show any clear trend of systematic increase or decrease with target distance (val- ues ranging from 60% to 80%). Thus, there is no evidence that the ventriloquism effect measured in a reverberant room is stronger for the V-closer direction, as suggested for anechoic space by Gardner (1968). And, the data are consis- tent with the suggestion that auditory distance is represented in logarithmic units, as the observed VE was approximately constant at 72% of the imposed 30% AV disparity in all the VE measurements. immediate VAE is expressed as a proportion of VE, the average across-distances is 63% in the V-farther direction and only 43% in the V-closer direction. Similarly, when the persistent VAE is expressed as a portion of VE, it is on aver- age 29% and 22% in the V-farther and V-closer conditions, respectively (see Fig. 6 in Appendix B for the data without the correction for the visual baseline). There are at least two possible explanations of this farther-closer asymmetry. Either the neural distance representation adapted in the ven- triloquism aftereffect is more ﬂexible in one direction than the other, or the visual signals that cause the adaptation are stronger or more salient in the V-farther direction. Importantly, the fact that VAE observed here is constant as a function of target distance for both directions is consistent with the assumption that the neural representation of audi- tory distance is adaptable in logarithmic units, at least over the range of distances and for the AV disparity examined in the current study. Finally, no adaptation was observed in the V-aligned data, even though the V-aligned A-only baseline was shifted by up to 10%–20% re. the AV baseline in Figs. 2(F) vs 2(E). It would be expected that, if the baseline misalignment was perceptual, the shift would be corrected by the V-aligned signals during the VAE adaptation due to multisensory enhancement (Bruns et al., 2020). D. Ventriloquism aftereffect The ventriloquism aftereffect (VAE) was examined on two timescales. First, the immediate VAE was assessed for the A-only trials randomly interleaved with the AV trials during the adaptation runs [Fig. 3(H)]. This effect reﬂects adaptation on the timescale of seconds and tens of seconds. Second, persistent VAE was assessed by two post- adaptation A-only trial runs, reﬂecting adaptation on the timescale of minutes [Fig. 3(I)]. To evaluate the immediate VAE, data from Fig. 3(E) were replotted in panel H relative to the V-aligned data and scaled by the size of the physical AV disparity, using the same procedure and rationale as discussed above for the VE. These data were subjected to a mixed two-way ANOVA with the factors of target location and condition which showed that VAE was independent of the target location [F(7, 1022) ¼ 1.81; p ¼ 0.08; for targets < 1.5 m: F(4,584) ¼ 2.14, n.s.], but that it was clearly stronger for the V-farther condition (approximately 44% of the AV offset) than for the V-closer condition [approximately 31% of the AV offset; F(1,146) ¼ 5.48; p ¼ 0.02; for targets < 1.5 m: F(1,146) ¼ 4.40, p ¼ 0.037]. https://doi.org/10.1121/10.0007066 Since no correction occurred, the disparity in baselines is more likely caused by differences in response strategies than in perception, e.g., since the same LED array was used to present the V-stimuli and collect the responses, the responses might be more accu- rate for stimuli with V-components than for auditory-only stimuli, as the subjects might be able to directly compare the perceived location of the visual-stimulus and the response LED. C. Ventriloquism effect While it is not clear what drove this trend, it might be due to within-run accom- modation of subjects to the experimental procedure, or due to decreased attentiveness or increased tiredness during the experimental session, which, on average, is expected to result in more bias towards the middle of the response range. Also, note that the bias is slightly larger in the post- adaptation runs 9–10 [Fig. 3(F)] than in the adaptation runs 4–8 [Fig. 3(E)]. Importantly, this bias can be approximately eliminated when comparing the effect of displaced V- stimuli (V-closer or V-farther) to the V-aligned reference, as it tends to affect them equally. In summary, all of the results shown in Figs. 3(D)–3(F) show a clear ventriloquism effect and aftereffect in distance 3600 J. Acoust. Soc. Am. 150 (5), November 2021 Hladek et al. Hladek et al. https://doi.org/10.1121/10.0007066 FIG. 4. (Color online) Temporal proﬁle of ventriloquism adaptation. (A) Response biases as a function of run number within a session for the three different conditions, averaged across target distance and referenced to the pre-adaptation baselines (runs 1–3). (B) VE (open symbols) and VAE (ﬁlled symbols) as a function run number, computed from data in (A) by referencing the V-closer and V-farther data to the V-aligned data and scaling them by the physical dis- parity between the stimuli. (C) Dynamics of post-onset/offset VAE adaptation shown as an average of the build-up (runs 4 and 5) and inverse of break-down (runs 9 and 10) of VAE from (B), referenced to the ﬁnal runs prior to the onset (runs 1–3) and offset (run 8) of the AV stimuli. FIG. 4. (Color online) Temporal proﬁle of ventriloquism adaptation. (A) Response biases as a function of run number within a session for the three different conditions, averaged across target distance and referenced to the pre-adaptation baselines (runs 1–3). (B) VE (open symbols) and VAE (ﬁlled symbols) as a function run number, computed from data in (A) by referencing the V-closer and V-farther data to the V-aligned data and scaling them by the physical dis- parity between the stimuli. (C) Dynamics of post-onset/offset VAE adaptation shown as an average of the build-up (runs 4 and 5) and inverse of break-down (runs 9 and 10) of VAE from (B), referenced to the ﬁnal runs prior to the onset (runs 1–3) and offset (run 8) of the AV stimuli. by the physical disparity of the visual and auditory compo- nents (Secs. III C and III D). Figure 4(C) aims at estimating the average dynamics of the VAE in the V-closer vs V- farther condition by showing the average of the VAE build- up [runs 4–5 referenced to the average of preadaptation runs 1–3 from Fig. 4(B)] and the inverse of the VAE break-down [runs 9–10 referenced to run 8 from Fig. 4(B)]. approximately constant at around 35% in runs 5–10. A similar pattern was observed after the offset of the adaptation. V-farther data dropped much faster than the V-closer data (compare runs 9 vs 8 for the ﬁlled green dia- monds and blue triangles). To better visualize this differ- ence in adaptation rate, Fig. https://doi.org/10.1121/10.0007066 4(C) plots the average of the build-up and the inverse of the break-down data from the two runs post-onset and post-offset of adaptation (relative to the preceding runs). The panel clearly shows much faster growth in the V-farther adaptation in the ﬁrst run after its on/offset, while the V-closer adaptation grew more gradually. Figure 4(A) shows that V-aligned data, averaged across target locations, were stable near 0 log(cm) throughout the sessions (red open and ﬁlled diamonds are always near 0). The VE was fast and stable [open green diamonds and blue triangles show constant values throughout the adaptation runs 4–8 in Fig. 4(A)], while the VAE had a clear build-up and decay after the onset and offset of adaptation (full green diamonds and blue triangles grow gradually in runs 4–8 and decay gradually in runs 9–10). When expressed as a propor- tion of physical disparity [Fig. 4(B)], the VE magnitudes were approximately equal for the V-closer and V-farther conditions, at 70%–80% of the V-component offset (open symbols). The VAE expressed as a proportion of physical disparity was slower than the VE and had more complex dynamics. Conﬁrming the results described in Sec. III C, VAE was stronger for the V-farther than V-closer conditions during both the adaptation and the post-adaptation runs [green ﬁlled diamonds are mostly above blue triangles for runs 4–10 in Fig. 4(B)]. Importantly, results also showed a difference in the rate of adaptation. The V-farther data [ﬁlled green diamonds in Fig. 4(B)] were approximately 40% of AV disparity by the ﬁrst adaptation run (run 4), while the V-closer data [ﬁlled blue triangles in Fig. 4(B)] were only 22% of the AV disparity within the ﬁrst adapta- tion run. Also, the V-farther data continued to grow, reach- ing more than 50% by run 8, while the V-closer data were Statistical analysis conﬁrmed these results. First, an ANOVA performed on the VE data from Fig. 4(B) with the factors of run (4–8) and condition (V-closer vs V-farther) found no main effect or interaction of the condition and run factors. A similar ANOVA performed on the VAE data from Fig. 4(B) only found a signiﬁcant main effect of condition [F(4,584)¼ 4; p < 0.01]. Finally, an ANOVA performed on data from Fig. E. Build-up and break-down of the ventriloquism effect and aftereffect Although the observed effects of target distance showed signiﬁcance in the analysis presented in the preceding sec- tion, the magnitude of the variation between individual tar- get locations was relatively small compared to the magnitude of the overall effect. Therefore, the dynamics of the build-up and break-down of the visually induced adapta- tion were analyzed after collapsing the data across target location (while keeping the data from each experimental run separate). It was expected that the VE build-up and break- down would be immediate while the VAE build-up and break-down would have slower dynamics. Also, it was expected that, given the difference in the strength of the VAE for the V-farther vs the V-closer condition, there might be a difference in the rate at which the VAE builds up for the two directions. To evaluate the persistent VAE, the data from panel F were again rescaled and replotted in Fig. 3(I). The observed patterns are also similar to the immediate VAE, except that the effect is smaller (on average, 19% of the physical AV displacement). ANOVA performed on these data did not show any signiﬁcant effect or interaction (p > 0.1; same for targets < 1.5 m). However, the green diamond data tend to fall above the blue triangle data in Fig. 3(I), showing that there still is a trend for the aftereffect to be stronger in the V-farther condition. Figure 4(A) shows the localization biases in the AV (open symbols) and A-only (ﬁlled symbols) trials separately for each run as a function of the run number, referenced to the pre-adaptation runs 1–3. Figure 4(B) shows the magni- tudes of the VE and VAE computed by referencing the data from panel A to the V-aligned condition and scaling them In summary, the ventriloquism aftereffect induced by the AV disparity is similar to the ventriloquism effect in that it is constant as a function of distance. However, contrary to the VE, the VAE is asymmetrical, being considerably stron- ger in the V-farther direction. Speciﬁcally, when the 3601 Hladek et al. 3601 J. Acoust. Soc. Am. 150 (5), November 2021 IV. DISCUSSION the V-farther condition than in the V-closer condition. This asymmetry in the effect was not expected and it is not immediately clear why it occurred. It might be related to the “adjacency effect” reported in auditory distance perception by Min and Mershon (2005), which observed that manipu- lating the perceived distance of a nearby sound by a simulta- neously presented distal light (i.e., ventriloquism effect) caused an adjacent reference sound to be perceived as far- ther away (i.e., ventriloquism aftereffect), whereas the reverse setup did not cause an adjacent reference sound to be perceived as closer. The current results suggest that this asymmetry may be a general observation in the adaptation of auditory distance percepts by vision and that it also applies to the speed with which the aftereffect builds up. Alternatively, this effect might relate to the fact that the A baseline responses were overestimated even in the pre- adaptation in the current study, which means that the per- ceived A-only locations were much closer to the V-farther visual adaptors than V-closer visual adaptors, even when they were interleaved with V-aligned AV stimuli. Finally, the constant 30% disparity used in this study corresponds to a larger disparity in logarithmic units for the V-closer condition (0.36) than for the V-farther condition (0.26) [Fig. 1(B)]. In the analysis performed here, the scaling by the actual applied separation corrected for this difference. However, it is possible that the strength of AV binding was stronger in the V-farther condition, as the separation in log units was smaller, which might have resulted in the observed stronger adaptation. Future studies will need to independently control the physical and perceptual disparities in the auditory and visual components of the auditory-visual and auditory stimuli in order to disentangle these alternatives. The current study examined ventriloquism effect (VA) and aftereffect (VAE) in the distance dimension for auditory stimuli presented over a range of distances from 0.7 to 2.03 m in real reverberant environment in front of the lis- tener. It found, for a ﬁxed 30% relative shift of the visual component of the AV stimuli, that the induced ventrilo- quism effect is constant at approximately 72% of the V- displacement, with no systematic variation as a function of stimulus distance or the direction of the induced shift. Also, this VE strength was constant over time. IV. DISCUSSION On the other hand, the ventriloquism aftereffect induced by these stimuli, while still independent of target distance, was stimulus-direction dependent. The VAE was stronger when the V-component of AV stimuli was placed farther than the auditory compo- nent (reaching 44% of the V-displacement) than when it was placed closer (31%). Also, the VAE in the V-farther condition was faster than in the V-closer condition. The observed differences between VE and VAE con- ﬁrm that these effects are caused by two different processes, comparable with the VE and VAE in the horizontal dimen- sion (Bosen et al., 2017; Bruns et al., 2011; Kopcˇo et al., 2009). Speciﬁcally, VE is likely a consequence of an imme- diate cross-modal integration, while VAE is a result of adap- tation of some auditory spatial representation by the visual signals. For VE, the observed independence of the effect of the direction of shift is not consistent with the “proximity image effect” (Gardner, 1968) which showed that, in anechoic room, the ventriloquism effect is stronger in the V- closer direction. A possible explanation of this difference is that the current experiment was performed in a real rever- berant room and the subjects were provided with a level- independent auditory distance cue, the direct-to-reverberant energy ratio, whereas in the Gardner study no such cue was available, and participants likely relied on guessing. The VAE data of the current study show, in both V- closer and V-farther conditions, that participants adapt to a constant-ratio shift in visual-component distance over a range of distances by a shift in response that is, again, approximately constant over the examined distance range. Previous studies in the horizontal plane suggested that adap- tation in response to a non-linear transformation of space often leads to a linear response pattern that approximates the non-linear transformation (Shinn-Cunningham et al., 1998b). Similarly, the current results are consistent with the interpretation that the neural structure undergoing adapta- tion in distance ventriloquism also adapts linearly in the log- arithmic units in which distance is represented, a result that is also consistent with the variance in responses to auditory stimuli which is approximately constant across distances (Anderson and Zahorik, 2014; Kopcˇo et al., 2012). However, this interpretation does not explain why there is the observed closer-vs-farther asymmetry in the strength of the aftereffect. Also, the ﬁxed ratio scale was the only scale examined here. https://doi.org/10.1121/10.0007066 4(C) found a signiﬁcant main effect of run [F(1,73)¼ 16.94; p < 0.01], a signiﬁcant main effect of condi- tion [F(1,73)¼ 11.51; p < 0.01], and a signiﬁcant interaction of the two factors [F(1,73)¼ 10.22; p < 0.01]. Overall, the current VE data show that the weight used for each modality in auditory-visual distance integration is approximately constant even across multiple experimental runs. On the other hand, the VAE is slow because it entails neural adaptation of the perceptual map of auditory space guided by the visual signals. Moreover, the current results show that the neural adaptation is faster when the visual sig- nals guide perception of auditory targets as farther away compared to when perception is guided to shift perception of auditory targets to be nearer. 3602 J. Acoust. Soc. Am. 150 (5), November 2021 https://doi.org/10.1121/10.0007066 logarithmic (e.g., linear), or that it is ﬂexible enough to be capable of adapting to several various transformations evoked by visual stimuli, as observed in the horizontal domain, where ventriloquism adaptation can be obtained both by shifting the V-component by a constant amount or by varying the gain of the V-component displacement; see Zwiers et al. (2003). Finally, it is even possible that the adaptation is robust to vary- ing size of the AV displacement from trial to trial, as recently shown for horizontal ventriloquism (Bruns et al., 2020). logarithmic (e.g., linear), or that it is ﬂexible enough to be capable of adapting to several various transformations evoked by visual stimuli, as observed in the horizontal domain, where ventriloquism adaptation can be obtained both by shifting the V-component by a constant amount or by varying the gain of the V-component displacement; see Zwiers et al. (2003). Finally, it is even possible that the adaptation is robust to vary- ing size of the AV displacement from trial to trial, as recently shown for horizontal ventriloquism (Bruns et al., 2020). Finally, an unexpected result of the current study is that A-only responses were slightly overestimated in the pread- aptation baseline measurements and in the V-aligned condi- tion, even though multisensory enhancement would be expected to occur, reducing the biases in the A-only stimuli (Anderson and Zahorik, 2014; Bruns et al., 2020). It is difﬁ- cult to identify the cause of this disparity, as no A-only ini- tial runs were performed to serve as a baseline. Thus, it is for example possible that the enhancement has actually occurred and that the biases would have been much larger in the initial run if it was performed without any interleaved V- aligned AV stimuli, as supported by the auditory-only fol- low-up measurement described in Appendix A. However, even if that was the case, it still is not clear why the bias would not continue to reduce even during the ﬁve adaptation runs in the V-aligned condition. Future studies are needed to explore this effect. In terms of the temporal proﬁle, the large immediate VAE and smaller persistent VAE observed here are gener- ally consistent with the observations of multiple distinct cross-modal adaptation mechanisms reported in the horizon- tal VAE studies (Bosen et al., 2018; Watson et al., 2019). https://doi.org/10.1121/10.0007066 However, a single adaptation mechanism cannot be ruled out for the current results given that the current study was not speciﬁcally designed to examine this question. On the other hand, the closer-vs-farther asymmetry in the VAE observed here differs from horizontal ventriloquism in which no equivalent left-right asymmetries are observed. Future studies will need to examine whether the observed asymmetry in the adaptation rates is directly related to the observed difference in the strength of the induced VAE, or whether these two phenomena are separable. ACKNOWLEDGMENTS This work was supported by VEGA 1/0355/20 and UPJS VVGS-2020-1514 to N.K., International Visegrad Fund 51300869 to L.H., and by BCS-1057625 to A.S. This work was supported by VEGA 1/0355/20 and UPJS VVGS-2020-1514 to N.K., International Visegrad Fund 51300869 to L.H., and by BCS-1057625 to A.S. V. CONCLUSION The current results show that VE and VAE in the dis- tance dimension can be robustly induced in real reverberant environments and that they are approximately independent of target distance on a logarithmic scale. They also show that, while VE is immediate and independent of the direc- tion of induced shift, VAE is more complex, showing stron- ger and faster effects in the V-farther direction. Our experimental setup consisted of real auditory and visual sources which assured that the stimuli were well externalized and corresponded to true physical distances. On the other hand, it also poses some limitations. The main ones are that (1) the range of A and V stimulus locations was limited by the room size, (2) the sound received from individual loudspeakers varied slightly as loudspeakers cast an acoustic shadow affecting the more distant speakers when placed behind each other especially in the high fre- quency region which might have caused coloration of the sound [see Fig. 1(E)]. Although coloration might have shifted the response biases in the A baseline, such bias would be accounted for by the relative comparisons and would be minimized in the effects of vision on auditory dis- tance, (3) the visual stimuli were linearly spaced by 4.76 cm and offset by about 5 cm from the acoustic stimuli, (4) the responses were collected through the same LED array as the visual stimuli. However, (1) the room was of a common size and shape, in which listeners are expected to naturally judge distances in everyday situations, (2) the partial shadowing of sources is a common occurrence in normal listening situa- tions and it is mainly at high frequencies while low- frequencies dominate for distance localization of broadband stimuli (Kopcˇo and Shinn-Cunningham, 2011), (3) auditory- visual synchrony was expected to provide a strong binding cue, and (4) using visually guided response is common in ventriloquism literature (Razavi et al., 2007). Some of these limitations could be mitigated by conducting future experi- ments in virtual reality (VR) (Hendrikse et al., 2018; Postma and Katz, 2017; Seeber et al., 2010). On the other hand, our study could also have implications to auditory- visual VR experience since the perceptual effects are likely to generalize to these environments as well. IV. DISCUSSION I.e., we did not investigate whether the adap- tation was equally good for a linearly constant shift in the V-component and nor did we examine how accurately a lin- ear model would ﬁt the current data. Thus, it is also possible that the representation/adaptation has other forms than The observed constant 72% weight given to the V- component in the current study is much smaller than the weight typically observed in horizontal studies, in which this weight is commonly more than 90% (Bruns et al., 2011; Frissen et al., 2012; Kopcˇo et al., 2009; Kopcˇo et al., 2019; Lewald, 2002; Recanzone, 1998, 2009; Stekelenburg et al., 2004; Vroomen et al., 2001). Assuming that auditory and visual components were combined optimally (Alais and Burr, 2004) in the current study, this result suggests that auditory distance acuity is much more comparable to visual distance acuity than found in the horizontal dimension. However, more studies are needed to determine whether the observed 72% V-component weight generalizes to other AV disparities, other reference distances, and other types of stimuli (e.g., familiar three-dimensional objects might pro- vide more accurate visual distance information than the sin- gle LEDs used here, while familiar sounds like speech stimuli might provide more auditory distance information). Even though ventriloquism effect observed in this study was equally strong in the V-farther and V-closer conditions, the ventriloquism aftereffect evoked by it was stronger in Hladek et al. 3603 3603 J. Acoust. Soc. Am. 150 (5), November 2021 APPENDIX A The current study did not aim to address auditory dis- tance perception per se, i.e., without considering the effect of visual stimuli. However, it is important to determine the extent to which the current experimental methods are equiv- alent to those used in the available auditory distance litera- ture. To this end, this Appendix ﬁrst reports the results of an auditory-only follow-up experiment performed using the same setup as in the main study. Then, it compares the cur- rent baseline results to those reported in several previous studies. The main experiment always included auditory-visual stimuli, starting and ending with the V-aligned condition in which A-only and AV stimuli were interleaved. This included a run prior to A-only baseline measurement per- formed in runs 2 and 3, to improve the accuracy of partici- pants responses, as well as to avoid any auditory-visual carry-over effects. As a follow-up experiment was per- formed with the same methodology as used in the main experiment, with the exception that all 11 runs of two ses- sions were always A-only conditions (i.e., there was no visual component in the stimuli). This analysis, averaged 3604 J. Acoust. Soc. Am. 150 (5), November 2021 Hladek et al. FIG. 5. (Color online) Results of a follow-up auditory-only experiment compared to the auditory-only baseline from the main study and to several results from the literature. For the current experiments, across-subject geo- metric mean of the response distance (6SEM) as a function of actual target distance is reported. https://doi.org/10.1121/10.0007066 https://doi.org/10.1121/10.0007066 https://doi.org/10.1121/10.0007066 G 5 (C l li ) l f f ll di l i experiment, which conﬁrms the assumption that the V- aligned condition in the ﬁrst run of the main experiment made the responses better aligned with the true loudspeaker positions. For the distances 100–200 cm, the data of the cur- rent experiments (both main and the follow-up) are very close to the data of Zahorik and Wightman (2001) as well as to (Kopcˇo and Shinn-Cunningham, 2011). While Anderson and Zahorik (2014) and Rebillat et al. (2012) show overesti- mation. For the distance below 100 cm, the current data are less biased than Zahorik and Wightman (2001) or Kopcˇo and Shinn-Cunningham (2011), suggesting that the listeners were less biased in the current study. APPENDIX A The reason for this improvement might be that the acoustic high-frequency shadowing enhanced the distance-dependent spectral-color- ation cue, which counteracted the overestimation bias observed in the previous studies. Or, it is possible that the listeners were able to judge those sources much more accu- rately in the current study’s real environment than in the vir- tual environment employed previously. FIG. 5. (Color online) Results of a follow-up auditory-only experiment compared to the auditory-only baseline from the main study and to several results from the literature. For the current experiments, across-subject geo- metric mean of the response distance (6SEM) as a function of actual target distance is reported. Previous studies modeled perception of auditory dis- tance using power law function (r0 ¼ kra, r0 – estimate of perceived distance; r, physical sound source distance; k, a, parameters) (Anderson and Zahorik, 2014; Zahorik et al., 2005). As an additional effort to establish that the baseline performance here is comparable with corresponding condi- tions of previous studies, the model was also ﬁtted to the current A-only and AV responses in the main-experiment baseline runs. For the A-only data, the ﬁtted values of the parameters were k ¼ 1.04 and a ¼ 0.82, very near the ideal values of 1 (all values are across-subject means). For the responses to AV stimuli, the ﬁtted parameters were k ¼ 1.15, a ¼ 0.93. Both these ﬁts are in the range of typical values found across literature (Zahorik et al., 2005). A study which used an array of loudspeakers found values of k ¼ 0.78, a ¼ 0.9 (in condition with all speakers visible) and k ¼ 0.92, a ¼0.66 (in condition with the participants blindfolded) (Zahorik, 2001). The visible-speaker condition could be directly compared with our A-only condition, and the observed values of k and a are comparable. In another study that used head-phone presentation and video screen presen- tation the AV condition was ﬁtted with values of k ¼ 0.96, data across two sessions and across 22 subjects who partici- pated in the follow-up. Figure 5 presents the results of this follow-up along with the results of the baseline A-only measurement in the main study and with several comparable auditory distance experiments from the literature. The geometric mean of response distance from the auditory-only follow-up is shown by circles. APPENDIX A Data from the A-only runs 2 and 3 from the main experiment, averaged across groups and sessions are shown by squares. Data were for relevant egocentric distances for frontal broadband targets from Kopcˇo and Shinn- Cunningham (2011), Fig. 2(A) broadband condition (n ¼ 6), shown by diamonds, Anderson and Zahorik (2014), Fig. 2(A) (n ¼ 57), shown by asterisks, Rebillat et al. (2012), Fig. 8(B), position 1 (n ¼ 40), shown by down-pointing tri- angles, and Zahorik and Wightman (2001), Fig. 5 (n ¼ 5), shown by up-pointing triangles. The data of the follow-up experiment are slightly more biased towards larger distances than the data of the main FIG. 6. (Color online) Ventriloquism effect and aftereffect with and without the correction for the visual baseline. The corrected data (in gray with dashed lines) are re-plotted from Figs. 3(G)–3(I). The uncorrected data are plotted identically to the data in Fig. 3 but without the correction for the visual baseline. For the color version of the ﬁgure, please refer to the online version. FIG. 6. (Color online) Ventriloquism effect and aftereffect with and without the correction for the visual baseline. The corrected data (in gray with dashed lines) are re-plotted from Figs. 3(G)–3(I). The uncorrected data are plotted identically to the data in Fig. 3 but without the correction for the visual baseline. For the color version of the ﬁgure, please refer to the online version. J. Acoust. Soc. Am. 150 (5), November 2021 Hladek et al. 3605 Hladek et al. 3605 Hladek et al. 3605 https://doi.org/10.1121/10.0007066 TABLE I. Summary statistical analysis of the VE, VAE, and persistent VAE for the uncorrected data shown in Fig. 6. p < 0.05, *p < 0.001 (signiﬁcance levels modiﬁed by Geisser-Greenhouse Epsilons). VE [Fig. 6(A)] VAE [Fig. 6(B)] Persistent VAE [Fig. 6(C)] Target Location F(71 022) 2.7 2.58* 0.57 condition F(1,146) 0.89 4.67* 0.63 Target Location x condition F(71 022) 6.92*** 0.8 1.28 a ¼ 0.93 (Anderson and Zahorik, 2014). These ﬁtted values are comparable with our AV condition. On the other hand, a study which used VR and a method of triangulation for auditory-visual distance estimations found more compressed responses (a ¼ 0.41 or a ¼ 0.29, k ¼ 1.62, or k ¼ 2.13) (Rebillat et al., 2012). These values differ more consider- ably from the current results. APPENDIX A Since our results obtained in real environment approach the ideal values of 1, it is likely that the inferior performance in this study can be ascribed to the use of virtual environment, to different distance ranges, or to other differences in the experimental design. increasing egocentric distance. Therefore, it is likely that the signiﬁcant effect of target location is driven by the measure- ment artifacts eliminated by the correction. In terms of VAE, the change of the data were about ﬁve percentual points for the two farthest target targets in the V-farther con- dition. In other target locations it was even smaller. This is relatively small change when averaged across all positions, however, due to a high number of participants even small change could change the signiﬁcance levels of the statistical test because one number is added to the data of all partici- pants. Again, the change due to the correction was relatively small but it seems to correct for the effects that most likely relate to the setup of the current study. Overall, the comparison provided here shows that the current baseline auditory-only performance is comparable to the previous studies. The responses are more accurate for the observed range than in the previous studies, which could relate to the availability of the direct sound level cue, loud- speakers in a real room (as opposed to virtual environ- ments), or the additional spectral cues caused by shadowing of the loudspeakers. Importantly for the current audiovisual study, the fact that the auditory distance percepts are less biased here, i.e., that the auditory-only and visual-only per- cepts are closer to each other in the V-aligned condition, might mean that the audio-visual binding was stronger here than would occur in virtual environment. Bedford, F. L. (1993). “Perceptual and cognitive spatial learning,” J. Exp. Psychol. Hum. Percept. Perform. 19, 517–530. Bertelson, P., Frissen, I., Vroomen, J., and de Gelder, B. (2006). “The after- effects of ventriloquism: Patterns of spatial generalization,” Percept. Psychophys. 68, 428–436. Bertelson, P., Vroomen, J., de Gelder, B., and Driver, J. (2000). “The ven- triloquist effect does not depend on the direction of deliberate visual attention,” Percept. Psychophys. 62, 321–332. Berzborn, M., Bomhardt, R., Klein, J., Richter, J.-G., and Vorl€ander, M. (2017). 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https://openalex.org/W4214750875	https://www.mdpi.com/2504-3110/6/3/137/pdf?version=1646126393	English	null	Fractional Behaviours Modelling with Volterra Equations: Application to a Lithium-Ion Cell and Comparison with a Fractional Model	Fractal and fractional	2,022	cc-by	6,583	Citation: Tartaglione, V.; Farges, C.; Sabatier, J. Fractional Behaviours Modelling with Volterra Equations: Application to a Lithium-Ion Cell and Comparison with a Fractional Model. Fractal Fract. 2022, 6, 137. https:// doi.org/10.3390/fractalfract6030137 fractal and fractional fractal and fractional fractal and fractional fractal and fractional f fractal and fractional Article Fractional Behaviours Modelling with Volterra Equations: Application to a Lithium-Ion Cell and Comparison with a Fractional Model Vincent Tartaglione 1, Christophe Farges 1,* and Jocelyn Sabatier 2,* Vincent Tartaglione 1, Christophe Farges 1,* and Jocelyn Sabatier 2,* 1 IMS Laboratory, Bordeaux University, UMR CNRS 5218-351, Cours de la Libération, CEDEX, 33405 Talence, France; vincent.tartaglione@u-bordeaux.fr 1 IMS Laboratory, Bordeaux University, UMR CNRS 5218-351, Cours de la Libération, CEDEX, 33405 Talence, France; vincent.tartaglione@u-bordeaux.fr 2 Bordeaux Institute of Technology, 15, Rue Naudet-CS 10 207, CEDEX, 33175 Gradignan, France * Correspondence: christophe.farges@u-bordeaux.fr (C.F.); jocelyn.sabatier@u-bordeaux.fr (J.S.) g 2 Bordeaux Institute of Technology, 15, Rue Naudet-CS 10 207, CEDEX, 33175 Gradignan, Fran * Correspondence: christophe.farges@u-bordeaux.fr (C.F.); jocelyn.sabatier@u-bordeaux.fr (J.S Abstract: This paper proposes to model fractional behaviors using Volterra equations. As fractional differentiation-based models that are commonly used to model such behaviors exhibit several draw- backs and are particular cases of Volterra equations (in the kernel deﬁnition), it appears legitimate in a modeling approach to work directly with Volterra equations. In this paper, a numerical method is thus developed to identify the kernel associated to a Volterra equation that describes the input– output behavior of a system. This method is used to model a lithium-ion cell using real data. The resulting model is compared to a fractional differentiation-based model with the same number of tunable parameters. Keywords: fractional behaviours; fractional models; Volterra equations; lithium-ion cell 1. Introduction Fractional behaviors are induced by numerous physical phenomena, often of a stochastic nature, such as diffusion, collision, adsorption, freezing, aggregation, and fragmentation [1,2] Many examples of this type of behavior have been revealed in various areas, including electrochemistry [3,4], thermal science [5,6], biology [7,8], mechanics [9], acoustics [10], and electrical engineering [11]. Faced with the omnipresence of such behaviors, efﬁcient modeling tools are required. Academic Editor: Norbert Herencsar Academic Editor: Norbert Physical systems with fractional behaviors are often modeled using fractional differentiation-based models, or “fractional models” for short. Such models are indeed able to ﬁt the input–output behavior of this class of system accurately and with a limited number of parameters. However, it is now known that fractional differentiation-based models are associated to several drawbacks mainly related to the doubly inﬁnite dimension of these models [12–16]. Received: 8 January 2022 Accepted: 24 February 2022 Published: 1 March 2022 Received: 8 January 2022 Accepted: 24 February 2022 Published: 1 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. To overcome some of these drawbacks, new modeling tools must be introduced. Some were recently proposed in [12] among which Volterra equations are included. Volterra equations of the ﬁrst kind were introduced by Volterra in [17] and, in the linear and homogenous case, are deﬁned by the relation: Copyright: © 2022 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/). Z t 0 ˜η(t −τ)x(τ) d τ = v(t). (1) (1) Copyright: © 2022 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/) Assuming that v(t) is differentiable, differentiation of the relation (1) leads to the following relation, which is called a linear Volterra equation of the second kind: x(t) = u(t) + Z t 0 η(t −τ)x(τ) d τ, (2) (2) ps://doi.org/10.3390/fractalfract6030137 https://www.mdpi.com/journal/fractalfract https://www.mdpi.com/journal/fractalfract Fractal Fract. 2022, 6, 137. https://doi.org/10.3390/fractalfract6030137 Fractal Fract. 2022, 6, 137 2 of 14 where u(t) = 1 ˜η(0) d d tv(t) and η(t) = − 1 ˜η(0) d d t ˜η(t). where u(t) = 1 ˜η(0) d d tv(t) and η(t) = − 1 ˜η(0) d d t ˜η(t). 1. Introduction η( ) η( ) Volterra equations have been used in various applications: energy [18,19], sorption kinetics [20], and mechanical systems [21]. These equations are more general than the fractional differentiation-based models, since fractional pseudostate space descriptions are special cases of Volterra equations [22] involving a particular kernel. The Cole and Cole [23] modeling work considered by some as the ﬁrst application of fractional differentiation could thus have been done using Volterra equations as demonstrated in [22]. g This paper therefore proposes an answer to the problem of the direct determination of the kernel of a Volterra equation from input–output data. There is very little work in the literature that is interested in this problem, which is nevertheless an essential step in the modeling methodology. We can, however, cite the work of Brewer et al. [24], which relates to the identiﬁcation of Volterra equation with weakly singular kernels but for which the kernel has a predeﬁnite structure; and of Karuppiah et al. [25] which relates to the reconstruction of a time-independent parameter in an integrodifferential equation. We can also cite the work of Glentis et al. [26] in which Volterra kernels are estimated via statistics method, of Nemeth et al. [27] in which interpolating technics are used to approximate Volterra kernels, and of Lorenzi [28] in which some results on identiﬁcation of unknown terms in integrodifferential equations are gathered. None of these works allow the direct computation of an analytic function for the kernel in the Volterra model to be obtained. In this paper, it is ﬁrst highlighted that Volterra equations generalize a pseudostate space description often used in the literature to model input–output fractional behaviors, but also other fractional models. An identiﬁcation method that permits the direct deter- mination of the kernel involved in a ﬁrst-kind Volterra equation is then proposed. The method is applied to derive a model for a lithium-ion cell on real data. A comparison of the obtained model with a fractional differentiation based model is also carried out. 2. Volterra Equations as Generalizations of Fractional Models 2. Volterra Equations as Generalizations of Fractional Models For a fractional model, the pseudostate space descritpion is given by the relation For a fractional model, the pseudostate space descritpion is given by the relation ( Dν 0x = Ax(t) + Bu(t) y(t) = Cx(t) + Du(t) , ∀t < 0, x(t) = u(t) = 0, (3) (3) where Dν 0 is a fractional differentiation operator of order ν ∈R [29], x(t) ∈Rn is the pseudostate vector [30], u(t) ∈R is the control input, supposed differentiable and y(t) ∈R is the measured output. All these functions are supposed to be integrable over their domain of deﬁnition. Several deﬁnitions exist for this operator [31]. One of the most used deﬁnitions is that of Riemann–Liouville, denoted RLDν t0 with: RLDν t0 f (t) = dm d tm 1 Γ(m −ν) Z t t0 (t −τ)m−ν−1 f (τ) d τ , (4) (4) and m = ⌈ν⌉. and m = ⌈ν⌉. ⌈⌉ In the case 0 < ν < 1, a ﬁrst order integration on both sides of the ﬁrst equation of (3), leads to the following relation: Z t 0 ζ(t −τ)x(τ) d τ = Z t 0 Ax(τ) + Bu(τ) d τ, (5) (5) where ζ(t −τ) = (t−τ)m−ν−1 Γ(m−ν) multiplies each component of the vector x(τ). The relation (5) can therefore be rewritten as follows Z t 0 (ζ(t −τ)In −A)x(τ) d τ = Z t 0 Bu(τ) d τ, (6) (6) Fractal Fract. 2022, 6, 137 3 of 14 where In denotes the identity matrix of size n. And so the pseudo-state representation (3) can be rewritten as a Volterra equation of the ﬁrst kind: where In denotes the identity matrix of size n. And so the pseudo-state representation (3) can be rewritten as a Volterra equation of the ﬁrst kind: Z t 0 η(t −τ)x(τ) d τ = v(t), (7) (7) with v(t) = R t 0 Bu(τ) d τ and η(t) = ζ(t)In −A. The relation (7) shows that the pseudostate representation is a particular case of Volterra equation of the ﬁrst kind and thus of the second kind given relation (2). This analysis can be extended to another class of fractional models. 2. Volterra Equations as Generalizations of Fractional Models Consider the following fractional differential equation: Na X k=1 ak dνak d tνak x(t) = Nb X k=1 bk dνbk d tνbk u(t), (8) (8) with with with νaNa > νaNa−1 > · · · > νa1 νbNb > νbNb−1 > · · · > νb1 νaNa > νbNb and \|νaNa −νbNb \| < 1. νaNa > νaNa−1 > · · · > νa1 νbNb > νbNb−1 > · · · > νb1 d \| \| Laplace transform applied to relation (8) permits to write that Laplace transform applied to relation (8) permits to write that x(s) 1 F(s) = u(s), (9) x(s) 1 F(s) = u(s), (9) (9) where x(s) and u(s) are respectively the Laplace transform of x(t) and u(t), and where where x(s) and u(s) are respectively the Laplace transform of x(t) and u(t), and where F(s) = PNb k=1 bksνbk PNa k=1 aksνak . (10) (10) By multiplying the two sides of relation (9) by 1 s , it becomes By multiplying the two sides of relation (9) by 1 s , it becomes x(s) 1 sF(s) = 1 s u(s), (11) (11) in which transfer function 1 sF(s) is now proper. sF(s) Using the inverse Laplace transform, relation (11) becomes Z t 0 ϕ(t −τ)x(τ) d τ = v(t), (12) Z t 0 ϕ(t −τ)x(τ) d τ = v(t), (12) (12) with v(t) = R t 0 u(τ) d τ and where ϕ(t) = L−1{ 1 sF(s)}. with v(t) = R t 0 u(τ) d τ and where ϕ(t) = L−1{ 1 sF(s)}. with v(t) = R t 0 u(τ) d τ and where ϕ(t) = L−1{ 1 sF(s)}. ( ) Relation (12) has thus the same form as relation (1) and is a ﬁrst-kind Volterra equation with a kernel of speciﬁc structure. Note that the constraint \|νaNa −νbNb \| can be lifted. In the case m −1 < \|νaNa −νbNb \| < m (m ∈N), v(t) is the integral of order m in relation (12) and ϕ(t) = L−1{ 1 smF(s)}. ( ) As a consequence, in a modeling approach, it is better to work directly with a Volterra equation, because this avoids being constrained on the choice of the kernel, which is ﬁxed in the case of a fractional model. The direct use of a Volterra equation is therefore more general. Fractal Fract. 2022, 6, 137 4 of 14 3. A Numerical Method to Determine the Kernel of the Volterra Model Let us denote x(t) the output of a linear system in response to an input u(t). The signal x(t) is modeled with the Volterra equation of relation (2) which involves a kernel η that can be determined numerically, as follows. A discretization of relation (2) is done using trapezoid method (with sampling time Ts), which provides: x(Ts) = u(Ts) + Z Ts 0 η(τ)x(t −τ) d τ ≈u(Ts) + η(Ts)x(Ts −Ts) + η(0)x(Ts −0) 2 Ts x(2Ts) = u(2Ts) + Z Ts 0 η(τ)x(t −τ) d τ + Z 2Ts Ts η(τ)x(t −τ) d τ ≈u(2Ts) + η(Ts)x(2Ts −Ts) + η(0)x(2Ts −0) 2 Ts + η(2Ts)x(2Ts −2Ts) + η(Ts)x(2Ts −Ts) 2 Ts .. x(Ts) = u(Ts) + Z Ts 0 η(τ)x(t −τ) d τ ≈u(Ts) + η(Ts)x(Ts −Ts) + η(0)x(Ts −0) 2 Ts x(2Ts) = u(2Ts) + Z Ts 0 η(τ)x(t −τ) d τ + Z 2Ts Ts η(τ)x(t −τ) d τ Z Ts ≈u(2Ts) + η(Ts)x(2Ts −Ts) + η(0)x(2Ts −0) 2 Ts + η(2Ts)x(2Ts −2Ts) + η(Ts)x(2Ts −Ts) 2 Ts (13) whose matrix writing is whose matrix writing is g   x(Ts) x(0) 0 · · · · · · · · · x(2Ts) 2x(Ts) x(0) 0 · · · · · · x(3Ts) 2x(2Ts) 2x(Ts) x(0) 0 · · · ... ...   \| {z } X   η(0) η(Ts) η(2Ts) ...   \| {z } η =   x(Ts) −u(Ts) x(2Ts) −u(2Ts) x(3Ts) −u(3Ts) ...   \| {z } U (14) (14) Samples η(k) can then be obtained solving the linear equation system (14) with a least square method. An analytic function η(t) can then be used to ﬁt the samples η(k). As an example, let suppose that the signal x(t) corresponds to the step response of the fractional transfer function F(s) = 1 s ωb + 1 ν , with ωb = 10−3 and ν = 0.2. (15) (15) The resulting step response is given by The resulting step response is given by f (t) = e−t/2ωbtν ν(ν + 1) t ωb −ν/2 WM ν, ν/2 + 1/2, t ωb + (ν + 1)e−t/2ωb , (16) where WM(·, ·, ·) is the Whittaker M function. where WM(·, ·, ·) is the Whittaker M function. where WM(·, ·, ·) is the Whittaker M function. Fractal Fract. 3. A Numerical Method to Determine the Kernel of the Volterra Model 2022, 6, 137 5 of 14 The samples η(k) are then computed using relation (14). The following function, which is close to the impulse response of F(s), is ﬁrst considered to ﬁt the samples η(k) η1(t) = a0tν−1 exp(−t/ωb). (17) (17) The response of Equation (2) with kernel η1(t) is denoted x1(t) and is compared to the response x(t) of the fractional model (15) in Figure 1. 0 500 1000 1500 2000 2500 3000 0 0.5 1 1.5 2 2.5 3 3.5 4 x(t) x1(t) Figure 1. Fractional model (15) compared to the response x1(t) of model (2) with kernel η1(t) of Equation (17). Figure 1. Fractional model (15) compared to the response x1(t) of model (2) with kernel η1(t) of Equation (17). After optimization, the quadratic error between x(t) and x1(t) is ε1 = X \|x1(i) −x(i)\|2 = 5.5985. (18) (18) Kernel (17) is singular. To avoid this singularity and to have a more accurate ﬁtting of η(k), another kernel must be found. For this, it is assumed that a physical system has a low-pass-type behavior, and thus its low-frequency behavior can be captured by a transfer function of the form 1 H(s) = 1 pk(p + α), k ∈N . (19) (19) Let h(t) be the inverse Laplace transform of H(s). To take into account the behavior of the system in medium frequency (frequencies less than 1/Ts), function h(t) is completed by a function of general form g(t) = Pna k=1 aktk Pnb k=1 bktk , with a0 = 1. (20) (20) Thus for the considered problem (as k = 0 in relation (19)), a possible analytic function for samples η(k) ﬁtting is: Thus for the considered problem (as k = 0 in relation (19)), a possible analytic function for samples η(k) ﬁtting is: η2(t) = exp(−αt) 1 + a1t + a2t2 b0 + b1t + b2t2 . (21) (21) In Figure 2 are compared η(k) and the kernel η2(t) with parameters gathered in Table 1 after optimization. p The quadratic error between η2(k) and η(kTs) is p The quadratic error between η2(k) and η(kTs) is ε2 = X \|η2(i) −η(i)\|2 = 3.6892 × 10−3. (22) (22) Fractal Fract. 2022, 6, 137 6 of 14 Table 1. Parameters of η2(t) in the relation (21). 3. A Numerical Method to Determine the Kernel of the Volterra Model Thus, the proposed kernel of the general form The comparison reveals that the error ε3 obtained with the Volterra model is notably smaller than the error ε1 obtained with the fractional model. Thus, the proposed kernel of the general form η(t) = h(t)g(t) (24) (24) g η(t) = h(t)g(t) (24) is a possible and interesting candidate for the ﬁtting of the samples η(k). is a possible and interesting candidate for the ﬁtting of the samples η(k). 3. A Numerical Method to Determine the Kernel of the Volterra Model α 4.2697 × 10−5 a1 7.7946 × 10−3 a2 2.3453 × 10−5 b0 6.7661 b1 4.2179 b2 2.3728 × 10−1 α 4.2697×10−5 a1 7.7946×10−3 a2 2.3453×10−5 b0 6.7661 b1 4.2179 b2 2.3728×10−1 Table 1. Parameters of η2(t) in the relation (21). α The output of the model (2) with kernel η2(t), denoted x2(t) is given in Figure 3 and is compared with the response of the fractional model f (t). The output of the model (2) with kernel η2(t), denoted x2(t) is given in is compared with the response of the fractional model f (t). 0 500 1000 1500 2000 2500 3000 t 0 0.05 0.1 0.15 (k) 2(t) 0 20 40 60 80 100 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Figure 2. Comparison of η(k) with η2(t). 0 500 1000 1500 2000 2500 3000 0 0.5 1 1.5 2 2.5 3 3.5 4 x(t) x2(t) Fi 3 O (t) f d l (2) i h k l (t) (E i (21)) d h Figure 2. Comparison of η(k) with η2(t). 0 500 1000 1500 2000 2500 3000 0 0.5 1 1.5 2 2.5 3 3.5 4 x(t) x2(t) Figure 3. Output x2(t) of model (2) with kernel η2(t) (Equation (21)) compared to the step response f (t) (Equation (16)). 0 500 1000 1500 2000 2500 3000 t 0 0.05 0.1 0.15 (k) 2(t) 0 20 40 60 80 100 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Figure 2. Comparison of η(k) with Figure 2. Comparison of η(k) with η2(t). 0.5 1 1.5 2 2.5 3 3.5 4 x(t) x2(t) 0 500 1000 1500 2000 2500 3000 0 0.5 1 1.5 2 2.5 3 3.5 4 x(t) x2(t) 0 500 1000 1500 2000 2500 3000 0 Figure 3. Output x2(t) of model (2) with kernel η2(t) (Equation (21)) compared to the step response f (t) (Equation (16)). Fractal Fract. 2022, 6, 137 7 of 14 Th d ti b t (t) d f (t) i The quadratic error between x2(t) and f (t) is The quadratic error between x2(t) and f (t) is The quadratic error between x2(t) and f (t) is ε3 = X \|x2(i) −x(i)\|2 = 3.11 × 10−1. (23) (23) The comparison reveals that the error ε3 obtained with the Volterra model is notably smaller than the error ε1 obtained with the fractional model. 4. Application to Lithium-Ion Cell This section is dedicated to lithium-ion cell input–output behavior modeling. A wide variety of battery models have been proposed in the literature with different complexities and applications. These models can be classiﬁed in 4 categories [32] • Electrochemical models, which accurately describe electrochemical reactions that take place in the electrodes and the electrolyte [33]—Pseudo-Two-Dimensional model and Single Particle Model belong to this category; • Mathematical models, which are based on empirical equations or math-based stochas- tic models which only evaluate the charge-recovery effect and ignore all other factors. The number of equations is reduced and simpliﬁed compared to the electrochemical model [34]; • Circuit-oriented models, which are electrical-equivalent models or impedance models in which each component of the circuit is related to an electrochemical process of the battery to provide a good description of its internal behaviour [35]; y p g p • Combined models that consists of the combination of different electrical models and mathematical models in order to combine the best attributes of each model, such as the correct prediction of the battery lifetime, steady-state and transient responses, and accurate estimation of the state of charge [36]. In this section, the model proposed falls in this last category as it is an impedance- based model, resulting from the simpliﬁcation of Single Particle model, that combines several mathematical submodels, especially a Volterra equation, to take into the long- memory behaviour of a lithium-ion cell. This model will be compared to a similar model that includes a fractional order-transfer function [37]. The data comes from [38] and results from a collaboration with the society PSA Peugeot-Citroën. The input of the cell is the current and the output its voltage. The incoming current over time is plotted in Figure 4 and the resulting voltage U(t) over time is plotted in Figure 5. 0 50 100 150 200 250 300 350 400 450 500 Time (s) -100 -50 0 50 100 150 Current (A) 0 50 100 150 200 250 300 350 400 450 500 Time (s) -100 -50 0 50 100 150 Current (A) Figure 4. Input current applied to the cell. Figure 4. Input current applied to the cell. Fractal Fract. 4. Application to Lithium-Ion Cell 2022, 6, 137 8 of 14 8 of 14 Under the asumption of material spherical particle in the electrodes and considering that the positive electrode is limiting in terms of dynamic behavior, a lithium-ion cell can be modeled by the single electrode model of Figure 6. This model contains a resistor R for the internal and connections resistances that can be estimated through the voltage drop that appears when a step current is applied. The function ϕ links the open circuit voltage (OCV) to the lithium concentration at the surface of the sphere. It can be obtained by measuring the cell voltage at rest for various cell states of charge. For the considered cell, this nonlinear function is ﬁtted by a 12-degree polynomial (see [38] for the ﬁtting result of this polynomial). The initial state of the battery is deﬁned by CS(0) and the cell voltage is U(t). The function G(s) permits to model the diffusion of lithium inside the sphere. 0 50 100 150 200 250 300 350 400 450 500 Time (s) 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 Voltage (V) Figure 5. Cell output voltage. I(t) G(s) + + ϕ Cs(t) + + U(t) R Cs(0) Figure 6. Model of a lithium-ion cell proposed in [38]. 0 50 100 150 200 250 300 350 400 450 500 Time (s) 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 Voltage (V) Figure 5. Cell output voltage. I(t) G(s) + + ϕ Cs(t) + + U(t) R Cs(0) Figure 6. Model of a lithium-ion cell proposed in [38]. 4.1. Fractional Model 4.1. Fractional Model First, the diffusion part is modeled using the following fractional transfer function: G(s) = K s (1 + sn w1 )(1 + sn w2 ) (1 + sn w3 ) . (25) (25) After a least square optimization, aiming at minimizing the differences between the model and the cell output, the parameters gathered in Table 2 were obtained for transfer function G(s). The optimal value for parameter R is 1.836 × 10−3. The resulting response U1(t) of the model of Figure 6 is compared to the cell output voltage in Figure 7. Fractal Fract. 2022, 6, 137 9 of 14 Table 2. Parameters of G(s) from the relation (25) for modeling the outgoing battery voltage. 4. Application to Lithium-Ion Cell K 4.332 × 10−5 n 7.195 × 10−1 ω1 4.001 × 10−2 ω2 1.143 × 102 ω3 4.345 0 50 100 150 200 250 300 350 400 450 500 Time (s) 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 Voltage (V) Data U(t) U1(t) Figure 7. Voltage U(t) compared to the response of model in Figure 6 with the fractional transfer function (25). Table 2. Parameters of G(s) from the relation (25) for modeling the outgoing battery voltage. 0 50 100 150 200 250 300 350 400 450 500 Time (s) 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 Voltage (V) Data U(t) U1(t) Figure 7. Voltage U(t) compared to the response of model in Figure 6 with the fractional transfer function (25). The quadratic error between U(t) and U1(t) is given by: ε4 = X \|U(i) −U1(i)\|2 = 1.250 × 10−1. (26) (26) The error as a function of time is plotted in Figure 8. The error as a function of time is plotted in Figure 8. Time (s) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 \|U-U1\| ×10-4 0 1 2 3 4 5 6 7 8 Figure 8. Error between voltage U(t) and the response U1(t) of model in Figure 6 using the fractional transfer function (25). Figure 8. Error between voltage U(t) and the response U1(t) of model in Figure 6 using the fractional transfer function (25). Fractal Fract. 2022, 6, 137 10 of 14 10 of 14 The error as a function of time is plotted in Figure 10. 4.2. Volterra Model The diffusion part of the cell model described in Figure 6 is now modeled by the Volterra Equation (2), in which the kernel η(t) is deﬁned by the relation: η2(t) = −exp(−αt) 1 + a0t2 b0 + a1t2 + a2t3 , (27) (27) where function u(t) is deﬁned as K1 R I(t). Parameter b0 = 0.264 is not optimized but is a ﬁxed value deﬁned so that the ﬁrst value of U2(0) coincides with the voltage U(0), where U2(t) denotes the Volterra equation-based cell model output. Hence, this model involves the same number of parameters as transfer function (25). After optimization, aiming at minimizing the differences between U(t) and U2(t), the obtained parameters for η2(t) are gathered into Table 3. The optimal value for parameter R is 1.836 × 10−3. g p p The cell output voltage U(t) and the output U2(t) of the model of Figure 6 using the Volterra model (2) for the diffusion part are compared in Figure 9. Temps (s) 0 50 100 150 200 250 300 350 400 450 500 Tension (V) 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 Data Model Figure 9. Voltage U(t) compared to the response U2(t) of model in Figure 6 using the Volterra model (2) with kernel (27). Figure 9. Voltage U(t) compared to the response U2(t) of model in Figure 6 using the Volterra model (2) with kernel (27). Table 3. Parameters of η2(t) of the relation (27) for the modeling of the outgoing battery voltage U(t). a0 5.545 × 10−1 a1 3.639 × 10−2 a2 1.274 × 10−3 α 3.965 × 10−3 K1 4.486 × 10−3 The quadratic error between the voltage data U(t) and the cell model is then given by ε5 = X \|U(i) −U2(i)\|2 = 1.112 × 10−1. (28) (28) 11 of 14 Fractal Fract. 2022, 6, 137 Time (s) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 \|U-U2\| ×10-4 0 1 2 3 4 5 6 7 8 Figure 10. Error between voltage U(t) and the response U2(t) of model in Figure 6 using the Volterra model (2) with kernel (27). Figure 10. Error between voltage U(t) and the response U2(t) of model in Figure 6 using the Volterra model (2) with kernel (27). 4.3. Discussion This ﬁgure shows that for Tf ≥5/α the Bode diagrams of x(jω)/u(jω) are similar and that it is not useful to chose Tf > 5/α. Frequency responses of x(jω)/u(jω) for η(t) = η2(t) (relation (27)) and for Tf = 0.05/α Tf = 0.1/α, Tf = 1/α, Tf = 5/α and Tf = 10/α are represented by Figure 11. This ﬁgure shows that for Tf ≥5/α the Bode diagrams of x(jω)/u(jω) are similar and that it is not useful to chose Tf > 5/α. 10-4 10-3 10-2 10-1 100 101 -20 -15 -10 -5 0 Gain (dB) Tf=0.05/ Tf=0.1/ Tf=1/ Tf=5/ Tf=10/ 10-4 10-3 10-2 10-1 100 101 -20 -15 -10 -5 0 Gain (dB) Tf=0.05/ Tf=0.1/ Tf=1/ Tf=5/ Tf=10/ 10-4 10-3 10-2 10-1 100 101 Frequency (rad/s) 0 20 40 60 Phase (°) Tf=0.05/ Tf=0.1/ Tf=1/ Tf=5/ Tf=10/ Figure 11. Frequency responses of x(jω)/u(jω) for η(t) = η2(t), for various values of Tf . 5 Conclusions 10-4 10-3 10-2 10-1 100 101 Frequency (rad/s) 0 20 40 60 Phase (°) Tf=0.05/ Tf=0.1/ Tf=1/ Tf=5/ Tf=10/ Figure 11. Frequency responses of x(jω)/u(jω) for η(t) = η2(t), for various values of Tf . 4.3. Discussion In comparison with a simple ﬁrst-order or second-order model, the Volterra model permits the system memory to be taken into account. The application of the previous section shows that both fractional and Volterra models accurately capture the dynamics of a system known for its fractional behaviour, with the same number of parameters and with a slightly better quadratic error for the Volterra model; however, one advantage of Volterra-based models is their ability to overcome several drawbacks of the fractional models [12,13,15]. Moreover, the Volterra model used here is simpler than single-particle models and pseudo-2D Li-ion electrochemical models. Furthermore, the memory of a Volterra model can be limited by modifying one bound of the integral deﬁning the Volterra model without affecting its input–output behavior. Relation (2) thus becomes: x(t) = u(t) + Z Tf 0 η(τ)x(t −τ) d τ, x(t) = 0 for t < 0 (29) (29) where Tf characterizes the memory length. f By choosing Tf well, the behaviors of (2) and (29) are very close. In order to deﬁne a rule for the choice of Tf , the frequency responses of the transfer function x(s)/u(s) resulting from relation (29) for several values of Tf are now compared. f By choosing Tf well, the behaviors of (2) and (29) are very close. In order to deﬁne a rule for the choice of Tf , the frequency responses of the transfer function x(s)/u(s) resulting from relation (29) for several values of Tf are now compared. f p As the Laplace transform of relation (29) is f As the Laplace transform of relation (29) is x(s) = u(s) + Z Tf 0 η(τ)e−sτx(s) d τ (30) (30) the transfer function x(s)/u(s) is thus given by the transfer function x(s)/u(s) is thus given by x(s) u(s) = 1 1 − R Tf 0 η(τ)e−sτ d τ . (31) (31) For s = jω, integral in (31) can be computed numerically and thus, with ∆= Tf N : x(jω) u(jω) ≈ 1 1 −PN−1 k=0 η(k∆)e−jωk∆. (32) (32) Fractal Fract. 2022, 6, 137 12 of 14 12 of 14 Frequency responses of x(jω)/u(jω) for η(t) = η2(t) (relation (27)) and for Tf = 0.05/α, Tf = 0.1/α, Tf = 1/α, Tf = 5/α and Tf = 10/α are represented by Figure 11. 5. Conclusions Starting from the idea that a fractional differentiation-based model is a particular type of Volterra equation [22], this paper proposes a method to directly derive an explicit form for the kernel involved in a Volterra equation from real input–output data. This method is based on the discretization of the Volterra equation and on the ﬁtting of the discrete form of its kernel using an analytic function. The method is applied to the input–output modelling of a lithium-ion cell. The comparison of the resulting model response and the data reveals a very small error. This error is similar (but slightly better) to those obtained with a fractional differentiation-based models with the same number of tunable parameters but without the drawbacks associated to fractional differentiation-based models [12–16]. The authors now intend to continue working on fractional-behavior modeling using Volterra equations in order to improve the method presented in this paper (for instance, using a system of Volterra equations), and extend it in particular to multi-input multi-output systems. The authors also intend to work with other modelling tools, such as the one described in [39]. Author Contributions: Conceptualization, J.S. and C.F.; methodology, J.S. and C.F.; validation, V.T., C.F. and J.S.; writing original draft preparation, review and editing, V.T., C.F. and J.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. 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https://openalex.org/W2133129785	https://www.hal.inserm.fr/inserm-00950634/file/ar4468.pdf	English	null	Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies	Arthritis research & therapy	2,014	cc-by	9,151	To cite this version: Laurent Drouot, Yves Allenbach, Fabienne Jouen, Jean-Luc Charuel, Jérémie Martinet, et al.. Ex- ploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl- glutaryl-CoA reductase autoantibodies.. Arthritis Research and Therapy, 2014, 16 (1), pp.R39. 10.1186/ar4468. inserm-00950634 Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies. Laurent Drouot, Yves Allenbach, Fabienne Jouen, Jean-Luc Charuel, Jérémie Martinet, Alain Meyer, Olivier Hinschberger, Brigitte Bader-Meunier, Isabelle Kone-Paut, Emmanuelle Campana-Salort, et al. HAL Id: inserm-00950634 https://inserm.hal.science/inserm-00950634v1 Submitted on 21 Feb 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Open Access Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3- methyl-glutaryl-CoA reductase autoantibodies Laurent Drouot1, Yves Allenbach2, Fabienne Jouen1,3, Jean-Luc Charuel4, Jérémie Martinet1,3, Alain Meyer5, Olivier Hinschberger6, Brigitte Bader-Meunier7,8, Isabelle Kone-Paut9, Emmanuelle Campana-Salort10, Bruno Eymard11, Anne Tournadre12, Lucile Musset4, Jean Sibilia5, Isabelle Marie1,13, Olivier Benveniste2,11, Olivier Boyer1,3* and the French Myositis Network [CN] * Correspondence: olivier.boyer@chu-rouen.fr 1Inserm, U905 & Normandie Univ, IRIB, 22 Bd Gambetta, F-76000 Rouen, France 3Rouen University Hospital, Department of Immunology, Rouen, France Full list of author information is available at the end of the article © 2014 Drouot et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: olivier.boyer@chu-rouen.fr 1Inserm, U905 & Normandie Univ, IRIB, 22 Bd Gambetta, F-76000 Rouen, France 3Rouen University Hospital, Department of Immunology, Rouen, France Full list of author information is available at the end of the article © 2014 Drouot 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 For these anti-HMGCR-positive patients, the following parameters were recorded from their medical reports: age, sex, past medical history, statin exposure, characteristics of myopathy including clinical manifest- ation and muscular deficit evaluation by muscle manual testing using the Medical Research Council scale on five points, and creatine kinase (CK) level at the date of the anti-HMGCR assay. Special emphasis was performed by the clinician to check statin exposure during follow-up medical consultations. For correlation experiments, we additionally analyzed 25 anti-SRP-positive sera that were described previously [16]. The term SRP in text and figures refers to the 54 kDa subunit of the multimeric SRP complex. Control sera were collected from 100 healthy blood donors and from 142 patients with different inflamma- tory/autoimmune diseases, according to established clas- sification criteria: American College of Rheumatology (ACR) revised criteria for systemic lupus erythematosus (SLE) [18] with anti-double-stranded DNA (dsDNA) aAbs (n = 19), American Rheumatism Association (ARA) criteria for rheumatoid arthritis (RA) [19] with anti-cyclic citrullinated peptide (CCP) antibodies and/ or rheumatoid factor (n = 18), revised European criteria for primary Sjögren’s syndrome [20] with anti-SSA and/or anti-SSB aAbs (n = 15), Bohan and Peter criteria [21] for dermatomyositis (DM) (n = 8), Troyanov criteria for over- lap myositis [10] with anti-tRNA-synthetase Ab (anti-Jo-1, n = 27; anti-PL-7, n = 1; anti-PL-12, n = 2) and Griggs cri- teria for inclusion body myositis (IBM) [22] (n = 30). Another control consisted in sera from patients with poly- clonal hypergammaglobulinemia (serum immunoglobulin (Ig)G: 23.1 ± 6.9 g/L, mean ± standard deviation (SD)) (n = 24). All serum samples were stored at −80°C until use. The diagnosis of NAM may be difficult, notably in slowly progressive forms in young adults, since histopathological features mimic some forms of muscular dystrophies and some patients may remain without diagnosis for years. Hence, the detection of aAbs in patients with undeter- mined necrotizing myopathy is of utmost importance to indicate an autoimmune mechanism and therefore guide appropriate therapy. Currently, the detection of anti-SRP autoantibodies is based on indirect immunofluorescence on HEp-2 cells, followed by confirmation using a dot-blot test or a quantitative addressable laser bead immunoassay (ALBIA) [16,17]. Here, we developed a new ALBIA for the detection and titration of anti-HMGCR aAbs (ALBIA- HMGCR) as a biomarker of NAM. Introduction French Ministry of Research (AC 2008-87) for the col- lection, analysis, storage and reuse. All samples were ob- tained for diagnostic purposes only with no extra sampling and, in this situation of retrospective study, pa- tients’ consent was not required. The research protocol was approved by research ethics committee of Pitié- Salpêtrière University Hospital. Inflammatory myopathies are a heterogeneous group of acquired muscle disorders including polymyositis, dermatomyositis, inclusion body myositis and overlap myositis. Recently, necrotizing autoimmune myop- athies (NAM) have been defined as a distinct group of severe acquired myopathies, characterized by patho- logical features of prominent myofiber necrosis with- out significant inflammation [1]. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis with little if any inflammation [2-4]. With the emergence of reports describing clinical cases of necrotizing myopathy with microangiopathy and microvascular deposition of com- plement [5], they were progressively distinguished from myositis and finally classified as NAM by a collaborative study group in 2004 [6]. NAM may be associated with auto- antibodies (aAbs) such as anti-signal recognition particle (SRP) autoantibodies. Anti-SRP aAbs are present in a minor- ity (4 to 6%) of patients with acquired inflammatory and/or necrotizing myopathies [6-10] and are associated with severe clinical forms, particularly with heart involvement [11,12]. In 2007, Needham et al. reported eight patients who devel- oped a myopathy during statin therapy [13]. Histological analysis of muscle biopsies revealed necrotic and regenerat- ing myofibers. Later, aAbs against a 100 kDa protein were characterized and finally identified by Mammen et al. as di- rected against 3-hydroxy-3-methylglutaryl-coenzyme A re- ductase (HMGCR), a key enzyme of the cholesterol biosynthesis pathway, which is pharmacologically inhibited by statins [14,15]. Serum or blood samples (n = 150 patients) that were sent to the Laboratory of Immunology of Rouen University Hospital for analysis of anti-HMGCR aAbs because of suspicion of NAM were analyzed. These samples were ob- tained between August 2012 and January 2013 from 24 French centers and were all negative for anti-Jo-1 and anti-SRP aAbs. All patients who were found positive for anti-HMGCR aAbs were negative for anti-PL-7 and anti- PL-12 aAbs. Introduction Also, in order to pro- vide a unique test for the diagnosis of aAb-associated NAM, we further developed a multiplex assay (ALBIA- NAM) and determined whether it could discriminate pa- tients with anti-HMGCR and anti-SRP aAbs. Abstract Introduction: Necrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy- 3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA). Methods: Recombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies. Results: No controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA. Conclusions: Both monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins. © 2014 Drouot 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. Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 2 of 11 Immunoprecipitation of human recombinant HMGCR with patients’ sera Immunoprecipitation was performed by adding 20 μl of human serum sample or 1 μg of positive control rabbit anti-HMGCR Ab to 10 μg of recombinant HMGCR C-domain and bringing the volume to 0.5 ml of IP buffer (1% Nonidet P40, 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA) and rotating the mix for 2 h at 4°C. Protein G agarose beads (Pierce, Rockford, IL, USA) were added for an additional hour. Then, immunoprecipitates were boiled in the presence of Laemmli buffer and sub- sequently electrophoresed on 4 to 10% gradient SDS- PAGE under reducing conditions. Immunoprecipitated HMGCR was revealed with a rabbit anti-HMGCR Ab coupled to Alexa Fluor 680 and read on an Odyssey Li- Cor (Li-Cor Biosciences, Lincoln, NE, USA). The analytical sensitivity of detection of ALBIA- HMGCR was determined by triplicate dosage of serial dilutions of rabbit anti-HMGCR antibodies. For each well, after subtraction of negative control MFI value, mean MFI and standard deviation (SD) were calculated. The analytical specificity of detection of ALBIA- HMGCR was determined by using high-level human anti-HMGCR, anti-SRP or anti-intrinsic factor positive sera at 1/1,000 dilution. Sera were incubated with puri- fied HMGCR, SRP or intrinsic factor beads for 1 h at room temperature and MFI was determined as described above. Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Multiscreen 96-well plates (Millipore, Bedford, MA, USA). Plates were incubated for 2 h at room temperature in the dark on a plate shaker at 650 rpm. Blank (no serum, sec- ondary antibody only), negative control (anti-HMGCR negative serum) and positive controls (highly positive hu- man anti-HMGCR Ab serum or rabbit anti-HMGCR Abs with appropriate secondary antibody) were included in every assay. Beads were collected by filtration under vac- uum and washed twice with 150 μl DPBS containing 0.1% Tween-20. Biotinylated mouse anti-human IgG- or isotype (IgG1, IgG2, IgG3 or IgG4)-specific secondary Ab (Southern Biotech, Birmingham, AL, USA) were added at 1/2,000 dilution and incubated for 1 h at room temperature under shaking. After washing, beads were incubated with 50 μl of streptavidin-R-phycoerythrin (Qiagen, Venlo, The Netherlands) at 1/1,000 dilution for 15 min. Finally, beads were resuspended in 100 μl of DPBS and mean fluorescence intensity (MFI) was determined on a Bio-Plex™apparatus using the Bio-Plex™ Manager software 4.0 (Bio-Rad). (GST) tag was obtained from Sigma-Aldrich (St Louis, MO, USA). Purity of this recombinant HMGCR protein was first determined by 4 to 10% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, followed by Coomassie Blue staining. Western blot analysis was further per- formed by transfer of proteins separated by nonreduc- ing SDS-PAGE to a nitrocellulose membrane followed by incubation with rabbit anti-HMGCR Abs (Abcam, Cambridge, MA, USA) or a human anti-HMGCR- positive serum and revelation with corresponding sec- ondary antibody coupled to Alexa Fluor 680 (Invitrogen, Cergy Pontoise, France). Western blot analysis of recombinant C-terminal domain of the HMGCR protein In this medical research, where we used identifiable hu- man sera and data, we obtained agreement from the The catalytic C-domain (amino acids 441 to 880) of hu- man HMGCR protein fused to a glutathione S-transferase Page 3 of 11 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Multiplex ALBIA for the simultaneous detection and quantification of anti-HMGCR and anti-SRP aAbs in NAM patients (ALBIA-NAM) To detect simultaneously anti-SRP or anti-HMGCR aAbs from a single sample, we used two different beads harboring a special mixture of red/infrared dyes so that they are endowed with a specific spectral signature. Color codes of HMGCR- and SRP-coupled beads were number 55 and 28 (Bio-Rad), respectively. After coup- ling with HMGCR or SRP, 10 μl of each bead prepar- ation were mixed and incubated with serum samples in the same conditions than described previously. Analyt- ical specificity of ALBIA-NAM was further determined by analyzing sera from patients with anti-HMGCR or anti-SRP aAbs or from healthy controls or patients with different inflammatory/autoimmune conditions including RA, systemic sclerosis, SLE, DM, anti-tRNA synthetase aAb positive myositis and IBM, or poly- clonal hypergammaglobulinemia. Levels of anti-SRP and anti-HMGCR sera were compared with those ob- tained with the corresponding monoplex ALBIA-HMGCR or ALBIA-SRP. HMGCR was coupled through a covalent link to fluor- escent beads and used to measure the levels of anti- HMGCR Abs. The development of ALBIA-HMGCR first required optimizing concentration of recombinant HMGCR protein used to coat beads, incubation times, washing steps and revelation with the secondary anti- body (not shown, see Material and Methods for details). This yielded a method allowing detection of as low as 5 ng/mL anti-HMGCR Abs (Figure 1C), indicating a very good analytical sensitivity of the assay. The specificity of HMGCR-coated beads was next evaluated. For this, HMGCR, SRP and intrinsic factor- specific beads were prepared and analyzed using a hu- man anti-HMGCR, anti-SRP or anti-intrinsic factor positive serum. Only anti-HMGCR but not irrelevant anti-SRP or anti-intrinsic factor Ab-positive sera reacted with HMGCR beads (Figure 2A). Similarly, beads coated with SRP or intrinsic factor recombinant proteins that were produced and purified using the same process that for HMGCR protein reacted only with the correspond- ing aAb-positive serum. HMGCR line immunoassay and immunofluorescence analysis The Myositis Euroline 7 (IgG) line immunoassay was kindly provided by Euroimmun (Lübeck, Germany). It consists in a membrane strip coated with different anti- gens, including two different recombinant HMGCR pro- teins. The first consists of the HMGCR C-domain fused to GST and the second a human cDNA expressed in Escherichia coli. The assay was used according to the manufacturer’s instruction and the intensity of color- ation was graded from one to four. Addressable laser bead immunoassay (ALBIA) for the detection and quantification of anti-HMGCR aAbs (ALBIA-HMCGR) Specific inhibition of ALBIA-HMGCR was performed using increasing concentrations of recombinant HMGCR protein, with 1/2,000 dilution of the anti-HMGCR serum used as 100% reference. Percent inhibition was calculated as (1 - (MFI pre-adsorbed serum/MFI serum)) × 100. Homologous and heterologous inhibition of ALBIA- HMGCR was further performed by pre-absorption of three anti-HMGCR, three anti-SRP or three anti-intrinsic factor positive sera with 100 μg/ml of recombinant HMGCR, SRP or intrinsic factor protein. We previously described an ALBIA for anti-SRP anti- bodies (ALBIA-SRP) [16]. Here, to develop immunoassay for anti-HMGCR antibodies (ALBIA-HMGCR), a similar strategy was applied with some modifications. Five to 12 μg of recombinant HMGCR C-domain were coupled to 1.25 × 106 fluorescent Bio-Plex™COOH-microspheres (Bio-Rad, Hercules, CA, USA) with the Bio-Plex™amine coupling kit (Bio-Rad) according to manufacturer’s proto- col. After optimization to maximize the signal/noise ratio, the quantity of HMGCR protein per coupling reaction was set at 10 μg. After coupling, coated beads were either used immediately or stored at −20°C in the dark. When indicated, a human recombinant SRP or intrinsic factor protein (Diarect AG, Freiburg, Germany) was used in place of HMGCR for controls. All patients' sera were initially assayed at a 1/500 dilu- tion. The anti-HMGCR Ab levels were determined at a 1/D dilution using the following formula: (MFIserum/ MFIcalibrator) × (level of calibrator) × D/500. The cali- brator is a human highly positive anti-HMGCR serum (the same throughout the study) whose level was arbitrarily set to 100 arbitrary units (AU)/mL). When sample MFI at 1/500 dilution was higher than 80% of calibrator MFI, further dilutions were per- formed and the first dilution yielding a MFI inferior to 80% of calibrator MFI was retained for calculation of level. Immediately prior to use, HMGCR-coated beads were vigorously agitated for 30 s. Then, a 10 μl volume con- taining 1,250 beads was added to 100 μl of serum from patients or controls (diluted in Dulbecco's phosphate- buffered saline (DPBS) plus 1% fetal bovine serum) in Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 4 of 11 characteristic (ROC) curve was determined using Prism software (GraphPad Software, La Jolla, CA, USA). Reproducibility of ALBIA-HMGCR was determined using three independent anti-HMGCR Ab positive sera (low, medium and high level). Intra-assay variation was determined by measurements of the same samples within the same run. Sensitivity and specificity of ALBIA-HMGCR Sensitivity and specificity of ALBIA-HMGCR To allow quantitative analysis of anti-HMGCR aAbs in patients, we developed an addressable laser bead im- munoassay (ALBIA-HMGCR) [16,23]. For this, we used as antigen the recombinant catalytic C-domain of the human HMGCR protein fused to GST. Purity of this protein was confirmed by Coomassie blue staining after SDS-PAGE, revealing a unique band (Figure 1A) that was specifically recognized by rabbit anti-HMGCR Abs or a human anti-HMGCR positive serum in Western blot (Figure 1B). Addressable laser bead immunoassay (ALBIA) for the detection and quantification of anti-HMGCR aAbs (ALBIA-HMCGR) Inter-assay variation (assay to assay) was determined by measurements of the same samples in separate runs. Inter-assay variation (batch to batch) was determined by measurement of the same samples in the same run using different batches of coupled beads. Coefficients of variation (CV) were computed as SD/mean. Determination of anti-HMGCR aAb levels The method used for calculating anti-HMGCR levels is illustrated in an additional figure (see Additional file 1). The serum used in this example showed a saturating sig- nal at the 1/500 screening dilution. A further 1/4,000 di- lution was retained to calculate the level by reference to Multiplex ALBIA for the simultaneous detection and quantification of anti-HMGCR and anti-SRP aAbs in NAM patients (ALBIA-NAM) Addition of free homologous protein, that is HMGCR, resulted in a dose-dependent inhibition of the HMGCR signal (Figure 2B) while no inhibition was observed when using a heterologous protein, that is SRP or intrin- sic factor, at the concentration of 100 μg/mL (Figure 2C). To further confirm the antigenic specificity of the assay, five sera that scored positive for anti-HMGCR aAbs using ALBIA-HMGCR were used to immunoprecipitate the recombinant HMGCR protein. This yielded a posi- tive result in 5/5 cases but not when using an irrelevant serum (Figure 2D). Indirect immunofluorescence was performed on HEp- 2000™cells (ImmunoConcepts, Sacramento, CA, USA) and Kallestad HEp-2 cells (Bio-Rad). Sera were tested at 1/80 screening dilution in PBS buffer, using a FITC- coupled antibody against human IgG. Reproducibility and repeatability of the ALBIA-HMGCR Reproducibility and repeatability of the ALBIA HMGCR Reproducibility and repeatability of the test were deter- mined by calculating both intra- and inter-assay variation for sera with high, medium and low anti-HMGCR levels. The intra-assay coefficients of variation (CV) ranged from 5% for high/medium levels to 13% for low levels, as indi- cated in an additional table (see Additional file 2). Statistical analysis HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. a highly positive anti-HMGCR serum used as calibrator whose level was arbitrarily set to 100 AU/mL. Similarly, the mean CV for inter-assay variation, as deter- mined from plate-to-plate or batch-to-batch variations ranged from 9 to 11%, and 2.8 to 5.6%, respectively. To- gether, these data indicate a good reproducibility and re- peatability of the ALBIA-HMGCR assay. Statistical analysis The normality of data distribution was analyzed using the D'Agostino and Pearson test, and the receiver operating Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 5 of 11 Figure 1 Development of a quantitative assay for the detection of anti-HMGCR antibodies. (A) SDS-PAGE analysis of the recombinant HMGCR catalytic C-domain after Coomassie blue staining. (B) Western blot analysis of this recombinant HMGCR using a specific rabbit anti-HMGCR antibody or a human anti-HMGCR-positive serum. (C) Addressable laser bead immunoassay (ALBIA-HMGCR) using serial dilutions of rabbit anti-HMGCR antibody. The mean fluorescence intensity (MFI) values are the mean of triplicate determinations. Standard deviation (SD) errors bars are not visible because of very low variability (coefficients of variation SD/mean lower than 1%, range (0.0025 to 0.7525)). Insert, higher magnification for the low anti-HMGCR antibody concentrations (horizontal dotted line depicts mean + 2 SD of 20 negative controls). HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Figure 1 Development of a quantitative assay for the detection of anti-HMGCR antibodies. (A) SDS-PAGE analysis of the recombinant HMGCR catalytic C-domain after Coomassie blue staining. (B) Western blot analysis of this recombinant HMGCR using a specific rabbit anti-HMGCR antibody or a human anti-HMGCR-positive serum. (C) Addressable laser bead immunoassay (ALBIA-HMGCR) using serial dilutions of rabbit anti-HMGCR antibody. The mean fluorescence intensity (MFI) values are the mean of triplicate determinations. Standard deviation (SD) errors bars are not visible because of very low variability (coefficients of variation SD/mean lower than 1%, range (0.0025 to 0.7525)). Insert, higher magnification for the low anti-HMGCR antibody concentrations (horizontal dotted line depicts mean + 2 SD of 20 negative controls). HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase Figure 1 Development of a quantitative assay for the detection of anti-HMGCR antibodies. (A) SDS-PAGE analysis of the recombinant HMGCR catalytic C-domain after Coomassie blue staining. (B) Western blot analysis of this recombinant HMGCR using a specific rabbit anti-HMGCR antibody or a human anti-HMGCR-positive serum. (C) Addressable laser bead immunoassay (ALBIA-HMGCR) using serial dilutions of rabbit anti-HMGCR antibody. The mean fluorescence intensity (MFI) values are the mean of triplicate determinations. Standard deviation (SD) errors bars are not visible because of very low variability (coefficients of variation SD/mean lower than 1%, range (0.0025 to 0.7525)). Insert, higher magnification for the low anti-HMGCR antibody concentrations (horizontal dotted line depicts mean + 2 SD of 20 negative controls). Validation of ALBIA-HMGCR We used ALBIA-HMGCR to screen serum samples for the presence of anti-HMGCR aAbs. First, a threshold of positivity was calculated using 100 control sera from Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 6 of 11 Figure 2 Specificity of HMGCR-coated beads. (A) HMGCR, signal recognition particle (SRP) and intrinsic factor-specific beads were coupled to the corresponding recombinant protein and analyzed by ALBIA using a human anti-HMGCR, anti-SRP or anti-intrinsic factor positive serum. (B) Dose-dependent inhibition of human anti-HMGCR antibodies binding to HMGCR-coated beads by free human recombinant HMGCR protein. Percent inhibition is given relative to the mean fluorescence intensity (MFI) value in the absence of free HMGCR. (C) Specific inhibition by homologous HMGCR but not heterologous proteins. Percent inhibition of three different human anti-HMGCR positive sera (referred to as #1, #2 and #3) by 100 μg/mL of free HMGCR, SRP or intrinsic factor proteins. (D) Immunoprecipitation of HMGCR protein by ALBIA-HMGCR positive or negative human sera. A rabbit anti-HMGCR polyclonal antibody was used as positive control. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Figure 2 Specificity of HMGCR-coated beads. (A) HMGCR, signal recognition particle (SRP) and intrinsic factor-specific beads were coupled to the corresponding recombinant protein and analyzed by ALBIA using a human anti-HMGCR, anti-SRP or anti-intrinsic factor positive serum. (B) Dose-dependent inhibition of human anti-HMGCR antibodies binding to HMGCR-coated beads by free human recombinant HMGCR protein. Percent inhibition is given relative to the mean fluorescence intensity (MFI) value in the absence of free HMGCR. (C) Specific inhibition by homologous HMGCR but not heterologous proteins. Percent inhibition of three different human anti-HMGCR positive sera (referred to as #1, #2 and #3) by 100 μg/mL of free HMGCR, SRP or intrinsic factor proteins. (D) Immunoprecipitation of HMGCR protein by ALBIA-HMGCR positive or negative human sera. A rabbit anti-HMGCR polyclonal antibody was used as positive control. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. minority (3% or less) of HEp-2000® cells (Figure 4). This immunoreactivity could be inhibited by previous incuba- tion with free HMGCR. Using Kallestad HEp-2 cells, only 30% of the ALBIA-HMGCR positive sera yielded this aspect (not shown). healthy blood donors. The values obtained with control sera did not follow a normal distribution (P <0.0001), their median was 0 AU/mL and the 99th percentile was 14 AU/mL. At a cutoff of 14 AU/mL, 99/100 control sera scored negative (Figure 3A). IgG isotypes of anti-HMGCR Abs IgG isotypes of anti-HMGCR Abs Fifteen ALBIA-HMGCR positive sera were tested for anti- HMGCR IgG subclasses. Only IgG1 were represented among anti-HMGCR aAbs in this series (not shown). IgG2, IgG3 or IgG4 subclasses were never found, neither as a single isotype nor in association with IgG1. Exploring necrotizing autoimmune myopathies with multiplex ALBIA-NAM In order to simultaneously quantify anti-HMGCR and anti-SRP aAbs, we next developed a multiplex assay (ALBIA-NAM) and compared its results to that of monoplex ALBIA-HMGCR and ALBIA-SRP [16]. For this, we assayed 26 anti-HMGCR positive patients from the present series plus 25 anti-SRP positive sera that were described previously [16]. Results from both assays were compared by linear regression and showed excel- lent correlation, with R values of 0.9942 and 0.9937 for Validation of ALBIA-HMGCR g g Next, we analyzed sera from 150 patients with suspi- cion of NAM. Of these, 37 patients (24%) were anti- HMGCR positive, with levels ranging from 24 to 2.656 AU/mL. ROC curve analysis revealed a specifi- city of 100% and a sensitivity of 100% when the cutoff was set at 20 AU/ml (Figure 3B). Patients with differ- ent inflammatory/autoimmune conditions including RA, systemic sclerosis, SLE, DM, anti-tRNA synthetase Ab positive myositis or IBM, as well as patients with polyclonal hypergammaglobulinemia all scored negative (Figure 3A). Anti-HMGCR positive and 16 negative sera from healthy donors were further tested by a recently de- veloped line immunoassay. All 37 ALBIA-HMGCR posi- tive sera were positive using this assay while 16/16 controls were negative (not shown). Yet, there was no correlation between band intensity and ALBIA-HMGCR level. Immunofluorescence aspect on HEp-2 cells Figure 3 Validation of ALBIA-HMGCR and determination of anti-HMGCR levels in patients with suspicion of NAM th Figure 3 Validation of ALBIA-HMGCR and determination of anti-HMGCR levels in patients with suspicion of NAM. (A) A positivity cutoff of the assay was first determined as the 99th percentile (14 arbitrary units (AU)/mL) of the healthy donors’ distribution (open circles) and depicted by a dotted line. Sera from patients with different inflammatory/autoimmune conditions including rheumatoid arthritis (RA), systemic sclerosis (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), anti-tRNA synthetase-positive myositis or inclusion body myositis (IBM), as well as patients with polyclonal hypergammaglobulinemia were assayed and revealed all negative. Insert is a magnification for low values. Thirty-seven out of 150 (24%) of serum samples from patients with suspicion of NAM scored positive using ALBIA-HMGCR. (B) Receiver operating characteristic (ROC) analysis was performed by comparing the 37 anti-HMGCR positive serum samples to those from healthy donors. Optimal results were obtained with a cutoff of 20 AU/mL, which was indicated as a plain line. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; NAM, necrotizing autoimmune myopathies. ALBIA-HMGCR versus ALBIA-NAM (Figure 5A) and ALBIA-SRP versus ALBIA-NAM (Figure 5B), respectively. ALBIA-NAM revealed able to perfectly discriminate the two populations of NAM patients, that is those with anti-HMGCR from those with anti-SRP aAbs (Figure 5C) and was negative for all other tested conditions, that is patients with different inflammatory/autoimmune condi- tions, DM, anti-tRNA synthetase Ab positive myositis or IBM, as well as patients with polyclonal hypergammaglo- bulinemia (see Additional file 3). The sensitivity of mono- plex and multiplex assays was equivalent. No NAM patient was positive for both aAbs. muscle weakness (92%), had elevated (mean >6,000 IU/L) CK levels and 60% had not taken statins. Muscle biopsies always showed regenerating and necrotic muscle fi- bers, with occasionally (28%) perivascular inflamma- tory infiltrates. Immunofluorescence aspect on HEp-2 cells Sixty-one percent (20/33) of ALBIA-HMGCR positive sera displayed a finely granular cytoplasmic pattern on a Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 7 of 11 Figure 3 Validation of ALBIA-HMGCR and determination of anti-HMGCR levels in patients with suspicion of NAM. (A) A positivity cutoff of the assay was first determined as the 99th percentile (14 arbitrary units (AU)/mL) of the healthy donors’ distribution (open circles) and depicted by a dotted line. Sera from patients with different inflammatory/autoimmune conditions including rheumatoid arthritis (RA), systemic sclerosis (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), anti-tRNA synthetase-positive myositis or inclusion body myositis (IBM), as well as patients with polyclonal hypergammaglobulinemia were assayed and revealed all negative. Insert is a magnification for low values. Thirty-seven out of 150 (24%) of serum samples from patients with suspicion of NAM scored positive using ALBIA-HMGCR. (B) Receiver operating characteristic (ROC) analysis was performed by comparing the 37 anti-HMGCR positive serum samples to those from healthy donors. Optimal results were obtained with a cutoff of 20 AU/mL, which was indicated as a plain line. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; NAM, necrotizing autoimmune myopathies. ion of ALBIA-HMGCR and determination of anti-HMGCR levels in patients with suspicion of NAM. (A) A positivity cut h Figure 3 Validation of ALBIA-HMGCR and determination of anti-HMGCR levels in patients with suspicion of NAM. (A) A positivity cutoff of the assay was first determined as the 99th percentile (14 arbitrary units (AU)/mL) of the healthy donors’ distribution (open circles) and depicted by a dotted line. Sera from patients with different inflammatory/autoimmune conditions including rheumatoid arthritis (RA), systemic sclerosis (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), anti-tRNA synthetase-positive myositis or inclusion body myositis (IBM), as well as patients with polyclonal hypergammaglobulinemia were assayed and revealed all negative. Insert is a magnification for low values. Thirty-seven out of 150 (24%) of serum samples from patients with suspicion of NAM scored positive using ALBIA-HMGCR. (B) Receiver operating characteristic (ROC) analysis was performed by comparing the 37 anti-HMGCR positive serum samples to those from healthy donors. Optimal results were obtained with a cutoff of 20 AU/mL, which was indicated as a plain line. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; NAM, necrotizing autoimmune myopathies. Discussion This report describes the first immunoassay which can detect and quantify simultaneously anti-HMGCR and anti-SRP aAbs in patients with NAM, a recently identified, severe form of inflammatory myopathy with important muscle necrosis/regeneration and little inflammation. Up to recently, the first aAb sought for in NAM patients was directed against SRP, a protein complex that guides the translocation of growing polypeptides into the endoplas- mic reticulum during protein synthesis. Currently, the Characteristics of anti-HMGCR positive patients Characteristics of anti-HMGCR positive patients are sum- marized in Table 1. These patients presented with proximal Characteristics of anti-HMGCR positive patients Table 1 Clinical characteristics of anti-HMGCR positive patients (n = 37) Age (mean ± SD): 44 ± 19 years Sex (M/F): 12/25 Muscle weakness: 34/37 (92%) Creatine kinase level (mean ± SD): 6,974 ± 4,970 IU/L Statin exposure: 15/37 (40%) HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; SD, standard deviation. Table 1 Clinical characteristics of anti-HMGCR positive patients (n = 37) correlation between the degree of myolysis and the level of anti-SRP aAbs, suggesting that these aAbs may be pathogenic. In the present study, we applied a similar strategy to provide a new immunoassay for anti-HMGCR aAbs, and also validated a multiplex immunoassay to de- tect and quantify both anti-HMGCR and anti-SRP aAbs using a single biological test. Whereas it cannot be excluded that some other epi- topes might exist, a mapping study showed that anti- HMGCR aAbs recognize the catalytic C-domain of HMGCR, but do not react with the N-terminal domain of the protein [15]. Hence, beads were coated with a re- combinant HMGCR C-domain protein. Rabbit anti- HMGCR Abs that were produced after immunization with a polypeptide corresponding to residues 550 to 650 of the human HMGCR C-domain could recognize the recombinant protein used herein for performing Western blot (Figure 1B) and ALBIA-HMGCR (Figure 1C). These results demonstrate the accessibility of recombinant HMGCR C-domain epitopes to anti-HMGCR Abs. The reactivity of the human anti-HMGCR positive serum against some smaller bands (approximately 40 to 45 kDa) in the Western blot (Figure 1B) presumably indicates that the recombinant HMGCR antigen has some breakdown products that are not recognized by the rabbit serum that was obtained by immunization with only a fragment of HMCGR C-domain. ALBIA-HMGCR detected anti-HMGCR aAbs in 37 individuals (24%) among a series of 150 patients with suspicion of NAM (Figure 3A). A perfect concordance with a recently developed line immunoassay was found. Yet, there was no correlation between band intensity in this assay and ALBIA-HMGCR level. Further, ALBIA- HMGCR remained negative when tested against sera from patients with different inflammatory/autoimmune conditions including RA, systemic sclerosis, SLE, DM, anti-tRNA synthetase-positive myositis or IBM, or in pa- tients with polyclonal hypergammaglobulinemia (Figure 3A). ROC analysis revealed a cutoff for positivity of 20 AU/mL, providing a high diagnostic value (Figure 3B). Figure 5 Comparison of monoplex ALBIA-HMGCR and ALBIA-SRP to multiplex ALBIA-NAM. Serum from anti-HMGCR positive (n = 26) or anti-SRP positive (n = 25) patients were compared. Characteristics of anti-HMGCR positive patients p p Characteristics of anti-HMGCR positive patients are sum- marized in Table 1. These patients presented with proximal Figure 4 Aspect of anti-HMGCR autoantibodies in indirect immunofluorescence on HEp-2 cells. Classical indirect immunofluorescence assay using the HEp-2000™line as cellular substrate. After incubation of serum at a 1/80 dilution, immunoreactivity was revealed by a FITC-labeled anti-human immunoglobulin (Ig)G secondary antibody. Example from two representative anti-HMGCR positive patients out of 20/33 sera with this immunofluorescence pattern shows a finely granular cytoplasmic staining with perinuclear reinforcement (left and middle). Inhibition by free HMGCR protein before immunofluorescence (right): the serum from example 2 was pre-incubated with free HMCGR before immunofluorescence assay. This yielded an extinction of immunoreactivity. HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Figure 4 Aspect of anti-HMGCR autoantibodies in indirect immunofluorescence on HEp-2 cells. Classical indirect immunofluorescence assay using the HEp-2000™line as cellular substrate. After incubation of serum at a 1/80 dilution, immunoreactivity was revealed by a FITC-labeled anti-human immunoglobulin (Ig)G secondary antibody. Example from two representative anti-HMGCR positive patients out of 20/33 sera with this immunofluorescence pattern shows a finely granular cytoplasmic staining with perinuclear reinforcement (left and middle). Inhibition by free HMGCR protein before immunofluorescence (right): the serum from example 2 was pre-incubated with free HMCGR before immunofluorescence assay. This yielded an extinction of immunoreactivity. HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 8 of 11 Figure 5 Comparison of monoplex ALBIA-HMGCR and ALBIA-SRP to multiplex ALBIA-NAM. Serum from anti-HMGCR positive (n = 26) or anti-SRP positive (n = 25) patients were compared. (A) Correlation of the signals generated by ALBIA-NAM versus ALBIA-HMGCR. Mean fluorescence intensity (MFI) (B) Correlation of the signals generated by ALBIA-NAM versus ALBIA-SRP. (C) ALBIA-NAM discriminates anti-SRP positive from anti-HMGCR positive patients in a single assay. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; SRP, signal recognition particle. Table 1 Clinical characteristics of anti-HMGCR positive patients (n = 37) Age (mean ± SD): 44 ± 19 years Sex (M/F): 12/25 Muscle weakness: 34/37 (92%) Creatine kinase level (mean ± SD): 6,974 ± 4,970 IU/L Statin exposure: 15/37 (40%) HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; SD, standard deviation. Characteristics of anti-HMGCR positive patients (A) Correlation of the signals generated by ALBIA-NAM versus ALBIA-HMGCR. Mean fluorescence intensity (MFI) (B) Correlation of the signals generated by ALBIA-NAM versus ALBIA-SRP. (C) ALBIA-NAM discriminates anti-SRP positive from anti-HMGCR positive patients in a single assay. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; SRP, signal recognition particle. Figure 5 Comparison of monoplex ALBIA-HMGCR and ALBIA-SRP to multiplex ALBIA-NAM. Serum from anti-HMGCR positive (n = 26) or anti-SRP positive (n = 25) patients were compared. (A) Correlation of the signals generated by ALBIA-NAM versus ALBIA-HMGCR. Mean fluorescence intensity (MFI) (B) Correlation of the signals generated by ALBIA-NAM versus ALBIA-SRP. (C) ALBIA-NAM discriminates anti-SRP positive from anti-HMGCR positive patients in a single assay. ALBIA, addressable laser bead immunoassay; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; SRP, signal recognition particle. detection of anti-SRP aAbs may be suspected upon result of an indirect immunofluorescence test and the diagnosis must be confirmed by a specific assay, for example dot- blot. Recently, we described a new immunoassay, that is ALBIA-SRP, to quantify anti-SRP aAbs and investigated the relationship between serum CK levels, muscle strength and anti-SRP levels [16]. Results revealed a significant Anti-HMGCR IgG subclass distribution, which had remained unknown until now, was determined. Contrary to what was observed for anti-SRP positive patients in whom IgG1 and IgG4 were principally observed while Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Page 9 of 11 Together, the results of this study show that multiplex ALBIA-NAM allows efficient detection and quantifica- tion of anti-HMGCR or anti-SRP aAbs in patients with suspicion of NAM and that the diagnosis of anti- HMGCR positive NAM should not be excluded a priori in patients who have not been exposed to statins. all four subclasses could possibly be found depending on the patient [16], or to other autoimmune diseases such as myasthenia gravis, membranous glomerulonephritis or pemphigus, in which both IgG1 and IgG4 isotypes have been found [24-26], the only subclass detected in anti-HMGCR positive patients was IgG1 in the present study. all four subclasses could possibly be found depending on the patient [16], or to other autoimmune diseases such as myasthenia gravis, membranous glomerulonephritis or pemphigus, in which both IgG1 and IgG4 isotypes have been found [24-26], the only subclass detected in anti-HMGCR positive patients was IgG1 in the present study. Conclusions Diagnosis and clinical studies of necrotizing auto- immune myopathies (NAM) have been hampered by the paucity of biological assays for the assessment of specific biomarkers. Some NAM patients have autoantibodies (aAbs) against the statins drug target, that is 3-hydroxy- 3-methyl-glutaryl-coenzyme A reductase (HMGCR). Here, we developed a new immunoassay allowing efficient detec- tion and quantification of anti-HMGCR aAbs in patients with suspicion of NAM. In a series of 150 patients, 24% were found positive for anti-HMGCR aAbs. Only 40% of anti-HMGCR-positive patients had been exposed to statins. Hence, lack of statin exposure should not eliminate the diag- nosis of anti-HMGCR-positive NAM. This assay was multi- plexed with an anti-signal recognition particle (SRP) aAb immunoassay, facilitating the diagnosis of NAM using a sin- gle assay. Christopher-Stine et al. found a 7% prevalence of anti- HMGCR aAbs (as initially determined by a 100 to 200 kDa reactivity after immunoprecipitation) within a series of 225 patients with proximal muscle weakness, elevated CK, myopathic findings on electromyography, muscle edema on magnetic resonance imaging and/or features of myopathy on muscle biopsy [14]. This preva- lence was higher in a subgroup of 38 patients with nec- rotizing myopathy among whom 16 (42%) were found positive for anti-HMGCR aAbs. Using indirect immunofluorescence, some anti-HMGCR positive sera exhibited a finely granular cytoplasmic pattern with perinuclear reinforcement in a minority of cells. Nec- rotizing myopathies may also encompass some forms of muscular dystrophy. Together, the present assays will be helpful for the diagnosis of necrotizing myopathies since a positive result allows ascribing patients to an autoimmune form. In a general population of statin users, with or without self-limited musculoskeletal side effects, the prevalence of anti-HMGCR aAbs is extremely low [27]. In contrast, Mammen et al. reported that 67% of anti-HMGCR posi- tive NAM patients had been exposed to statins [14,15]. Here, we found that 40% of our anti-HMGCR positive patients had taken statins. Therefore, although anti- HMGCR aAbs are directed against the statins target it- self, the diagnosis of anti-HMGCR positive NAM should not be excluded a priori in patients who have not been exposed to statins. Characteristics of anti-HMGCR positive patients Using immunofluoresence on HEp-2 cells, 30 to 61% of anti-HMGCR positive sera exhibited a finely granular cytoplasmic pattern with perinuclear reinforcement in a minority of cells (Figure 4). Yet, the cellular substrates used for immunofluorescence appeared heterogeneous in terms of HMGCR expression levels and some com- mercial sources may provide HEp-2 cells less prone to allow detecting anti-HMGCR aAbs than others. Additional files Bronner IM, Hoogendijk JE, Wintzen AR, van der Meulen MF, Linssen WH, Wokke JH, de Visser M: Necrotising myopathy, an unusual presentation of a steroid-responsive myopathy. J Neurol 2003, 250:480–485. 2. Bronner IM, Hoogendijk JE, Wintzen AR, van der Meulen MF, Linssen WH, Wokke JH, de Visser M: Necrotising myopathy, an unusual presentation of a steroid-responsive myopathy. J Neurol 2003, 250:480–485. (IBM), as well as patients with polyclonal hypergammaglobulinemia were assayed. aAbs, autoantibodies; ALBIA, addressable laser bead immunoassay; AU, arbitrary units; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; NAM, necrotizing autoimmune myopathies; SRP, signal recognition particle. 3. van der Meulen MF, Bronner IM, Hoogendijk JE, Burger H, van Venrooij WJ, Voskuyl AE, Dinant HJ, Linssen WH, Wokke JH, de Visser M: Polymyositis: an overdiagnosed entity. Neurology 2003, 61:316–321. Authors’ contributions LD, OBe, OBo and FJ designed the study. LD and OBo developed the immunoassays. LD, FJ, YA, JLC, JM, LM, OBe, AM, OH, BBM, IKP, ECS, BE, AT, JS and IM participated to acquisition and interpretation of data. LD, OBe, OBo, FJ, YA, JLC and LM analyzed and interpreted the data. LD and OBo wrote the first draft of the manuscript. OBo 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. All authors read and approved the final manuscript. y 8. Hengstman GJ, ter Laak HJ, Vree Egberts WT, Lundberg IE, Moutsopoulos HM, Vencovsky J, Doria A, Mosca M, van Venrooij WJ, van Engelen BG: Anti-signal recognition particle autoantibodies: marker of a necrotising myopathy. Ann Rheum Dis 2006, 65:1635–1638. y 8. Hengstman GJ, ter Laak HJ, Vree Egberts WT, Lundberg IE, Moutsopoulos HM, Vencovsky J, Doria A, Mosca M, van Venrooij WJ, van Engelen BG: Anti-signal recognition particle autoantibodies: marker of a necrotising myopathy. Ann Rheum Dis 2006, 65:1635–1638. 9. Targoff IN, Johnson AE, Miller FW: Antibody to signal recognition particle in polymyositis. Arthritis Rheum 1990, 33:1361–1370. 9. Targoff IN, Johnson AE, Miller FW: Antibody to signal recognition particle in polymyositis. Arthritis Rheum 1990, 33:1361–1370. 10. Troyanov Y, Targoff IN, Tremblay JL, Goulet JR, Raymond Y, Senecal JL: Novel classification of idiopathic inflammatory myopathies based on overlap syndrome features and autoantibodies: analysis of 100 French Canadian patients. Medicine (Baltimore) 2005, 84:231–249. Collaborators (French Myositis Network) 12. Miller T, Al-Lozi MT, Lopate G, Pestronk A: Myopathy with antibodies to the signal recognition particle: clinical and pathological features. J Neurol Neurosurg Psychiatry 2002, 73:420–428. ( y ) Pierre Kieffer, Tanya Stojkovic, Alexandra Nadaj-Pakleza, Bénédicte Giroux-Leprieur, Claire Laroche, Pascal Laforet, Anthony Béhin, Patrice Cacoub, Mamy Ralazamahaleo, Xavier Ferrer, Marielle Roux, Anne-Laure Bedat-Millet, Jean-Michel Ristori, Anne-Sophie Deleplancque, Julie Cosserat, Aude Rigolet, Laurent Arnaud, Laurent Servais, Chafika Morati, Nathalie Costedoat, Olivier Fain, Bernard Grosbois, Raphael De Paz, Noémie Le Gouellec, Guillaume Gondran, François Maurier, François-Jérôme Authier, Romain Ghérardi, Dimitri Renard, Antoine Laudat, Cédric Ménard, Isabelle Bahon, Claude Lambert, Léonard Feasson, Jean-Claude Antoine, Alfred Mahr. 13. Needham M, Fabian V, Knezevic W, Panegyres P, Zilko P, Mastaglia FL: Progressive myopathy with up-regulation of MHC-I associated with statin therapy. Neuromuscul Disord 2007, 17:194–200. 14. Christopher-Stine L, Casciola-Rosen LA, Hong G, Chung T, Corse AM, Mammen AL: A novel autoantibody recognizing 200-kd and 100-kd proteins is associated with an immune-mediated necrotizing myopathy. Arthritis Rheum 2010, 62:2757–2766. 15. Mammen AL, Chung T, Christopher-Stine L, Rosen P, Rosen A, Doering KR, Casciola-Rosen LA: Autoantibodies against 3-hydroxy-3-methylglutaryl- coenzyme A reductase in patients with statin-associated autoimmune myopathy. Arthritis Rheum 2011, 63:713–721. Acknowledgments We are indebted to Andrew Mammen for critical reading of the manuscript and helpful discussions, and would like to thank Lucille Michel and Florence Voisin for their technical assistance. This work supported in part by a grant from the Association Française contre les Myopathies. 11. Kao AH, Lacomis D, Lucas M, Fertig N, Oddis CV: Anti-signal recognition particle autoantibody in patients with and patients without idiopathic inflammatory myopathy. Arthritis Rheum 2004, 50:209–215. Abbreviations 4. Sadeh M, Dabby R: Steroid-responsive myopathy: immune-mediated necrotizing myopathy or polymyositis without inflammation? J Clin Neuromuscul Dis 2008, 9:341–344. 4. Sadeh M, Dabby R: Steroid-responsive myopathy: immune-mediated necrotizing myopathy or polymyositis without inflammation? J Clin Neuromuscul Dis 2008, 9:341–344. aAbs: autoantibodies; ALBIA: addressable laser bead immunoassay; AU: arbitrary units; CK: creatine kinase; CV: coefficient of variation; DM: dermatomyositis; DPBS: Dulbecco's phosphate-buffered saline; GST: glutathione S-transferase; HMGCR: 3-hydroxy-3-methylglutaryl coenzyme A reductase; IBM: inclusion body myositis; Ig: immunoglobulin; MFI: mean fluorescence intensity; NAM: necrotizing autoimmune myopathies; RA: rheumatoid arthritis; ROC: receiver operating characteristic; SD: standard deviation; SLE: systemic lupus erythematosus; SRP: signal recognition particle. 5. Emslie-Smith AM, Engel AG: Necrotizing myopathy with pipestem capillaries, microvascular deposition of the complement membrane attack complex (MAC), and minimal cellular infiltration. Neurology 1991, 41:936–939. 5. Emslie-Smith AM, Engel AG: Necrotizing myopathy with pipestem capillaries, microvascular deposition of the complement membrane attack complex (MAC), and minimal cellular infiltration. Neurology 1991, 41:936–939. 6. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, Vencovsky J, de Visser M, Hughes RA: 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands. Neuromuscul Disord 2004, 14:337–345. 6. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, Vencovsky J, de Visser M, Hughes RA: 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands. Neuromuscul Disord 2004, 14:337–345. 6. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, Vencovsky J, de Visser M, Hughes RA: 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands. Neuromuscul Disord 2004, 14:337–345. Author details 1 Dalakas MC, Hohlfeld R: Polymyositis and dermatomyositis. Lancet 2003, 362:971–982. 20. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, Daniels TE, Fox PC, Fox RI, Kassan SS, Pillemer SR, Talal N, Weisman MH: Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002, 61:554–558. Reference 1. Dalakas MC, Hohlfeld R: Polymyositis and dermatomyositis. Lancet 2003, 362:971–982. Author details 1 1Inserm, U905 & Normandie Univ, IRIB, 22 Bd Gambetta, F-76000 Rouen, France. 2Pierre and Marie Curie University & Inserm, U974 & Assistance Publique - Hôpitaux de Paris, Pitié-Salpêtrière University Hospital, Department of Internal Medicine, Paris, France. 3Rouen University Hospital, Department of Immunology, Rouen, France. 4Assistance Publique - Hôpitaux de Paris, Pitié-- Salpêtrière University Hospital, Laboratory of Immunochemistry & Auto- immunity, Paris, France. 5Department of Physiology, Nouvel Hôpital civil, Strasbourg, France. 6Department of Internal Medecine, Mulhouse Hospital, Mulhouse, France. 7Inserm, U768, Paris, France. 8Department of Immunology and Hematology, Assistance Publique - Hôpitaux de Paris, Necker University Hospital, Paris, France. 9Assistance Publique - Hôpitaux de Paris, Bicêtre University Hospital, Department of Pediatrics, Le Kremlin-Bicêtre, France. 10Assistance Publique - Hôpitaux de Marseille, La Timone University Hospital, Marseille, France. 11Assistance Publique - Hôpitaux de Paris, Pitié-Salpêtrière University Hospital, Institute of Myology, Paris, France. 12Department of Rheumatology, Clermont-Ferrand University Hospital, Clermont-Ferrand, France. 13Rouen University Hospital, Department of Internal Medicine, Rouen, France. 16. Benveniste O, Drouot L, Jouen F, Charuel JL, Bloch-Queyrat C, Behin A, Amoura Z, Marie I, Guiguet M, Eymard B, Gilbert D, Tron F, Herson S, Musset L, Boyer O: Correlation of anti-signal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy. Arthritis Rheum 2011, 63:1961–1971. 17. Werner JL, Christopher-Stine L, Ghazarian SR, Pak KS, Kus JE, Daya NR, Lloyd TE, Mammen AL: Antibody levels correlate with creatine kinase levels and strength in anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase-associated autoimmune myopathy. Arthritis Rheum 2012, 64:4087–4093. 18. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ: The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982, 25:1271–1277. 19. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315–324. Received: 23 July 2013 Accepted: 20 January 2014 Published: 3 February 2014 20. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, Daniels TE, Fox PC, Fox RI, Kassan SS, Pillemer SR, Talal N, Weisman MH: Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002, 61:554–558. Reference 1. Additional files Additional file 1: Determination of the level of anti-HMGCR aAbs. Anti-HMGCR levels were determined by reference to the MFI value, in the same assay, of a calibrator that is, a highly positive anti-HMGCR + serum whose level was arbitrarily set to 100 AU/mL. The assay was first performed using a 1/500 screening dilution of the serum. In this example, the sample’s MFI at a 1/500 dilution was higher than 80% of the calibrator’s MFI. Thus, further dilutions were performed and the first dilution yielding a MFI inferior to 80% of the calibrator MFI was retained for calculation, yielding a 428 AU/mL anti-HMGCR level. aAbs, autoantibodies; AU, arbitrary units; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; MFI, mean fluorescence intensity. An advantage of the ALBIA methodology is to allow the concomitant detection of several aAbs in a single assay from a unique serum sample, therefore reducing number of biological tests, labor and time necessary between diag- nosis and therapy. In this study, we multiplexed ALBIA- HMGCR and ALBIA-SRP to provide the multiplex ALBIA-NAM. The performance of ALBIA-NAM was compared to that of the parental monoplex immunoassays. Linear regression analysis showed an excellent correlation between monoplex and multiplex assays (Figure 5A,B). None of the sera found positive by monoplex assays scored negative when using ALBIA-NAM and vice versa. Further- more, all anti-HMGCR positive sera were negative for anti-SRP aAbs, suggesting that patients with anti-SRP or anti-HMGCR aAb might belong to different NAM sub- groups (Figure 5C). Yet, a fraction of NAM patients re- mains without any known aAb, suggesting that new auto-antigenic specificities are still to be discovered. Additional file 2: Reproducibility of ALBIA-HMGCR. Intra- and inter- assay reproducibility as determined by the coefficients of variation (CV %) for repeated measures of high, medium and low anti-HMGCR level samples. ALBIA, addressable laser bead immunoassay; HMGCR, 3- hydroxy-3-methylglutaryl-coenzyme A reductase. Additional file 3: Validation of ALBIA-NAM. A positivity cutoff of the multiplex assay (dotted line) was determined as the 99th percentile of the healthy donors’ distribution (open circles) for both (A) anti-SRP (9 AU/mL) and (B) anti-HMGCR aAbs (8 AU/mL). Sera from patients with different inflammatory/autoimmune conditions including rheumatoid arthritis (RA), systemic sclerosis (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), anti-tRNA synthetase aAb-positive myositis or inclusion body myositis Page 10 of 11 Page 10 of 11 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 2. Competing interests The authors declare that they have no competing interests. 7. Brouwer R, Hengstman GJ, Vree Egberts W, Ehrfeld H, Bozic B, Ghirardello A, Grondal G, Hietarinta M, Isenberg D, Kalden JR, Lundberg I, Moutsopoulos H, Roux-Lombard P, Vencovsky J, Wikman A, Seelig HP, van Engelen BG, van Venrooij WJ: Autoantibody profiles in the sera of European patients with myositis. Ann Rheum Dis 2001, 60:116–123. 7. Brouwer R, Hengstman GJ, Vree Egberts W, Ehrfeld H, Bozic B, Ghirardello A, Grondal G, Hietarinta M, Isenberg D, Kalden JR, Lundberg I, Moutsopoulos H, Roux-Lombard P, Vencovsky J, Wikman A, Seelig HP, van Engelen BG, van Venrooij WJ: Autoantibody profiles in the sera of European patients with myositis. Ann Rheum Dis 2001, 60:116–123. The authors declare that they have no competing interests. Received: 23 July 2013 Accepted: 20 January 2014 Published: 3 February 2014 Reference Page 11 of 11 doi:10.1186/ar4468 Cite this article as: Drouot et al.: Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies. Arthritis Research & Therapy 2014 16:R39. Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 Drouot et al. Arthritis Research & Therapy 2014, 16:R39 http://arthritis-research.com/content/16/1/R39 21. Bohan A, Peter JB: Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975, 292:344–347. 22. Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, Rowland LP: Inclusion body myositis and myopathies. Ann Neurol 1995, 38:705–713. 22. Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, Rowland LP: Inclusion body myositis and myopathies. Ann Neurol 1995, 38:705–713. 23. Fritzler MJ, Behmanesh F, Fritzler ML: Analysis of human sera that are polyreactive in an addressable laser bead immunoassay. Clin Immunol 2006, 120:349–356. 23. Fritzler MJ, Behmanesh F, Fritzler ML: Analysis of human sera that are polyreactive in an addressable laser bead immunoassay. Clin Immunol 2006, 120:349–356. 24. Makker SP, Tramontano A: Idiopathic membranous nephropathy: an autoimmune disease. Semin Nephrol 2011, 31:333–340. p , 25. Shigemoto K: Myasthenia gravis induced by autoantibodies against MuSK. Acta Myol 2007, 26:185–191. 26. Sitaru C, Mihai S, Zillikens D: The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases. Arch Dermatol Res 2007, 299:1–8. 26. Sitaru C, Mihai S, Zillikens D: The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases. Arch Dermatol Res 2007, 299:1–8. 27. Mammen AL, Pak K, Williams EK, Brisson D, Coresh J, Selvin E, Gaudet D: Rarity of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies in statin users, including those with self-limited musculoskeletal side effects. Arthritis Care Res (Hoboken) 2012, 64:269–272. doi:10.1186/ar4468 Cite this article as: Drouot et al.: Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies. Arthritis Research & Therapy 2014 16:R39. 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https://openalex.org/W4306847652	https://www.mdpi.com/2071-1050/14/20/13428/pdf?version=1666094386	English	null	Connecting Social Enterprises and Sustainable Consumption: Systematic Review, Bibliometric Analysis, and Conceptual Framework	Sustainability	2,022	cc-by	13,713	Citation: Salido-Andres, N.; Garcia-Rodriguez, N.; Cachero-Martinez, S. Connecting Social Enterprises and Sustainable Consumption: Systematic Review, Bibliometric Analysis, and Conceptual Framework. Sustainability 2022, 14, 13428. https://doi.org/10.3390/ su142013428 Keywords: social enterprises; sustainable consumption; systematic review; bibliometric analysis; conceptual framework; antecedents; product attributes; customer behavior sustainability sustainability sustainability sustainability Systematic Review Noelia Salido-Andres 1,* , Nuria Garcia-Rodriguez 2 and Silvia Cachero-Martinez 2 1 School of Economics and Business, University of A Coruña, 15071 A Coruña, Spain 2 School of Economics and Business, University of Oviedo, 33006 Oviedo, Spain * Correspondence: noelia.sandres@udc.es; Tel.: +34-881-014-402 1 School of Economics and Business, University of A Coruña, 15071 A Coruña, Spain 2 School of Economics and Business, University of Oviedo, 33006 Oviedo, Spain * Correspondence: noelia.sandres@udc.es; Tel.: +34-881-014-402 Abstract: The purpose of this study is to explore the meeting point between social enterprises (SEs) and sustainable consumption, given the proven potential of these hybrid organizations in the achievement of sustainable development. Paradoxically, scholarly attention has been scarce to this ﬁeld of research, particularly from the perspectives of SE products and (potential) customers. Aiming to shed some light, a systematic literature review was conducted, resulting in 24 scientiﬁc publications descriptively and thematically explored based on a bibliometric analysis. The ﬁndings show that the link between SEs and sustainable consumption is very recent and that empirical articles using quantitative methodologies prevail focused on the analysis of capabilities and performances of SEs aiming to positively inﬂuence customers’ response. Nevertheless, the attention to the identiﬁcation of product attributes and the individual determinants effective enough to press the buy button is still limited. In response to this shortcoming, the originality of this study consists of assembling the ﬁndings in this regard into an integrated conceptual framework that paves the way for future analysis in this ﬁeld of study. Systematic Review Connecting Social Enterprises and Sustainable Consumption: Systematic Review, Bibliometric Analysis, and Conceptual Framework res 1,* , Nuria Garcia-Rodriguez 2 and Silvia Cachero-Martinez 2 1. Introduction Current models of production and consumption are at the origin of multiple environ- mental, social, and economic challenges that stand in the way of achieving the Sustainable Development Goals (SDGs) included in the United Nations 2030 Agenda [1]. Given that sustainable development is a fundamental purpose for 21st century societies [2], different actors (civil society, policy makers, international organizations, and representatives of the business sectors, among others) have intensiﬁed the search for alternative approaches to conduct economic activities [3]. In this search for alternative approaches, social enterprises (SEs) have recently gained prominence. An SE is an “operator of the social economy whose main objective is to have a social impact rather than to make a proﬁt for its owners or shareholders. It operates by providing goods and services to the market in an entrepreneurial and innovative way and uses its proﬁts primarily to achieve social objectives. It is managed in an open and responsible manner and, in particular, involves employees, consumers and stakeholders affected by its commercial activities” [4] (p. 2). This deﬁnition highlights the hybrid character of these types of organizations in which the purpose is to achieve social value through market interaction. Therefore, the authors must distinguish these companies from non-proﬁt organizations (NPOs) and their social missions. The beneﬁciaries of SEs may be their clients, employees, partners, or owners, but they also orient value creation activities to have a positive impact on social well-being and on the environment as a whole. However, SEs are similar to commercial companies, since their main source of income comes from commercial or market activities, not from donations or subsidies [5]. Social cooperatives, Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sustainability Sustainability 2022, 14, 13428. https://doi.org/10.3390/su142013428 Sustainability 2022, 14, 13428 2 of 20 WISEs (i.e., Social Employment Centers for Social Initiative, and Employment Integration Enterprises), or new innovative SEs deploying technology-driven solutions to address social needs are examples of SE models [6–8]. 1. Introduction Recent literature presents SEs not only as promising vehicles for promoting sustainable development [2,9–13], but also as one of the actors with great potential to solve current social and environmental problems [14–16]. The different dimensions that conﬁgure sustainable development (economic, social, environmental, and cultural) are all linked to the daily activities of SEs, whose DNA incorporates an intrinsic ability to contribute to SDGs: “social enterprises are created to foster integrated and holistic approaches to sustainable development, notwithstanding the complexities of managing them, including the various resource types mobilized, the inclusive governance and participation of stakeholders, etc.” [17] (p. 184). g p p f p More speciﬁcally, the European Commission [17] stresses the importance of companies that adopt a shared value model, commercializing products with a double perspective on value, one internal (ﬁnancial proﬁtability) and the other external (positive impact for society and the environment). In this manner, SEs are aligned with OSD12 (sustainable production and consumption) by offering innovative goods and services that help allevi- ate social and/or environmental problems [18]. Following this line of reasoning, Kovaˇc Vujasinovi´c et al. [10] (p. 3) concluded that, “[ . . . ] it is now clear that more and more people are willing to align their economic choices with their values and are searching for ways that would allow the to do so [ . . . ]. Among the models that have appeared as alternatives to business-as-usual, social entrepreneurship has been one of the most prominent, receiving notable attention from the general public, researchers and policy makers”. Similarly, the UN Inter-Agency Task Force on Social and Solidarity Economy maintains that “business-as-usual is no longer an option” and focuses on the role of the “innovations and practices at work within the Social and Solidarity Economy (SSE) as crucial for connecting economic activity and sustainable development” [17] (p. 183); [19]. f g y p p Despite the importance of their commercial role, most research about SEs has concen- trated on organizational issues, analyzing models of innovative services, or operational and managerial variables [20–23]. Only a few studies have focused on the consumer’s perspec- tive [24,25]. Research that incorporates the demand viewpoint is thus needed [26,27] and may provide a better understanding of customer decision-making and its driving forces (personal, situational, or commercial), within the framework of sustainable consumption. 1. Introduction p p Research on sustainable consumption is increasingly capturing the interest of schol- ars [28,29] with the characteristics of sustainable products as a central topic [30]. Sustainable products deﬁned as those “that have positive social and/or environmental attributes” [31]. As such, they incorporate more value since they produce a social and/or environmental improvement in addition to satisfying customer needs. The systematic review of the lit- erature carried out by Bangsa and Schlegelmilch [28] highlights that, although literature on sustainable consumption behavior contains valuable ﬁndings, there is a persistent gap in terms of sustainable product attributes and their role in consumer decision-making. Therefore, this is a central research topic to contribute to from several perspectives. In the particular case of SE research, the identiﬁcation of the key sustainability attributes of the products marketed by these companies is of special interest. With the purpose to ﬁll these gaps, this paper aims to shed light on the unexplored common ground where SEs and sustainable consumption converge, with special atten- tion to both, enhancing the comprehension of SE customers’ behaviors and identifying the attributes of SEs’ sustainable products that customers value when making purchase decisions. Therefore, the authors conducted a systematic review and bibliometric analysis and posed the following three guiding research questions: RQ 1: What antecedents of sustainable consumption in SEs does the literature address? This question aims to identify drivers and barriers of the sustainable consumption of SEs’ products. p RQ 2: Which types of sustainable products of SEs dominate the literature, and what attributes do customers value the most? This question seeks to identify the prevailing products of the RQ 2: Which types of sustainable products of SEs dominate the literature, and what attributes do customers value the most? This question seeks to identify the prevailing products of the Sustainability 2022, 14, 13428 3 of 20 3 of 20 SEs’ commercial offer within the literature and the set of attributes valued to a greater degree by SEs’ customers. SEs’ commercial offer within the literature and the set of attributes valued to a greater degree by SEs’ customers. g y RQ 3: What are the prevalent individual factors of sustainable customer behavior of SE clients reﬂected within the literature? This question aims to identify the customer behavior factors determining their purchase decisions in relation to SEs’ sustainable products. The paper is structured as follows. 1. Introduction The next section details methodological aspects of the systematic review of the literature, highlighting the design of the review protocol and the data search process. Section 3 presents the main results from both a descriptive analysis and a keywords analysis, also providing an overview of the main topics of the ﬁnally selected publications. An overview of the main ﬁndings in relation to research questions posed above is included in Section 4, together with a proposal for a conceptual framework that paves the way for future analysis in this conﬂuent ﬁeld of research. Finally, conclusions and gaps to be covered through future lines of research are presented in Section 5. 2. Methodology Sustainability 2022, 14, 13428 4 of 20 ither an Figure 1. Boolean search equation and search settings of the literature extraction. Source: Author own elaboration. Figure 1. Boolean search equation and search settings of the literature extraction. Source: Authors’ own elaboration. Figure 1. Boolean search equation and search settings of the literature extraction. Source: Author own elaboration. Figure 1. Boolean search equation and search settings of the literature extraction. Source: Authors’ own elaboration. The combination of words and connectors (AND; OR) employed proved to be th most effective within a hitherto unexplored field of study, bringing together, first, the u of wide-ranged but specific terminology related to topics of interest (i.e., social enterpris sustainable; product; consumption) and, second, the gathering of sufficient publication to conduct a systematic review with assurance of robustness. In the same line, previou theoretical delimitations of main terms were useful to identify similar and/or compleme tary words and concepts to be finally included in the equation in order to amplify th search boundaries. SEs, for instance, are regularly accepted within the previous literatu as “social businesses”, since they develop business activities for a social purpose or mi sion [8]. Similar is the case of “responsibility” and “ethics” with regard to “sustainability as specific studies on sustainable consumption, social responsibility, and (corporativ management regularly approach these terms as related even interconnected [41–46] The combination of words and connectors (AND; OR) employed proved to be the most effective within a hitherto unexplored ﬁeld of study, bringing together, ﬁrst, the use of wide-ranged but speciﬁc terminology related to topics of interest (i.e., social enterprise; sustainable; product; consumption) and, second, the gathering of sufﬁcient publications to conduct a systematic review with assurance of robustness. In the same line, previous theoretical delimitations of main terms were useful to identify similar and/or complemen- tary words and concepts to be ﬁnally included in the equation in order to amplify the search boundaries. SEs, for instance, are regularly accepted within the previous literature as “social businesses”, since they develop business activities for a social purpose or mission [8]. Similar is the case of “responsibility” and “ethics” with regard to “sustainability”, as speciﬁc studies on sustainable consumption, social responsibility, and (corporative) management regularly approach these terms as related, even interconnected [41–46]. management regularly approach these terms as related, even interconnected [41 46]. 2. Methodology In order to obtain comprehensive insights into the scholarly literature merging SEs and sustainable consumption to date, the authors conducted a systematic literature review. The practicality of this review method lies in synthesizing the existing evidence within a particular topic or ﬁeld of knowledge, boosting the codiﬁcation and analysis of literature results, and, consequently, identifying gaps for further research [32–34]. This systematic approach builds on the delimitation of a research question(s), the clear deﬁnition of the inclusion criteria to accurately select the target publications, and the analysis of the resulting outputs, reducing potential bias risks [35]. p g p Review Protocol and Data Search Review Protocol and Data Search To conduct a precise systematic review, a review protocol was designed [36,37], including information on the inclusion criteria delimiting the search of publications, the selected databases, the extraction process, the search settings, and the screening constraint guidelines. g The inclusion criteria resulted from a three-fold delimitation: (i) Publication type-based constraint: peer-reviewed scholarly articles and proceedings published in academic journals; (ii) Thematic approach-based constraint: papers directly addressing the relation between SEs and sustainable/responsible/ethical consumption, regardless of the type of SEs and the category of product; and (iii) Research and methodology types-based constraint: theoretical/conceptual and empir- ical papers with quantitative, qualitative, mixed, or hybrid methodologies. Considering the novel and interdisciplinary nature of a topic like SEs and sustainable consumption, the target literature was selected from the ISI Web of Science (WoS) and Sco- pus databases, respectively. WoS and Scopus provide the most comprehensive compilation of documents published in indexed, peer-reviewed multidisciplinary academic journals, reducing the bias produced from searching speciﬁc databases. In addition, both databases allowed the fulﬁlment of the mentioned inclusion criteria, in response to a systematic review aiming at robustness and rigor, and the effective application of a searching discrimination process by type of publication, indexing range, scientiﬁc ﬁelds, or peer-reviewed scientiﬁc journals. This decision allowed the method to skip the random blending from academic and grey literature outputs in which (no) peer-reviewed publications are mixed with (no) indexed journals, among other types of publications like sectorial reports, dissertations, or policy statements [38–40]. For the sake of covering the scope of SEs and sustainable consumption ﬁeld of research, an algorithm-based Boolean search equation is applied in the topic, title, abstract, and author-provided keywords of publications written in English, limited by neither any time speciﬁcation nor any speciﬁc subject area (Figure 1). 2. Methodology Aiming to refine but also to complete the final output as precisely as possible, a snow ball search was conducted among WoS results. This database provided the largest varie of effectively valid publications between the initially extracted potential ones. A total Aiming to reﬁne but also to complete the ﬁnal output as precisely as possible, a snowball search was conducted among WoS results. This database provided the largest variety of effectively valid publications between the initially extracted potential ones. A total of 77 potential documents were initially identiﬁed (Figure 2). y p y p 77 potential documents were initially identified (Figure 2). After the identiﬁcation of duplicates considering the title and/or abstracts, 22 publica- tions were removed (77.2% of those extracted from Scopus), and 55 documents followed into the screening phase in which off-topic documents were removed considering the title and abstracts. The volume of off-topic results was not negligible (n = 31, 56.6% of the total), and the main reasons behind the drop were (1) publications that revolved around entities erroneously referred to as SEs when in fact they were non-governmental organizations (NGOs), nonproﬁts (NPOs), or simply for-proﬁt businesses practicing cause-related mar- keting campaigns; (2) publications that addressed (corporative) social responsibility-based policies/measures in private businesses; (3) publications that dealt with SEs but not in relation to sustainable consumption; and (4) publications that included “social enterprises” and/or “sustainability” in the title and/or abstract but that did not directly address these issues in the rest of the work. Once discarding the off-topic results identiﬁed, the authors obtained 24 papers for ﬁnal review. Sustainability 2022, 14, 13428 Sustainability 2022, 14, x FOR P 5 of 20 5 of he tit Figure 2. Flow diagram, PRISMA. Source: Authors' own elaboration based on [47]. Figure 2. Flow diagram, PRISMA. Source: Authors’ own elaboration based on [47]. ( p into the screening phase in which off-topic documents were and abstracts. The volume of off-topic results was not neglig and the main reasons behind the drop were (1) publications erroneously referred to as SEs when in fact they were non (NGOs), nonprofits (NPOs), or simply for-profit businesses keting campaigns; (2) publications that addressed (corpo based policies/measures in private businesses; (3) publicatio in relation to sustainable consumption; and (4) publication prises” and/or “sustainability” in the title and/or abstract bu these issues in the rest of the work. After 3. Results sparse l b After the identification of duplicates considering the title and/or abs cations were removed (77.2% of those extracted from Scopus), and 55 docu 3.1. Descriptive Analysis of the Literature on SEs and Sustainable Consumption being said, it is important to point out a remarka cations were removed (77.2% of those extracted from Scopus), and 55 documents followe into the screening phase in which off-topic documents were removed considering the tit and abstracts. The volume of off-topic results was not negligible (n = 31, 56.6% of the tota and the main reasons behind the drop were (1) publications that revolved around entiti erroneously referred to as SEs when in fact they were non-governmental organization (NGOs), nonprofits (NPOs), or simply for-profit businesses practicing cause-related ma keting campaigns; (2) publications that addressed (corporative) social responsibilit based policies/measures in private businesses; (3) publications that dealt with SEs but n SEs and sustainable consumption are very recently connected topics within a still sparse literature, according to the distribution of publications over time (Figure 3). This being said, it is important to point out a remarkable increase of the scholarly literature (+87.5%) since 2015. Not accidentally, this increase of literature coincided with the adoption of the 2030 Agenda and the corresponding publication of the 17 SDGs, and it ampliﬁed the wake of previous literature anticipating the potential of social economy—and SEs in particular—to achieve a more sustainable development [10–16,18,19]. g p p (+87.5%) since 2015. Not accidentally, this increase of literatu tion of the 2030 Agenda and the corresponding publication o fied the wake of previous literature anticipating the potential in particular—to achieve a more sustainable development [1 p p ( ) p in relation to sustainable consumption; and (4) publications that in prises” and/or “sustainability” in the title and/or abstract but that did these issues in the rest of the work. Once discarding the off-topic r authors obtained 24 papers for final review. 3. Results 3.1. Descriptive Analysis of the Literature on SEs and Sustainable Consum SEs and sustainable consumption are very recently connected sparse literature, according to the distribution of publications over being said, it is important to point out a remarkable increase of th p p p in relation to sustainable consumption; and (4) publications that included prises” and/or “sustainability” in the title and/or abstract but that did not di these issues in the rest of the work. 2. Methodology Once discarding the o authors obtained 24 papers for final review. 3. Results 3.1. Descriptive Analysis of the Literature on SEs and Sustainabl SEs and sustainable consumption are very recently co lit t di t th di t ib ti f bli ti e drop were (1) publicatio when in fact they were n n to sustain d/ “ t ults scriptive Analysis Figure 2. Flow diagram, PRISMA. Source: Authors' own elaboration based on [47 Figure 2. Flow diagram, PRISMA. Source: Authors’ own elaboration based on [47]. SEs and sustainable consumption are very re Figure 2. Flow diagram, PRISMA. Source: Authors' own elaboration based on [47 Figure 2. Flow diagram, PRISMA. Source: Authors’ own elaboration based on [47]. 3 R lt SEs and sustainable consumption are very r sparse literature, according to the distribution of p Figure 2. Flow diagram, PRISMA. Source: Authors' own elaboration based on [47 Figure 2. Flow diagram, PRISMA. Source: Authors’ own elaboration based on [47]. SEs and sustainable consumption are very re lit t di t th di t ib ti f After 3. Results sparse l b Once discarding the off-topic results authors obtained 24 papers for final review. 3. Results 3.1. Descriptive Analysis of the Literature on SEs and Sustainable Consumption SEs and sustainable consumption are very recently connected topics sparse literature, according to the distribution of publications over time (F being said, it is important to point out a remarkable increase of the schol (+87.5%) since 2015. Not accidentally, this increase of literature coincided w ti f th 2030 A d d th di bli ti f th 17 SDG Figure 3. Time distribution of publications. Source: Authors’ own elaboration. (+87.5%) since 2015. Not accidentally, this increase of literature coinc ti f th 2030 A d d th di bli ti f th 17 Figure 3. Time distribution of publications. Source: Authors’ own elaboration. tion of the 2030 Agenda and the corresponding publication of the 17 SDGs, and it amp fied the wake of previous literature anticipating the potential of social economy—and SE in particular—to achieve a more sustainable development [10–16,18,19]. According to the type of source from which the documents were extracted (Figure 4), a vast majority are peer-reviewed, indexed journals contributing 22 papers versus only two conference proceedings (see Appendix A for more details). Sustainability 2022, 14, 13428 6 of 20 ng 22 Figure 4 Relation of peer-reviewed indexed journals Source: Authors’ own Figure 4. Relation of peer-reviewed, indexed journals. Source: Authors’ own elaboration. Figure 4. Relation of peer-reviewed, indexed journals. Source: A Regarding the geographical distribution, at least eigh 73 authors involved in the literature reviewed (Figure 5). Fi 4 R l ti f i d i d d j l S A th Figure 4. Relation of peer-reviewed, indexed journals. Source: Authors’ own elaboration. 73 authors involved in the literature reviewed (Figu Fi 4 R l ti f i d i d d j l S A th Figure 4. Relation of peer-reviewed, indexed journals. Source: Authors’ own elaboration. 73 authors involved in the literature reviewed (Figu Figure 4. Relation of peer reviewed, indexed journals. Source: Authors own Regarding the geographical distribution, at least eight countries p 73 authors involved in the literature reviewed (Figure 5). Three geogr be distinguished: Asian affiliation represented 30% of the total, follow Regarding the geographical distribution, at least eight countries produced 82% of the 73 authors involved in the literature reviewed (Figure 5). After 3. Results sparse l b Three geographical poles could be distinguished: Asian afﬁliation represented 30% of the total, followed by American afﬁliation, with 28% of the documents (with the USA leading by a wide margin), and authors belonging to European institutions, with 24% of the results. be distinguished: Asian affiliation represented 30% of the filiation, with 28% of the documents (with the USA lead thors belonging to European institutions, with 24% of the filiation, with 28% of the documents (with the USA leading by a thors belonging to European institutions, with 24% of the results. Figure 5. Geographical distribution of authors’ affiliation. Source: Autho Figure 5. Geographical distribution of authors’ affiliation. So Figure 5. Geographical distribution of authors’ afﬁliation. Source: Authors’ own elaboration. Figure 5 Geographical distribution of authors’ affiliation Source: Autho Figure 5. Geographical distribution of authors’ affiliation. So Figure 5. Geographical distribution of authors’ afﬁliation. Source: Authors’ own elaboration. 7 of 20 Sustainability 2022, 14, 13428 7 of 20 Within the Asian pole, (South) East Asia afﬁliations prevailed; among these, Taiwanese institutions provided more than half (17%) of the total Asian contributions, followed by South Korea and Malaysia. In the case of Taiwan, SEs are ﬂourishing in recent years with the objective to solve working exclusion and poverty effects of vulnerable social groups. The SEs generally come from family businesses moving to self-help groups that scale up to institutional collectivism forms, even becoming inﬂuential in the country’s social and political issues [48,49]. In this context, the announcement of the Social Enterprise Action Plan by the Taiwan Government in 2014 marked an important turning point, boosting the establishment and progress of SEs in the country for the following three years. In 2018, the Taiwanese SE ecosystem was formed by nearly 400 SEs and more than 11,000 potential ones, most of which are cooperatives and associations for local development [50]. Action Plan by the Taiwan Government in 2014 marked ing the establishment and progress of SEs in the countr 2018, the Taiwanese SE ecosystem was formed by nea potential ones, most of which are cooperatives and as [50]. Regarding the research approaches and methodolo prevalent were empirical articles (n = 22, 91.6%) using q 6) p p Regarding the research approaches and methodologies within the literature, the most prevalent were empirical articles (n = 22, 91.6%) using quantitative methodologies (Figure 6). 6). Figure 6 Empirical research distribution per methods and too Methods and tools No. After 3. Results sparse l b Papers % Survey 15 68.2% Case study 3 13.6% Experimentation 2 9.2% In-depth interviews 1 4.5% Mathematical model 1 4.5% Total 22 100% Figure 6. Empirical research distribution per methods and tools used. Source: Authors’ own elaboration. Fi 6 E i i l h di t ib ti th d d t Figure 6. Empirical research distribution per methods and tools used. Source: Authors’ own elaboration. Figure 6. Empirical research distribution per methods and too ration. Among these, the use of surveys was prominent in tical techniques: structural equation model (SEM) (n = 7) (MANOVA) (n = 1); multivariate logistic regression (n Among these, the use of surveys was prominent in combination with different statisti- cal techniques: structural equation model (SEM) (n = 7); multivariate analysis of variance (MANOVA) (n = 1); multivariate logistic regression (n = 1); combination of factor analysis and correlation analysis (n = 1); combination of SEM and regression (n = 1); hierarchical linear model (n = 1); descriptive analysis (n = 1); multiple regression (n = 1); and T-test and analysis of variance (ANOVA) (n = 1). Only 8% of publications reviewed were of a theoretical/conceptual approach, including one literature review. and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption y ( ) linear model (n = 1); descriptive analysis (n = 1); multipl analysis of variance (ANOVA) (n = 1). Only 8% of pub retical/conceptual approach, including one literature re In order to identify the most common terms within the 24 ﬁnal publications reviewed, bibliometric or scientiﬁc mapping was employed. Bibliometric analyses are helpful to statistically calculate the literature on a speciﬁc topic from scholarly publications [51,52]. Among the different computerized methods for treating data, bibliometric mapping has been gaining relevance in recent years to conduct bibliometric analysis. p pp g 3.2. Keyword Analysis of the Literature on SEs and Sustaina In order to identify the most common terms wi viewed, bibliometric or scientific mapping was employe In the case of literature on SEs and sustainable consumption, a bibliometric map allows for the visualization of a range of bibliometric networks, such as networks of citation, of co-authorship, or of co-occurrent keywords from the title, abstract, and list of author(s)-provided keywords [53,54]. Considering all of the valid approaches available, the co-occurrence of keywords within the text data reviewed was selected. This speciﬁc co-occurrence analysis is useful to delimitate the topics of research and how the scholarly literature is structured based on links among the prevalent keywords [55]. ful to statistically calculate the literature on a specific [51,52]. Among the different computerized methods fo ping has been gaining relevance in recent years to cond In the case of literature on SEs and sustainable con The authors employed VOSviewer, a bibliometric software that provided a relevance score-based automatic selection of the 23 most co-occurrent keywords via 91 links. The resulting bibliometric map (Figure 7) allowed us to graphically visualize the strength of the co-occurrence through colored networks, distributed in four clusters. In this ﬁgure, nodes’ size indicates the importance of an item, and network relationships indicate the most closely related topics. This representation allows getting a sense of the keywords Sustainability 2022, 14, 13428 8 of 20 most used by the authors of the extracted publications, as well as the links between the topics analyzed in the papers. most used by the authors of the extracted publications, as well as the links between the topics analyzed in the papers. As expected, the most used keyword is “social enterprise”, a ﬁeld of study on which this literature review is focused (red node). and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption Source: Authors’ own The study of “social enterprise” represents the cornerstone 24 l d h thi t i k d) Si it i th The study of “social enterprise” represents the cornerstone of this cluster (18 of the 24 papers analyzed have this topic as a keyword). Since it is the unit of analysis of this bibliometric study, the word "social enterprise" is related to the rest of the clusters. However, the links are stronger with the words from the ﬁrst cluster. ever, the links are stronger with the words from the first cluster. Social entrepreneurship (7 papers of the 24 papers reviewed) is recognized as a pro ising vehicle for solving social and environmental challenges and fostering sustainab development. In this respect, the documents highlight the importance of sustainable d velopment (3 papers of the 24 papers analyzed) as a central objective of 21st century so eties. The papers related to this topic deal in some cases with certifications, highlighti their important role as an instrument to foster more sustainable habits among consume The use of certifications by SEs affects the evaluation that consumers make of their pro ucts and, consequently, their consumption behavior. The next topic identified in this clu ter is “marketing research” (3 papers of the 24 papers reviewed). This topic focuses on t communication actions carried out by companies. Some examples are information p vided on the website or social networks, advertising, or the messages that can be tran mitted by the salesperson. In addition, some papers are linked to usual marketing pr tices, which focus on addressing the challenges that SEs face in comparison with the m 24 papers analyzed have this topic as a keyword). Since it is th bibliometric study, the word "social enterprise" is related to the r ever, the links are stronger with the words from the first cluster. Social entrepreneurship (7 papers of the 24 papers reviewed) ising vehicle for solving social and environmental challenges a development. In this respect, the documents highlight the impo velopment (3 papers of the 24 papers analyzed) as a central obje eties. and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption Main Topics Addressed in the Literature on SEs and Sustainable Consumption y p 24 papers, combined with the results of the previous analysis 3 3 Main Topics Addressed in the Literature on SEs and Sustainable Consumptio 3.3. Main Topics Addressed in the Literature on SEs and Sustainable Consumption 24 papers, combined with the results of the previous a 3.3. Main Topics Addressed in the Literature on SEs and Sustainable Consumption The next level of analysis focuses on the topics studied. The detailed reading of t 24 papers, combined with the results of the previous analysis of keywords (see Secti 3 2) ll id if j h i The next level of analysis focuses on the topics studied. The detailed reading of the 24 papers, combined with the results of the previous analysis of keywords (see Section 3.2), allow us to identify major research topics. 3.2), allow us to identify major research topics. The first cluster is the one with the most weight of the tota 3.2), allow us to identify major research topics. The first cluster is the one with the most weight of the total number of papers an lyzed. Fifty percent of the selected papers are related to topics that belong to this clus y j p The ﬁrst cluster is the one with the most weight of the total number of papers analyzed. Fifty percent of the selected papers are related to topics that belong to this cluster [56–67] (Figure 9). lyzed. Fifty percent of the selected papers are related to topics t [56–67] (Figure 9). [56 67] (Figure 9). Figure 9. Distribution of publications by cluster. Source: Authors’ own elaboration. Figure 9. Distribution of publications by cluster. Source: Authors’ own elaboration. Figure 9. Distribution of publications by cluster. Source: Authors’ own elaborati Figure 9. Distribution of publications by cluster. Source: Authors’ own elaboration. The study of “social enterprise” represents the cornerstone of this cluster (18 of t 24 papers analyzed have this topic as a keyword). Since it is the unit of analysis of t bibliometric study, the word "social enterprise" is related to the rest of the clusters. Ho ever the links are stronger with the words from the first cluster Figure 9. Distribution of publications by cluster. and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption From this central keyword, numerous categories of analysis are born, which advance the subsequent study of the main themes (social entrepreneurship, sustainability, and behavior, among others). Keyword co-occurrence analysis identiﬁes four clusters in which keywords associate. In each cluster, there are one or more items related to behavior and sustainability (i.e., sustainable development, purchase behavior, sustainability), so that the main themes can be visualized, which will be analyzed in detail in the following section. Cluster 1 (red color), which is the most studied of all (see Figure 8), is mainly composed of keywords such as "social enterprise", "social entrepreneurship", "entrepreneurship", "sus- tainable development", “marketing research” and "customer satisfaction". In cluster 2 (purple color), keywords such as “purchase intention”, “perceived value”, “credibility”, “responsibility”, “attitude”, “intention”, “behavior” and “performance” are identiﬁed. Cluster 3 (green color) is made up of keywords such as “consumers”, “consumption”, “sus- tainability”, “strategies”, and “impact”. Finally, cluster 4 (yellow color) -that contains the least number of keywords -, is related to “consumer behavior”, “planned behavior”, “de- composed theory”, and “governance”. This cluster analysis allows us to see the recurrence of the consumer theme, as will be detailed below. REVIEW Figure 7. Co-occurrence based bibliometric map of keywords. Source: Authors’ own with VOSviewer. Figure 7. Co-occurrence based bibliometric map of keywords. Source: Authors’ own elaboration with VOSviewer. Figure 7. Co-occurrence based bibliometric map of keywords. Source: Authors’ own with VOSviewer. Figure 7. Co-occurrence based bibliometric map of keywords. Source: Authors’ own elaboration with VOSviewer. Sustainability 2022, 14, 13428 Sustainability 2022, 14, x FOR 9 of 20 9 of Figure 8. Prevalent topics within the literature on SEs and sustainable consumption. Source: Au thors’ own elaboration. Figure 8. Prevalent topics within the literature on SEs and sustainable consumption. Source: Authors’ own elaboration. Figure 8. Prevalent topics within the literature on SEs and sustainable c thors’ own elaboration. 3.3. Main Topics Addressed in the Literature on SEs and Sustainable C The next level of analysis focuses on the topics studied. Th Figure 8. Prevalent topics within the literature on SEs and sustainable consumption. Source: Au- thors’ own elaboration. Figure 8. Prevalent topics within the literature on SEs and sustainable consumption. Source: Authors’ own elaboration. 3.3. Main Topics Addressed in the Literature on SEs and Sustainable C The next level of analysis focuses on the topics studied. Th 3 3 Main Topics Addressed in the Literature on SEs and Sustainable Consumption 3.3. and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption The papers related to this topic deal in some cases with ce their important role as an instrument to foster more sustainable h The use of certifications by SEs affects the evaluation that consum ucts and consequently their consumption behavior The next top Social entrepreneurship (7 papers of the 24 papers reviewed) is recognized as a promis- ing vehicle for solving social and environmental challenges and fostering sustainable development. In this respect, the documents highlight the importance of sustainable devel- opment (3 papers of the 24 papers analyzed) as a central objective of 21st century societies. The papers related to this topic deal in some cases with certiﬁcations, highlighting their important role as an instrument to foster more sustainable habits among consumers. The use of certiﬁcations by SEs affects the evaluation that consumers make of their products and, consequently, their consumption behavior. The next topic identiﬁed in this cluster is “marketing research” (3 papers of the 24 papers reviewed). This topic focuses on the com- munication actions carried out by companies. Some examples are information provided on the website or social networks, advertising, or the messages that can be transmitted by the salesperson. In addition, some papers are linked to usual marketing practices, which focus on addressing the challenges that SEs face in comparison with the marketing practices adopted by conventional companies. In addition, they also study how an SE can grow Sustainability 2022, 14, 13428 10 of 20 10 of 20 through new distribution channels and differentiate itself through a reliable product. The last relevant topic in this cluster is customer satisfaction. In this topic, the papers analyzed focused on studying expectations and perceived value as antecedents of satisfaction in the ﬁeld of SE. The authors found that 21% of the papers selected in this bibliometric analysis belong to the second thematic cluster [68–72]. The most important topic studied in this cluster was purchase intentions, contained in 4 of the 24 selected papers. Some authors focus their studies on the social mission with which an SE may be born, because it can affect the competitiveness of the enterprise and consumer behavior. The second of the topics highlights the importance of perceived value as a key factor in determining consumer behavior in SEs. These consumers especially appreciate the social value of their prod- ucts, but also consider their utilitarian and emotional value. Credibility and responsibility are other topics identiﬁed. 4. Overview of Findings on Sustainable Consumption of SE Products To shed light on the unexplored common ground where SEs and sustainable con- sumption converge—with special orientation to enhance the comprehension of customers’ behaviors as well as the identiﬁcation of product attributes inﬂuencing customers’ deci- sion making—the results obtained allow for some initial considerations guided by the aforementioned research questions to be stated. and correlation analysis (n = 1); combination 3.2. Keyword Analysis of the Literature on SEs and Sustainable Consumption These studies analyze whether SEs have greater credibility than traditional enterprises or whether the image of corporate social responsibility (CSR) and credibility of both the SE and the social entrepreneur can be a determinant of con- sumer behavior. Additionally, consumers’ intention to purchase social products is related to attitudes. The third thematic cluster is related to 16.5% of the analyzed papers [73–76]. The ﬁrst topic is the consumer, with 3 of the 8 papers in this cluster. In this cluster, a topic as important as the information or knowledge that consumers have has been studied, given the lack of knowledge they have in the relationship between SE and sustainability. In this regard, comparisons have been made between the consumer behavior of SEs and traditional businesses. Although it seems that SEs have better perceptions in terms of social responsibility and credibility, this does not translate into higher sales, with all the risks that this entails for an SE. Another theme of this cluster is sustainability (3 of the 8 papers). This is closely linked to the previous topic, since it is studied whether, for example, meta- sustainability labels help reduce consumer confusion and provide valuable information in these terms. The last of the topics in this cluster is strategies (2 of the 8 papers), related to aspects which a company can inﬂuence in order to achieve its objectives and improve its results. Research in this area is related to the achievement of a competitive advantage, obtained through factors such as product, price, quality, and distribution. g p p q y Finally, 12.5% of the reviewed papers are linked to the fourth cluster [77–79]. In this cluster, the main topic is the consumer behavior (5 of the 6 papers deal with this topic). The studies highlight insufﬁcient attention given to understanding the behaviors of consumers, which are a key stakeholder of SEs. To do this, at least one of the papers relied on Ajzen’s Theory of Planned Behavior (TPB) to examine consumer behavior when purchasing SE products. 4.1. Antecedents of Sustainable Consumption Addressed in the Literature (RQ 1) In relation to the antecedents of sustainable consumption, the literature presents a symmetric coverage between drivers and barriers. Whereas 12 papers pay attention to those factors prompting sustainable consumption of SE products, 11 are the sources from which speciﬁc obstacles emerge in this regard. Drivers of sustainable consumption of SEs’ commercial offers at a macro level mainly have institutional, social, and market natures. Institutional-based drivers relate to the potential of public (municipal) awareness-raising campaigns communicating the advantages and beneﬁts of participating in sustainable consumption models boosted by SEs through, for instance, Sustainability 2022, 14, 13428 11 of 20 11 of 20 the collection and treatment of wastes to amplify their life cycle, as in the case of Prepared- for-Reuse items [61]. The utility of public authorities’ incentives also works as a driver when intermingling the meeting of customers’ (basic) needs and appealing to their playfulness through the stimulus to learn [60]. The existence of institutional support programs within the public procurement strategies for the provision of sustainable food items in public schools also plays a relevant role propelling the sustainable consumption of this type of hybrid organization. y g From the side of social-based drivers, the social awareness of the existence of sustainable (and affordable) solutions for meeting common good causes emerged as a relevant starting point, also for the shape of the subjective norm (i.e., opinions of friends and relatives), which, in turn, determines potential customers’ intentions to a great extent [69,73]. Market-based drivers, from the other side, refer to the positive inﬂuence of effective cross-sectoral partnerships through which actors involved—for-proﬁts, public authorities, third-sector organizations, civil society organizations (e.g., customer associations), and SEs, among others—interact to adopt solutions in the form of effective tools and schemes like sustainable labels and certiﬁcations [73,75]. With respect to the antecedents acting as barriers at a macro level to the adoption of sustainable consumption, scholarly literature raises institutional obstacles with implications for the market ecosystem. In particular, the lack of governmental funding support, data access, and a speciﬁc regulative framework weakens the position of SEs to overcome the resistance of competitors (i.e., private enterprises) to develop sustainable schemes or tools based on veriﬁable third-party standards or certiﬁcation (e.g., the case of the Trafﬁc Light Index in the food sector) [61,75]. 4.1. Antecedents of Sustainable Consumption Addressed in the Literature (RQ 1) Finally, optimal management of the stakeholder relationship by SEs also emerges as a driver of sustainable consumption, in the sense of promoting valued-based and close relationships with local communities to Sustainability 2022, 14, 13428 12 of 20 12 of 20 enhance social cohesion or collaborating in cross-sector partnerships with other reputed actors [61]. The prominence of barriers to the adoption of sustainable consumption at a meso level is, however, scarcer within the literature. They relate speciﬁcally to i) SEs’ malprac- tices, behaving irresponsibly on competence and effectiveness through the use of social washing-based advertising strategies [2,61]; ii) the dependence on public funding in the work integration organizations and on an exclusive customer perspective (instead of a multistake- holder perspective) [75]; and 3) the lack of reliable and veriﬁable third-party standards and certiﬁcations [61]. ﬁ Finally, the scholarly attention on antecedents of sustainable consumption at a micro level is limited and unequal, with drivers having a larger presence within the literature than the antecedents acting as barriers. A signiﬁcant majority of drivers are perception-based antecedents acting at a micro (customer) level, so much so that these drivers relate speciﬁcally to the perceived contribution of a purchase to the common good, increasing the customer motivation to consume in a more sustainable way [77]; the perception of quality, positively affecting the perception of functional, emotional, and social value [72]; the perception of satisfying personal needs through sustainable consumption [61]; the perception of SEs’ effectiveness [76]; and the perception of SEs’ social responsibility, credibility, and competence [75]. Other drivers emerging from the literature are the identiﬁcation with the brand [68] and the customer attitude, intention, and behavior towards sustainable consumption in the context of SEs [69,76,77]. Antecedents impeding the sustainable consumption at a micro level hardly have presence within the literature, being reduced to the perception of (product) risk [69,77], the risk arising from possible information saturation [63], and the low conﬁdence of customers in product quality [77]. 4.1. Antecedents of Sustainable Consumption Addressed in the Literature (RQ 1) Social barriers involve cultural obstacles when persuading potential customers to be actively engaged in sustainable consumption through the purchase or the alternative uses of sustainable products provided by SEs, as Gelbmann and Hammerl claim to be the case of re-use ECO-WISEs products [60]. From an entirely market perspective, tensions generated along the supply chains when SE products experience fast-growing demand emerge in the literature as an important barrier to be considered in the particular case of the food distribution sector [73]. Drivers and barriers at a meso level also attracted academic attention, albeit to varying degrees. Several antecedents are boosting or impeding sustainable consumption under the umbrella of SEs’ organizational performance and capabilities. From the side of drivers, scholarly literature shows the pivotal role of communicational capabilities of SEs through visual graphic-based advertising actions appealing to positive emotions of customers (e.g., pride and joy), rather than a narrative on complex information about social im- pact [61] to increase consumers’ awareness and retention. In addition, and as part of SEs’ marketing activities, the literature shows that effective communication investment on dissemination of central aspects such as social mission—if congruently aligned to customers’ needs—has beneﬁts on intangible assets like brand image and product competitiveness, positively determining the perceptions of purchase intentions of potential customers [63,70]. The potential role of brands and products levering sustainable consumption of SEs’ com- mercial offer is of great relevance when these organizations are capable to position them as trust-to-go alternatives, in the sense of brands and products with strong identity and image (i.e., reputed brands or products) [68,69,73]. Other effective dimensions driving sustainable consumption relate to the capability of SEs when transmitting to potential cus- tomers favorable attributes on quality [58,72], competitiveness [70], uniqueness [58], price [58], quality certiﬁcation (e.g., displaying social certiﬁcation logos on product labels) [65], or the meaningfulness and superiority of products [64], positively affecting purchase intentions. The potential of meso-level drivers also falls on the capacity of SEs to enrich the shopping experience in order to increase customer loyalty through the provision of incentives [60], the assistance of trained salespersons and a friendly customer service [58,61], the pleasant atmosphere of the SEs, and/or a convenient location [58]. 4.2. Sustainable Products and Product Attributes Valued by SE Customers (RQ 2) SEs’ commercial offer within the literature is predominantly based on physical prod- ucts that belong to a set of distinctive categories, among which the grocery category prevails. Some studies revolving around SEs’ supplying–manufacturing–distribution– commercialization activities in the food sector refer in particular to organic food items [68], coffee [59,70], grab-and-go snacks, breakfast, meal kits, raw ingredients [73], cookies, cakes, and frozen food [69]. Other products and categories are smartphones made from recyclable mate- rials [77] in the tech category; different electric and electronic devices and appliances, furniture pieces, and other decorative stuff in the cases of product multicategory studies [60,61,69,77]; art pieces [56]; salt, in the context of mineral extraction with medical purposes [74]; traditional clothing, craft-made fabrics, bags, and shoes in the textile, apparel, fashion complements, and footwear product categories [60,64,67,69,77]; and cosmetic items for personal use [65,77]. Valued product attributes in the eye of SE customers coexist within the literature in relation to the utilitarian–hedonic–ethical beneﬁts perceived. Previous literature highlights the assortment, ease of seeking, price comparison, information attainment and availability, quality, functionality, reputed or well-known brand, packaging easy to open/use, use of recycled materials in the products; ready availability in the stores; and easiness to buy as valued attributes built on utilitarian beneﬁts perceived [62,64,67,68,73,77]. From the side of attributes resulting from hedonic beneﬁts perceived, the literature addresses being trendy among the members of the reference group, product attractiveness appealing to customers’ sense of style, visual appearance, stimulation, stress relief, and social interaction [61,67,68,73]. Finally, ethical beneﬁts perceived gather valued attributes underlying the social engagement and orientation of SEs, brands, and products, such as the image of CSR performance in relation to ethics, law, and economic dimensions [68]; trust in social performance; and the attainment of certiﬁcations and information on socially vulnerable groups [65]. Sustainability 2022, 14, 13428 13 of 20 13 of 20 4.3. Individual Factors Determining Sustainable Customer Behavior (RQ 3) 4.3. Individual Factors Determining Sustainable Customer Behavior (RQ 3) Specialized literature shows evidence of a range of socio-demographic, psychographic, and sociographic factors determining customers’ behavior referring to purchase decisions regarding SEs’ sustainable products. 4.2. Sustainable Products and Product Attributes Valued by SE Customers (RQ 2) In regard to socio-demographics inﬂuencing favorable customer behavior towards SE products, studies pay attention to the potentiality of con- sumers’ level of income, the strengthened relationship between consumers’ intention and behaviors [76], and the role of genre, since, in correspondence with their traditional caring nature, women regularly have more information on SEs, positively affecting their willing- ness to buy from SEs. In the same line, age and educational level also appear as relevant socio-demographics, as customers between 16 and 25 and between 26 and 35 years old tend to have less information on SEs, a trait shared with those with secondary education. The informative level seems to also play a remarkable role. Studies have conﬁrmed the positive relation between information and willingness to buy, meaning that the more information on SEs customers have, the greater their willingness to conduct frequent purchases [63]. g g q p [ ] Scholarly literature also echoes the pivotal role of psychographics, in regards to those personality traits, attitudes, interests, values, beliefs, ideology, and expectations being the backbone of customers’ lifestyle. In this respect, previous research proved the role of customers’ political ideology [76]; their common-good orientation, being the main purchasing goal beyond products’ performance; and their ethical self-identity and moral identity [77]. Attitude towards SE products also inﬂuences purchase intentions [69,77]. A positive attitude is conditioned, in turn, by a high compatibility to customer values towards the support of individual/common-good causes, by low quality or safety perceived risk, and by a favorable brand perception towards SEs’ commercial offer [69]. Perceived behavioral control and self-efﬁcacy are positively associated with intention to purchase SEs’ products [69]. Customers trust in the scheme also inﬂuences the decision-making; for instance, eco-labels are perceived as trustworthy tools by customers when endorsed by NGOs and consumer organizations, as far as consumers prefer these entities as a trusted and independent source of environmental information [75,76]. Information on consumers’ behavioral factors affecting, in particular, the repurchase of SEs’ products is lacking in the scholarly literature, being reduced to the potential of brand trust for repurchasing intentions and the role of utilitarian beneﬁts positively inﬂuencing costumers’ brand trust [68]. ﬁ p y g Finally, sociographics gather the set of relations, trends, risks, needs, proﬁles, and experiences of individuals, understood as part of social groups or targets. 4.2. Sustainable Products and Product Attributes Valued by SE Customers (RQ 2) Sociographics that emerged from the literature refer to the SE customers’ sense of belonging and involvement in social causes supported through purchases [58] and to the relevance of reference groups’ perception-based subjective norms (i.e., the opinions of friends and relatives) inﬂuencing customer behavior [69,77]. 5. A Proposal for a Conceptual Framework Based on the aforementioned ﬁndings, a conceptual model is proposed (Figure 10) on sustainable consumption of SEs’ commercial offers. This conceptual framework reﬂects signiﬁcant relationships between antecedents in the form of drivers and barriers, indi- vidual determinants of sustainable customer behavior, and valued product attributes in the eyes of SE customers. Antecedents at the macro, meso, and micro levels of analysis may inﬂuence customers’ sustainable behavior, which, in turn, is modeled by individ- ual factors of sociodemographic, psychographic, and sociographic natures. At the same time, customers’ sustainable behavior and (valued) product attributes perceived can be mutually conditioned. 14 of 20 4 of 20 Sustainability 2022, 14, 13428 Sustainability 2022, 14, x FOR 6. Conclusions This study compiles the main ﬁndings in the ﬁeld of academic research on SEs and sustainable consumption, given the scholarly prominence of SEs in the pursuit of sustain- able development. A systematic literature review was conducted, resulting in 24 scientiﬁc publications analyzed. To the best of our knowledge, this is the ﬁrst literature review shedding light on the crossroads between SEs and sustainable consumption with special orientation to customer behavior and attributes of SE products, a domain that remained largely obscure. g y The incipient literature with a majority of publications from 2015 onward in the form of empirical articles using quantitative methodologies is mainly focused on the antecedents of sustainable consumption at the macro and meso levels, in which institutional drivers (and to a lesser degree, barriers) and organizational capabilities of SEs prevail, respectively. The literature on sustainable consumption of SE products at a micro level mainly explored the individual perceptions towards the social causes supported and the organizational performances of SEs’ commercial activities in terms of effectiveness, credibility, or competence. In order to ﬁll the persistent gap within the literature on the inﬂuence of sustainable product attributes on SE consumers’ decision-making and behavior [28], this research provides an integrative conceptual framework. This framework, susceptible to being empir- ically proved in future research, reﬂects signiﬁcant relationships between antecedents—in the form of drivers and barriers—at different levels, individual determinants of sustainable customer behavior, and valued product attributes in the eyes of SE customers. In particular, antecedents may inﬂuence customers’ sustainable behavior, which in turn, is modeled by individual factors of sociodemographic, psychographic, and sociographic natures. Simulta- neously, customers’ sustainable behavior and (valued) product attributes perceived can be mutually conditioned. Moreover, this framework may pave the way for future analysis in this ﬁeld of research, where several gaps and weaknesses emerged after systematically reviewing the literature. Further research would deepen the knowledge about the inﬂuence of product attributes on SE customers’ behavior in the pre- and post-purchase phases. Similarly, it is necessary to shed light on the inﬂuence of product attributes on customer behavior in the context of the economic, social, or environmental dimensions featuring SEs’ commercial activity, especially after the pandemic. The exploration of actors, enablers, and effects of the sustainable consumption of SE products from a multistakeholder perspective, together with those acting as obstacles to its adoption at the micro (individual) level, are lines of research needing scholarly attention. Figure 10. Conceptual framework on sustainable consumption of SEs’ products. Source: Autho own elaboration. Figure 10. Conceptual framework on sustainable consumption of SEs’ products. Source: Aut own elaboration. Figure 10. Conceptual framework on sustainable consumption of SEs’ products. Source: Authors’ own elaboration. Figure 10. Conceptual framework on sustainable consumption of SEs’ products. Source: Authors’ own elaboration. 15 of 20 Sustainability 2022, 14, 13428 15 of 20 6. Conclusions Some methodological limitations can be drawn from this study. Firstly, in light of the results and despite of having used WoS and Scopus as databases, it would be interesting to expand the body of literature searching in more geographically and/or sectoral specialized databases. This would contribute to a better understanding of the implications of sustainable consumption of SE products in regions where the literature in this regard is profuse (e.g., SEs in speciﬁc Asian countries). Secondly, and in relation to the keyword co-occurrence analysis conducted through the software VOSviewer, only those keywords included in the extracted publications have been considered to determine nodes and connections, forming the thematic clusters analyzed. This could limit the accurate description of the SEs and sustainable consumption landscape for two reasons: ﬁrst, due to the novel and emergent dimension of this topic within the scholarly literature and, secondly, due to the still vague conceptual arena of an emergent and evolving topic like SEs, where civil society organizational proﬁles like NGOs or NPOs, or even for-proﬁt businesses implementing CSR practices, are confused with SEs in the literature. Sustainability 2022, 14, 13428 16 of 20 16 of 20 Author Contributions: Conceptualization, N.S.-A. and N.G.-R.; methodology, N.S.-A.; software, N.S.-A.; validation, N.S.-A., N.G.-R. and S.C.-M.; formal analysis, N.S.-A.; investigation, N.S.-A., N.G.-R. and S.C.-M.; resources, N.S.-A., S.C.-M. and N.G.-R.; writing—original draft preparation, N.S.-A.; writing—review and editing, N.S.-A., N.G.-R. and S.C.-M.; supervision, N.S.-A.; project administration, N.S.-A., N.G.-R. and S.C.-M.; funding acquisition, N.S.-A., N.G.-R. and S.C.-M. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, N.S.-A. and N.G.-R.; methodology, N.S.-A.; software, N.S.-A.; validation, N.S.-A., N.G.-R. and S.C.-M.; formal analysis, N.S.-A.; investigation, N.S.-A., N.G.-R. and S.C.-M.; resources, N.S.-A., S.C.-M. and N.G.-R.; writing—original draft preparation, N.S.-A.; writing—review and editing, N.S.-A., N.G.-R. and S.C.-M.; supervision, N.S.-A.; project administration, N.S.-A., N.G.-R. and S.C.-M.; funding acquisition, N.S.-A., N.G.-R. and S.C.-M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the MICINN National Project CO-CRESEO: The co-creation of value in social enterprises. Effects of the omnichannel strategy (PID2019-109580RB-I00/AEI/10.13039/ 501100011033). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Appendix A Appendix A Appendix A Fi A1 R l ti f fi l i d bli ti Title of publication (co)Authorship Source The effects of customer socialization on customer behavior in social enterprises: role of organizational legitimacy in the eyes of customers. Chen et al. (2021) Management Decision Multi-Stakeholder Perspective on Food Labelling for Environmental Sustainability: Attitudes, Perceived Barriers, and Solution Approaches towards the “Traffic Light Index” Gröfke et al.(2021) Sustainability Determinants of Customer Intention to Purchase Social Enterprise Products: A Structural Model Analysis Lee et al.(2021) Journal of Social Entrepreneurship Consumer Behavior and Social Entrepreneurship: The Case of South Korea Lee et al.(2021) Journal of Social Entrepreneurship A study on the consumer’s intention to purchase a social enterprise’s product Lin et al.(2021) European Journal of International Management Driving forces of repurchasing social enterprise products Wu et al.(2021) Journal of Business & Industrial Marketing Social enterprise marketing: review of literature and future research agenda Bandyopadhyay & Ray (2020) Marketing Intelligence & Planning Marketing Communications of Latvian Social Enterprises from a Consumer Perspective: Practical Suggestions for Improvement Casno et al. (2020) European Integration Studies The power of information: a key component for the successful performance of Latvian social enterprises Casno et al. (2020) Proceedings of 12th International Scientific Conference New Challenges in Economic and Business Development The new competitive environment of social enterprises: an experimental study on perceptions and consumer intentions for social vs. traditional enterprises Lin-Hi et al. (2020) Int. Journal of Entrepreneurial Venturing How to meet reuse and preparation for reuse targets? Shape advertising strategies but be aware of ‘‘social washing” Rizzi et al. (2020) Waste Management In pursuit of goodwill? The cross-level effects of social enterprise consumer behaviors. Tsai et al.(2020) Journal of Business Research Factors that motivate Latvian consumers to purchase products and services from social enterprises in Latvia: The case of socially responsible consumption. Casno et al. 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https://openalex.org/W3207549851	https://europepmc.org/articles/pmc8583247?pdf=render	English	null	COVID-Transformer: Interpretable COVID-19 Detection Using Vision Transformer for Healthcare	International journal of environmental research and public health/International journal of environmental research and public health	2,021	cc-by	7,445	Abstract: In the recent pandemic, accurate and rapid testing of patients remained a critical task in the diagnosis and control of COVID-19 disease spread in the healthcare industry. Because of the sudden increase in cases, most countries have faced scarcity and a low rate of testing. Chest X-rays have been shown in the literature to be a potential source of testing for COVID-19 patients, but manually checking X-ray reports is time-consuming and error-prone. Considering these limitations and the advancements in data science, we proposed a Vision Transformer-based deep learning pipeline for COVID-19 detection from chest X-ray-based imaging. Due to the lack of large data sets, we collected data from three open-source data sets of chest X-ray images and aggregated them to form a 30 K image data set, which is the largest publicly available collection of chest X-ray images in this domain to our knowledge. Our proposed transformer model effectively differentiates COVID-19 from normal chest X-rays with an accuracy of 98% along with an AUC score of 99% in the binary classiﬁcation task. It distinguishes COVID-19, normal, and pneumonia patient’s X-rays with an accuracy of 92% and AUC score of 98% in the Multi-class classiﬁcation task. For evaluation on our data set, we ﬁne-tuned some of the widely used models in literature, namely, EfﬁcientNetB0, InceptionV3, Resnet50, MobileNetV3, Xception, and DenseNet-121, as baselines. Our proposed transformer model outperformed them in terms of all metrics. In addition, a Grad-CAM based visualization is created which makes our approach interpretable by radiologists and can be used to monitor the progression of the disease in the affected lungs, assisting healthcare. Citation: Shome, D.; Kar, T.; Mohanty, S.N.; Tiwari, P.; Muhammad K.; AlTameem, A.; Zhang Y.; Saudagar, A.K.J. COVID-Transformer: Interpretable COVID-19 Detection Using Vision Transformer for Healthcare. Int. J. Environ. Res. Public Health 2021, 18, 11086. https://doi.org/10.3390/ ijerph182111086 Academic Editors: Alireza Daneshkhah, Amin Hosseinian-Far, Samer A. Kharroubi and Vasile Palade Received: 23 September 2021 Accepted: 17 October 2021 Published: 21 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: vision transformer; COVID-19; deep learning; data science; healthcare; interpretability; transfer learning; grad-CAM Article COVID-Transformer: Interpretable COVID-19 Detection Using Vision Transformer for Healthcare Debaditya Shome 1 , T. Kar 1 , Sachi Nandan Mohanty 2 , Prayag Tiwari 3 , Khan Muhammad 4,* , Abdullah AlTameem 5, Yazhou Zhang 6 and Abdul Khader Jilani Saudagar 5,* 1 School of Electronics Engineering, KIIT Deemed to be University, Odisha 751024, India; 1 School of Electronics Engineering, KIIT Deemed to be University, Odisha 751024, India; 1804372@kiit ac in (D S ); tkarfet@kiit ac in (T K ) ( ); ( ) 2 Department of Computer Science & Engineering, Vardhaman College of Engineering (Autonomous Hyderabad 501218, India; sachinandan09@gmail.com 3 Department of Computer Science, Aalto University, 02150 Espoo, Finland; prayag.tiwari@aalto.ﬁ 4 Visual Analytics for Knowledge Laboratory (VIS2KNOW Lab), School of Convergence, College of Computing and Informatics, Sungkyunkwan University, Seoul 03063, Korea g y y 5 Information Systems Department, College of Computer and Information Sciences, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11432, Saudi Arabia; altameem@imamu.edu.sa 6 Software Engineering College, Zhengzhou University of Light Industry, Zhengzhou 450001, China; yzzhang@zzuli.edu.cn y g * Correspondence: khan.muhammad@ieee.org (K.M); aksaudagar@imamu.edu.sa (A.K.J.S.) International Journal of Environmental Research and Public Health Citation: Shome, D.; Kar, T.; Mohanty, S.N.; Tiwari, P.; Muhammad K.; AlTameem, A.; Zhang Y.; Saudagar, A.K.J. COVID-Transformer: Interpretable COVID-19 Detection Using Vision Transformer for Healthcare. Int. J. Environ. Res. Public Health 2021, 18, 11086. https://doi.org/10.3390/ ijerph182111086 1. Introduction For instance, in [13], Mittal et al. developed an unsupervised learning-based technique for COVID-19 diagnosis from multiple modalities of chest imaging. They used a novel clustering based Gravitational Search algorithm for labeling the images into covid and non-covid. They achieved an accuracy of 99.36% on the ultrasound dataset but their approach achieved only 64.41% on CT dataset. In [14], Rui et al. used a pulmonary opacity detecting model trained using unsupervised learning over a small dataset of COVID CT scan images and they achieved an accuracy of 95.5% for detection of COVID-19. g y y A few object detection-based approaches have also been used for detecting COVID- 19. For example, the authors of [15] proposed a YOLO-based object detection model for detecting and differentiating COVID-19 from other thoracic diseases. Their model achieved a detection accuracy of 90.67%. In [16], Fatima et al. used single shot multi-box detector based object detection technique which creates bounding boxes over the areas of the chest X-rays and each bounding box is classiﬁed as normal or COVID-19. They report an accuracy of 92% for classifying COVID-19. y g The most used approach for solving this task has been using deep convolutional neural networks (CNNs) with supervised learning [17–19]. As an example, a work by Mukherjee et al. [20] proposed a deep CNN-based architecture and trained it on a com- bined data set of chest X-ray and CT images where they achieved an overall accuracy of 96.28%. In [21], Li et al. proposed a stacked autoencoder model where the ﬁrst four layers consist of four autoencoders to extract better features from CT images. The ﬁnal model is built by chaining together these four autoencoders and linking them to the dense layer and the softmax classiﬁer. The authors report achieving an accuracy of 94.7% on the CT images data set. In [22], Chakraborty et al. proposed Corona-Nidaan, a lightweight deep CNN architecture trained on chest X-ray data set with three classes which achieved an accuracy of 95%. In [23], the authors used transfer learning with a VGG-16 model pre-trained on pneumonia detection and further ﬁne-tuned it over a COVID-19 detection data set achiev- ing an accuracy of 93%, although they sometimes misclassify COVID-19, viral pneumonia, and normal cases. In [24], Khan et al. 1. Introduction As of June 2021, there have been 173 million COVID-19 cases worldwide, with new cases rapidly increasing at an alarming rate and showing no signs of abating [1]. If COVID- 19 infection is not detected early enough, it can induce a ﬂu-like sickness that can proceed to acute respiratory distress syndrome (ARDS), which can be deadly [2–8]. Due to limited resources and the amount of data accessible to the scientiﬁc community, early diagnosis of COVID-19 remains a tough challenge despite recent worldwide research efforts in health- care [9]. RT-PCR has been the standard and approved diagnostic approach for COVID-19, however it has a number of drawbacks. It is costly, risky to medical staff, and there are Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 11086. https://doi.org/10.3390/ijerph182111086 Int. J. Environ. Res. Public Health 2021, 18, 11086 2 of 14 just a few diagnostic test kits accessible. Medical imaging techniques such as X-ray and CT-based screening, on the other hand, are relatively safe, faster, and more widely available. X-ray imaging has been frequently utilized for COVID-19 screening in comparison to CT imaging as it takes less imaging time, is less expensive, and X-ray scanners are commonly available even in remote regions [10]. Because of the complicated morphological patterns of lung involvement, which can ﬂuctuate in degree and appearance over time, the accuracy of a COVID-19 infection diagnosis using chest imaging is strongly reliant on radiological proﬁciency. The scarcity of competent radiologists, particularly in developing countries, affects the reliability of sophisticated chest examination interpretation. In a study by Cozzi et al. [11], it was found that chest X-ray imaging achieved a balanced real-world diagnostic performance with accuracy in the range of 76% to 86%, and 89% sensitivity. They showed that the speciﬁcity was much greater for experienced radiologists than that of less experienced ones. Deep Learning and Data Science are widely employed in many ﬁelds of medical imaging, and they have shown excellent results in Thoracic Imaging [12]. There have been many approaches for diagnosing COVID-19 from CT and X-ray images that made use of Deep Learning/Data Science recently. There have been some efforts on using unsupervised learning based approaches for this task. 1. Introduction presented a Xception network-based architecture with pre-trained weights from ImageNet, which they ﬁne tuned over a 1.2 K images data set, and they report an overall accuracy of 89.5% on multi-class classiﬁcation. In [25], the authors proposed a two-level pipeline with an image segmentation block made up of a fully connected DenseNet backbone and a classiﬁcation block where Resnet-18 was used patch-wise which achieved an accuracy of 91% upon training on a 354 images data set and upon evaluating over 99 images. In [26], Mishra et al. presented an approach with a two neural network-based system and reported a maximum accuracy of 96%. The authors of [27] used an attention based pre-trained VGG-16 model and ﬁne-tuned it over three data sets with 1125, 1638, and 2138 images, respectively. Upon evaluation, they achieved an Int. J. Environ. Res. Public Health 2021, 18, 11086 3 of 14 accuracy of 79.58%, 85.43%, 87.49% over the three data sets, respectively. Shankar et al. [28] proposed a BMO-CRNN algorithm for the covid-19 detection efﬁciently. In [29], Xueyu et al. ﬁne-tuned multiple pre-trained models over a 2500 CT scan images data set and achieved 82.5% accuracy. Luz et al. [30] proposed models based on the EfﬁcientNet family with a hierarchical classiﬁer and achieved an overall accuracy of 93.9%. Pham et al. [31] presented a comprehensive study on transfer learning for COVID-19 detection from CT images by training and comparing 16 pre-trained models. g p g p After reviewing the relevant literature, it is clear that despite the effectiveness of deep learning-based frameworks in COVID-19 identiﬁcation, there are a few ﬂaws. Most of the models have been trained and evaluated on data sets with few samples which can lead to improper generalization due to which the model might perform very poorly in real world and having a small test set might result in missing out on false positives or negatives. With this motivation, we have conducted this research with main contributions highlighted as follows: • Due to lack of large public data sets, we collected and merged three standard data sets (https://data.mendeley.com/datasets/9xkhgts2s6 (accessed on 1 May 2021)), (https: //data.mendeley.com/datasets/8h65ywd2jr/3 (accessed on 1 May 2021)), (https: //www.kaggle.com/endiqq/largest-covid19-dataset) (accessed on 1 May 2021) to form a 30 K chest X-ray images COVID-19 data set for multi-class classiﬁcation and a 20 K images data set for binary classiﬁcation. 1. Introduction These two data sets have equal number of images in each class making it the largest and balanced data set on COVID- 19 imaging-based detection available as open-source, which can help the research community in training much more accurate and generalizable models in the future. • We implemented a model based on Vision Transformer (ViT) architecture on both data sets and achieved a state-of-the-art overall accuracy of 98.4% in distinguishing COVID-19 positive from normal X-rays, and an accuracy of 92.4% in distinguishing COVID-19 from pneumonia and normal X-ray images. • For evaluation, we ﬁne-tuned multiple state-of-the-art baseline models which are widely used in literature such as Inception-V3, Resnet-V2, EfﬁcientNet-B0, MobileNet- V2, VGG-16, Xception, and DenseNet-121 on both of the data sets and compared these with our proposed model on multiple standard metrics. p p p • For better model interpretability and ease of diagnosis, we created Grad-CAM-based visualizations of COVID-19 progression in the lungs, which assists the diagnosis process for healthcare. The rest of this paper is divided into four sections, with multiple subsections within each of them. Section 2 is focused on the proposed model’s architecture and training pipeline. Section 3 discusses the ﬁne-grained details of our data collection and prepro- cessing pipeline, followed by performance evaluation, comparison with baselines, and interpretable visualizations. Section 4 presents an overview on the real-world utility of our methodology in order to assist health services during emergency times. Section 5 concludes this work. 2. Model Architecture and Pipeline This section covers information about the proposed transfer learning model as well as critical parameter evaluations, ﬁne-tuning steps, and model comparisons. 2.1. Architecture After the success of Transformers in solving natural language processing problems [32], Dosovitskiy et al. in [33] presented the Vision Transformer (ViT) model. When trained on enough data, ViT beats state-of-the-art CNN with approximately four times less computing resources. ViT tries to resemble the original transformer architecture [34] as much as possible. We designed a COVID-19 detection pipeline utilizing the Vision Transformer model and ﬁne-tuned it on our dataset with a custom MLP block. The initial part of the network has a Patch Encoder layer which reshapes the input image into multiple Int. J. Environ. Res. Public Health 2021, 18, 11086 4 of 14 ﬂattened patches. Along with the patches, positional embeddings are added to form a sequence, because only sequential data are compatible with the Transformer encoders. The Transformer encoder used is the same as that in [34] and contains multi-headed self- attention layers and multiple Multi-layer Perceptron (MLP) blocks. ViT’s self-attention layer enables it to integrate information globally throughout the full picture. To recreate the visual structure from the training data, ViT learns to encode the relative placement of the patches. Self-attention has a quadratic cost as each pixel in the image is given as input, self-attention requires each pixel to pay attention to every other pixel. Because the quadratic cost of self-attention is prohibitively expensive and does not scale to a reasonable input size, the image is separated into patches. Because it does not establish any additional dependencies between the training images, Layer Norm is used before each block which assists in reducing training time and improving generalization performance. The overall architecture has been illustrated in Figure 1. Figure 1. The proposed ViT model for COVID-19 detection. Figure 1. The proposed ViT model for COVID-19 detection. 2.2. Fine-Tuning Procedure 2.2. Fine-Tuning Procedure We used the ViT L-16 model for the initial stage of our model, which is the “Large” variant with a patch size of 16 × 16. The ViT model had pre-trained weights from training on ImageNet data [35]. This initial stage consists of 23 transformer encoder layers stacked on top of each other. We removed the pre-trained MLP prediction block and attached an untrained set of feed-forward layers constituting the custom MLP block which can be seen in Figure 2. The ﬂattened output of the ﬁnal transformer encoder is passed through two sets of batch normalization and dense layers constituting the MLP block. Batch normalization is a neural network layer that allows the model’s other layers to learn more independently [36]. It is used to make the output of the preceding layers more natural and to make the activations scale the input layer. Learning becomes more efﬁcient when batch normalization is utilized, and it may also be employed as a regularization to prevent model overﬁtting. The ﬁrst dense layer consists of a Gaussian error linear unit (GELU)-based activation with 120 neurons. GELU has been widely used in revolutionary transformer models such as GPT-2 [37], BERT [38], and also in vision transformers [33] due to its deterministic nonlinearity that encapsulates a stochastic regularization effect [39], which leads to a major performance boost in most models with complex transformer architectures. Int. J. Environ. Res. Public Health 2021, 18, 11086 5 of 14 The last dense layer has softmax activation, and we use L2 regularization [40] to minimize overﬁtting as much as possible. The last dense layer has softmax activation, and we use L2 regularization [40] to minimize overﬁtting as much as possible. Figure 2. Custom MLP block attached to the Vision Transformer. Figure 2. Custom MLP block attached to the Vision Transformer. 2.3. Model Training Mechanism 2.3. Model Training Mechanism We use the NovoGrad optimizer with a categorical cross-entropy loss function to train our model for multi-class classiﬁcation, and binary cross-entropy loss in the case of binary classiﬁcation. In each case, label smoothing of 0.3 was added which helps to make the neural network generalize on unseen data by adding noise to the labels [41]. NovoGrad performs similarly to SGD but with gradient normalization per layer, making the optimizer more robust to initial learning rate selection. When compared to Adam, NovoGrad uses less memory and is more numerically stable due to which the training time of our model reduced without a drop in performance. It broadens Adam and decouples weight decay from regularization. It also has half the memory cost of Adam and similar memory needs to SGD with momentum. We also use the adaptive learning rate scheduler and callbacks from the Keras library [42] which automatically reduces the learning rate and stops overtraining the model if the validation accuracy does not improve. The data set was randomly split into train/validation/test sets with 75%/15%/10% of all the images, respectively. g p y As seen in Figure 3, multiple metrics were monitored during the training process where all of those showed a progressively increasing curve even on validation. 1. Accuracy: The most common performance metric in any classiﬁcation problem is the accuracy metric. For the multi-class classiﬁcation, the categorical accuracy was chosen which resembles the average accuracy over all the three classes of chest X-ray images. The binary classiﬁcation involved the binary accuracy metric which measures how many times the predicted label matches the true label for the chest X-ray image. y p y g 2. AUC score: The area under the ROC curve (AUC) score shows how well predictions are ranked across all the classes and how much the model can distinguish between each class. It ensures that performance across all feasible categorization criteria is aggregated. It has been proved in the literature that AUC score is a more robust metric to measure the ability of a classiﬁer than the accuracy [43]. 3. Precision: Precision is deﬁned as the number of true positives divided by the number of true positives plus the number of false positives. Int. J. Environ. Res. Public Health 2021, 18, 11086 6 of 14 4. 2.3. Model Training Mechanism Recall: Recall is deﬁned as the number of true positives divided by the number of true positives plus the number of false negatives. 4. Recall: Recall is deﬁned as the number of true positives divided by the number of true positives plus the number of false negatives. 4. Recall: Recall is deﬁned as the number of true positives divided by the number of true positives plus the number of false negatives. Figure 3. Performance measures during training. Figure 3. Performance measures during training. Figure 3. Performance measures during training. 7 of 14 Int. J. Environ. Res. Public Health 2021, 18, 11086 3. Experimental Results and Discussion 3.1. Data Set We constructed a three-class data set of 30 K chest X-ray pictures with labels COVID-19—for patients with COVID-19 infection, normal—for stable patients, and pneumonia—for patients with viral and bacterial pneumonia, following the pattern of likely classes reported in the literature. We took 5500 COVID images and 4044 normal images from El-Shafai et al. [44]. Another 1281 COVID-19 images, 3270 normal images, and 4657 pneumonia images were taken from Sait et al. [45]. Finally, we took 3000 normal images, 6045 pneumonia images, and 4038 COVID-19 images from the COVID-Ti data set by Qi et al. [46]. The distribution of the aggregated data has been visually illustrated in Figure 4. Figure 4. Data set distribution and description Figure 4. Data set distribution and description Figure 4. Data set distribution and description To make our data set completely balanced, we sampled top 10 K images from each class by ranking them based on resolution, making it a chest X-ray data set of 30 K images for COVID-19 detection. This is, to the best of our knowledge, the largest open source collection of chest X-ray images for the detection of COVID-19 and pneumonia till date. 3.2. Preprocessing 3.2. Preprocessing Each image in the gathered data set is passed through a minimal image pre-processing pipeline which ensures to make all images compatible for the model training. The following are the steps in the pipeline: 1. Resize: As neural network models have a ﬁxed-size input layer, all images must be scaled to the same size. Therefore, we resize all the images in the data set to 224 × 224 pixels. p 2. Interpolation: There are a few images in the data set which are of size lesser than 224 × 224. While increasing their size, the estimation of new pixels needs to be done efﬁciently in order to retain quality. This process is termed “Interpolation” of images. For our pipeline we used the nearest neighbor interpolation, in which the closest pixel value to the supplied input coordinates is used to approximate the output pixel value. This approach is straightforward to implement, and there is no bogus data in the end result [47]. 3.3. Data Augmentation In order to develop accurate and generalizable deep learning models, supervised learning requires large amounts of data. In our training pipeline, we employed a variety Int. J. Environ. Res. Public Health 2021, 18, 11086 8 of 14 of data augmentation techniques such as random rotation, width shift, height shifts, and ﬂipping which have been demonstrated in the literature to be beneﬁcial in increasing deep learning model performance [48,49]. of data augmentation techniques such as random rotation, width shift, height shifts, and ﬂipping which have been demonstrated in the literature to be beneﬁcial in increasing deep learning model performance [48,49]. 3.4. Testing Environment All the training and testing pipelines for the proposed models, as well as baselines were implemented using TensorFlow 2.4 framework [50] in a Python 3.8 virtual envi- ronment. The graphics processing unit used in the training pipeline was a 4.1 TFLOPS Tesla K80. The net available RAM was 24 GB. Jupyter notebooks were utilized to conduct the experiments. 3.6. Ablation Experiments In order to ensure that our transfer learning architecture is optimal, we conduct a comprehensive ablation study on the multi-class classiﬁcation data set. We ﬁrst experiment by modifying the custom block using different number of layers, activations, and order of layers. First, we observe that using only one dense layer with Batch normalization and ReLU activation, the accuracy drops down to 90%. Upon removing the Batch normalization the accuracy further degrades to 89%. However, if we replace ReLU with the GeLU activation function, a single dense layer with Batch normalization achieves an accuracy of 91% and 90% without Batch normalization. This shows that GeLU activation is slightly more effective in processing the outputs of the stacked transformer encoders compared to the ReLU activation. Next, we further experiment with a custom MLP block of two GeLU activated dense layers with and without batch normalization, where the accuracy increases to 92% and 91%, respectively. However, if we further add another dense layer with and without batch normalization, the training accuracy increases whereas the testing accuracy drops to 88% and 89%, respectively. This is a clear indication that our model results in overﬁtting beyond 2 dense layers, thus we decide to keep a custom MLP block with two batch normalization and dense layers in the ﬁnal architecture. 3.5. Model Evaluation Our proposed COVID-Transformer was evaluated over the test set of both of our multi-class and binary classiﬁcation data sets. It can be observed from Figure 5a that our model is capable of distinguishing between all the three classes very accurately. Although the number of false positives and true negatives is lower, the model sometimes confuses between COVID-19 and other types of pneumonia, which is acceptable as COVID-19 is itself a form of pneumonia and it is very tough even for expert radiologists to distinguish between the two. As observed in Figure 5b, our proposed model performs extremely well over the binary classiﬁcation data set with only 21 out of 1000 images misclassiﬁed. The overall performance metrics over the test data sets have been outlined in Table 1. The multi-class classiﬁcation model works well, with an accuracy of 92% and an AUC score of 98%. In this situation, the accuracy is lower than the AUC score because only images projected as pneumonia but really COVID-19 are misclassiﬁed, while all other categories are correctly classiﬁed, hence the AUC score is not much affected. The binary classiﬁcation model achieves an accuracy of 98% and an AUC score of 99% which is suitable for real- world deployment as a diagnosis tool for detecting COVID-19 as it has signiﬁcantly higher performance than standard RT-PCR tests. 9 of 14 Int. J. Environ. Res. Public Health 2021, 18, 11086 Figure 5. Cont. Figure 5. Confusion matrix for both types of classiﬁcation. Figure 5. Cont. Figure 5. Cont. Figure 5. Cont. Figure 5. Cont. Figure 5. Confusion matrix for both types of classiﬁcation. Figure 5. Confusion matrix for both types of classiﬁcation. Int. J. Environ. Res. Public Health 2021, 18, 11086 10 of 14 Table 1. Evaluation of the proposed model. Model Accuracy Precision Recall F1 Score AUC Binary-class 0.98 0.97 0.97 0.97 0.99 Multi-class 0.92 0.93 0.89 0.91 0.98 3.6. Ablation Experiments Table 1. Evaluation of the proposed model. 3.7. Comparison with Baseline Models As our data set has not been evaluated using other models in the literature, we ﬁne- tuned some of the widely used state-of-the-art models on both variants of our data set. All the data preparation and image augmentation steps are same for the baselines, ex- cept for some of the preprocessing functions which are necessary for input to the models. For Inception-V3 and Xception ﬁne-tuning, the images were re-sized to 299 × 299 pixels. The same data augmentation techniques as for our proposed COVID-Transformer model were used. The MobileNet-V2, ResNet-V2-50, DenseNet-121, VGG-16, and EfﬁcientNet-B0 models have a standard size requirement of 224 × 224 pixels, which is same as of our COVID-Transformer model, thus we used the same data preprocessing and augmenta- tion steps for ﬁne-tuning them. From Table 2, it can be noted that among the baselines MobileNet-V3 and Xception perform the best with 90% Accuracy on the multi-class classi- ﬁcation problem. Our COVID-Transformer model outperforms all the baselines in terms of accuracy, precision, F1 score, and AUC score. Table 2. Performance comparison of our COVID-Transformer with baseline models on the multi-class classiﬁcation problem. Model Accuracy Precision Recall F1 Score AUC Inception-V3 [31] 0.90 0.89 0.91 0.89 0.92 EfﬁcientNet-B0 [30] 0.89 0.88 0.89 0.88 0.92 MobileNet-V2 [31] 0.90 0.90 0.89 0.90 0.92 ResNet-V2 [29,31] 0.88 0.87 0.86 0.86 0.93 VGG-16 [23,27,29,31] 0.87 0.87 0.85 0.86 0.90 Xception [24,31] 0.90 0.92 0.87 0.90 0.93 DenseNet-121 [25,29,31] 0.88 0.90 0.85 0.87 0.92 COVID-Transformer (Ours) 0.92 0.93 0.89 0.91 0.98 3.8. Grad-Cam Visualization Table 2. Performance comparison of our COVID-Transformer with baseline models on the multi-class classiﬁcation problem. 3.8. Grad-Cam Visualization For better visual representation and model interpretability, the Grad CAM Map-based illustration introduced by Selvaraju et al. [51] is shown in Figure 6. The Grad CAM Map Int. J. Environ. Res. Public Health 2021, 18, 11086 11 of 14 11 of 14 visualization has the capability to highlight affected areas of the lungs that are signiﬁcant for disease predictions as well as disease development. The images are obtained by passing the output of the embedding layer present in our model at the beginning just after the input layer. visualization has the capability to highlight affected areas of the lungs that are signiﬁcant for disease predictions as well as disease development. The images are obtained by passing the output of the embedding layer present in our model at the beginning just after the input layer. Figure 6. Grad-CAM visualization for the three classes. Figure 6. Grad-CAM visualization for the three classes. Figure 6a shows a normal patient with no disease having no highlighted region in the lungs. Figure 6b shows pneumonia patient’s lungs with affected regions highlighted in blue and green. Figure 6c shows a COVID-19 infected patient with mostly yellow and red highlighted regions which indicate severe infection. The ﬁgure clearly shows that our suggested methodology recognizes and differentiates relevant impacted areas from COVID-19 and other pneumonia images. COVID-19 impacts the lungs considerably more intensively than other types of pneumonia, thus our model emphasizes this by highlighting yellow and red areas in the COVID patient’s X-ray image. 4. Case Study in Medical Services Health systems in both rich and poor nations were overburdened by the COVID-19 outbreak. Sustainable Development Goals (SDGs) planned for 2025 will be affected by the pandemic-related losses, there is no question about it. As a result of the epidemic, there was a window of opportunity to take use of current digital solutions and discover new ones. These solutions can aid in the fulﬁllment of the SDGs, particularly those that pertain to health. In this sense, achieving global health coverage is an important SDG. Early diagnosis is an important factor in reducing the number of deaths from COVID-19, which almost becomes impossible when there is a steep rise in infections concentrated in a particular location. If an infected individual is isolated at the right time, multiple infections from further transmissions can be prevented. Our proposed method for X-ray based detection of COVID-19 would be an efﬁcient addition to the healthcare system boosting the global health coverage. It can be used as an aid for radiologists to reduce human errors in their diagnosis, as well as can be used as a single tool to detect COVID-19 in places where radiologists are not adequate due to infections rising at a breakneck pace. Figure 7 shows the typical ﬂow of our method when deployed in a real-world setting to have zero human error diagnosis. 12 of 14 12 of 14 Int. J. Environ. Res. Public Health 2021, 18, 11086 Figure 7. Flow of deployable solution. Figure 7. Flow of deployable solution. Data Availability Statement: https://github.com/DebadityaShome/COVID-Transformer. Acknowledgments: The authors extend their appreciation to the Deputyship for Research & Inno- vation, Ministry of Education in Saudi Arabia for funding this research work through the project number 959. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions In this research, we proposed a robust and interpretable deep learning model that can efﬁciently diagnose COVID-19 infection at scale in real-world situations for healthcare. For this objective, a 30 K chest X-ray image collection was produced by combining several open-source data sets. The model architecture chosen was based on the Vision Transformer and it showed high performance with accuracy and AUC score as high as 98% and 99%, respectively. For making our model trustworthy, we made an interpretable inference pipeline with Grad-CAM based visualizations per image. We believe that with the help of our proposed approach, chest X-ray images can also be used as a crude and low-cost bedside diagnostic tool for detecting COVID-19. This may be extremely valuable in areas where quick testing is unavailable, and it may also be used as a second screening method after the standard RT-PCR test to verify that any true negative or false positive cases do not occur. Our future work will focus on proposing another variant of the Vision Transformer for further improving the performance, given the availability of larger data sets. Author Contributions: D.S., T.K., S.N.M., P.T., K.M. and Y.Z. contributed to the concept and method- ological parts. D.S. did the experimental analysis along with T.K., S.N.M., P.T., K.M. and Y.Z. assisted in experiments and also provided the feedback. 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https://openalex.org/W4389574837	https://bmcmusculoskeletdisord.biomedcentral.com/counter/pdf/10.1186/s12891-023-06923-8	English	null	Positive outcomes following Autologous Matrix-Induced Chondrogenesis (AMIC) in the treatment of retropatellar chondral lesions: a retrospective analysis of a patient registry	BMC musculoskeletal disorders	2,023	cc-by	6,301	Positive outcomes following Autologous Matrix‑Induced Chondrogenesis (AMIC) in the treatment of retropatellar chondral lesions: a retrospective analysis of a patient registry J. Gille1* , E. Reiss2, P. Behrens3, R. P. Jakob4 and T. Piontek5 J. Gille1* , E. Reiss2, P. Behrens3, R. P. Jakob4 and T. Piontek5 © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background The patellofemoral joint is a challenging environment for treating chondral defects. Among the sur- gical options for the treatment of chondral defects, the single-stage Autologous Matrix-Induced Chondrogenesis (AMIC) procedure uses a porcine collagen I/III membrane to enhance bone-marrow stimulation. However, longer term outcomes data are rare for this specific indication. In order to provide real-world information, an ongoing registry has been established to record patient data and outcomes when AMIC is used to treat chondral and osteochondral lesions. Methods Patient data were retrieved from an ongoing, prospective, multisite registry of patients who had under- gone AMIC treatment of chondral defects. We identified 64 patients who had undergone AMIC for patellofemoral chondral defects and for whom pre-operative and at least 1 post-operative score were available were included in this retrospective data analysis. Outcomes were assessed via the KOOS, VAS pain, and the Lysholm scores. Outcomes at the post-operative time-points were analysed using a factorial ANOVA with post-hoc testing while linear regression was used to assess associations between the change in the Lysholm score and lesion size. Results There was a significant improvement in Lysholm, VAS pain, and KOOS scores from pre-operative to the 1st year post-operative (p < 0.001), and this was maintained during the follow-up. Conclusions The forces exerted on the patellofemoral joint make this a challenging scenario for chondral repair. Our data demonstrates that the AMIC procedure with a collagen I/III membrane is an effective treatment for retropatel- lar cartilage lesions, and provides reliable results, with decreased pain and improved function. Importantly, these improvements were maintained through the follow-up period. Keywords Cartilage, Patella, AMIC, Autologous matrix-induced chondrogenesis, Chondral justus_gille@usa.net Full list of author information is available at the end of the article Open Access Open Access Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 https://doi.org/10.1186/s12891-023-06923-8 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 https://doi.org/10.1186/s12891-023-06923-8 BMC Musculoskeletal Disorders © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 2 of 9 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Introduction undergone repair of chondral lesions via this procedure [17, 18]. Documentation was made on electronic case report forms, with surgeons having access to the reg- istry via a web interface. Surgeons had access to their own patients’ data, whereas the summary and overall performance data were anonymized. All patients were educated and informed in detail about the AMIC tech- nique as well as alternative procedures. Thereafter, the patients who chose to undergo AMIC were enrolled in the registry. All patients signed an informed con- sent to participate in the registry, and all treatment and follow-up examinations followed the standard of care, with no additional visits imposed on the patients. Ethical approval for this study was obtained from the ethics review board of the University of Lübeck (file no. 19–178). Because the registry has no provision for radiographic follow-up, data regarding the develop- ment of radiographically verified osteoarthritis were not available. Articular cartilage is essential to the proper function- ing of synovial joints, as it transmits loads and provides an articulating surface with a low coefficient of friction [1, 2]. Unfortunately, articular cartilage has minimal regenerative capacity due to its avascular and hypocel- lular character [3]. In the patellofemoral joint (PFJ), this low friction environment is essential as the patella glides in the trochlear groove, enhancing the moment arm of the quadriceps [4] as well as contributing to the optimization of control strategies in locomotion [5]. While patellofemoral compressive forces have been cal- culated to be somewhat more than 4 times body weight [6] there is also data that shows these can be as high as 11 times body weight [7]. Thus, the gliding movement and the high compressive forces create a challenging environment for articular cartilage. In considering the biomechanical complexity and the forces exerted on the articular surfaces, it is not surpris- ing that the patellofemoral (PF) joint is a common site of pathology, whether chronic or acute. A systematic review had noted that PF defects comprised 37% of the knee chondral lesion sites [8]. This is consistent with the conditions that had been recorded for over 5000 arthro- scopic surgeries, in which it was reported that PF defects accounted for the majority of the lesions [9]. Patientsh The data was based on a prospective registry for AMIC patients, which has previously been published concern- ing a larger cohort [18]. Patients were included in this analysis if their retropatellar chondral lesion had been treated via AMIC and they had completed pre- and post-operative measures of knee function and pain. The indication for chondral repair was a symptomatic, circumscribed cartilage lesion on the retropatellar sur- face, with an Outerbridge classification of grade III or IV. The main exclusion criteria were concomitant surgery at the time of the index procedure (e.g. ACL reconstruction), advanced osteoarthritis, significant narrowing of the joint lines, underlying rheumatic dis- ease, total meniscectomy, or deviation of the mechani- cal axis of the affected compartment. Baseline data collection included surgical history, lesion size, concur- rent procedures, age, BMI, and sex. Data were collected at baseline and at each subsequent folow-up visit, which had followed the standard of care. As this was a registry-based study, including all patients who fulfilled the inclusion and exclusion criteria, we did not perform an a priori power analysis. While first-line treatment will often be conservative management [10], persistent symptoms may indicate a need for surgery [11]. Among surgical options, a single- stage procedure known as autologous matrix induced chondrogenesis (AMIC) uses bone marrow stimulation techniques and then covers the treated site with a bilayer collagen I/III membrane (Chondro-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) in order to protect the blood clot that contains the factors released from the bone marrow stimulation, thus providing a biological chamber for repair tissue to mature [12, 13]. However, the data that has focused specifically on patellar lesions has been limited, with relatively short follow-up [14] or small cohorts [15, 16]. With a paucity of data on the repair of PF lesions, we conducted a retrospective review of patient outcomes, using data that had already been entered into a registry, in order to assess the role that this procedure can play in treating focal chondral defects on the retropatellar surface. Treatmenth The operative procedure was performed through either a mini-open approach or arthroscopically, at the discretion of the surgeon. Study designh After debridement and careful removal of degenera- tive and loose cartilage fragments bone marrow stimu- lation was performed using a 1.2mm drill or k-wire in order to perforate the subchondral bone plate, typically to a depth of approximately 1 cm, resulting in bleeding This study is an analysis of the data of patients who had been treated with AMIC and were prospectively enrolled in a registry. The registry is an ongoing, mul- ticentre database designed to longitudinally track changes in function and symptoms in patients who have Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 3 of 9 and release of bone marrow stem cells and other fac- tors into the defect. The prepared chondral defect was then covered with a collagen I/III membrane of por- cine origin (Chondro-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) that was previously cut to fit the defect size by using an aluminium template. Next, the membrane was fixed either by sutures or a fibrin sealant, depending on the surgeon’s preference. Previ- ous research has shown that these 2 fixation methods provide equivalent results. [19, 20]. The knee joint was held in an extended position for 5 min before the joint was flexed ten times to test the stability and position of the matrix. Figure 1 illustrates the steps during an open AMIC procedure. The incision was then closed in layers with standard techniques, and a drainage without suction was applied. The knee was immobi- lized for the first few days followed by continuous pas- sive motion with restricted knee angles and limited weight bearing for approx. 6 weeks. worst pain the patient has known, while functional outcomes were assessed using the Lysholm score and the KOOS, both of which are validated, functional scores. [21, 22]. Because the data from this study were based on a registry, which followed standard of care, there were no additional, predefined, clinical follow- up visits. Each clinic had maintained contact with their patients, and actively motivated them to adhere to the follow-up protocol as well as sending follow-up questionnaires to patients. There was no radiographic follow-up after AMIC in the registry, and additional radiographic exams had not been described in the original consent and were not part of the standard of care follow-up. Consequently, data regarding the development of radiographically verified osteoarthritis are not available. Study designh Patients were not financially com- pensated for their time in completing the data-collec- tion forms. Patient demographicsh The most common surgical approach was mini-open, the most common fixation method was using fibrin glue and the mean length of follow-up was 5.8 years with a range from 1 – 10 years. Of the 64 patients included in this analysis, 56 patients have a follow-up of 2 or more years. The patient demographics are presented in Table 2. There were no significant differences when the lesion size, age or BMI were compared relative to the sex of the patient. Of these patients, sport was the most commonly listed cause of the injury. Table 2 The characteristics of the patients that were included in this study Table 2 The characteristics of the patients that were included in this study N Lesion size (cm2) Age (years) BMI Male 32 2.9 ± 1.4 36.1 ± 15.4 24.9 ± 3.2 Female 33 3.1 ± 1.4 33.7 ± 11.6 23.9 ± 5.2 Table 2 The characteristics of the patients that were included in this study N Lesion size (cm2) Age (years) BMI Male 32 2.9 ± 1.4 36.1 ± 15.4 24.9 ± 3.2 Female 33 3.1 ± 1.4 33.7 ± 11.6 23.9 ± 5.2 Outcomes assessment We did not conduct a power analysis to estimate the sample size, as this registry study included all patients who had undergone AMIC surgery for the repair of a At each follow-up, patients rated their pain using the VAS, with 0 indicating no pain and 10 indicating the Fig. 1 a Prepared retropatellar defect with a stable, perpendicular cartilage shoulder surrounding the defect; b bone marrow stimulation using a k-wire; c inserting cut membrane into prepared defect; d Chondro-Gide membrane in place ig. 1 a Prepared retropatellar defect with a stable, perpendicular cartilage shoulder surrounding the defect; b bone marrow s k-wire; c inserting cut membrane into prepared defect; d Chondro-Gide membrane in place Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 4 of 9 Page 4 of 9 Table 1 The number of patients with available data at each time point Visit n Pre-operative 64 Year 1 40 Year 2 24 Year 3 17 Year 4 12 Year 5 7 Year 6 11 Year 7 4 Year 8 6 Year 9 11 Year 10 10 Resultsh The current registry database was queried in order to find all patients who had been treated for a patellar, chondral lesion by one of the 5 co-authors. Of the 86 patients that were identified, only those who had pre-operative and at least 1 post-operative outcome measure were included in the statistical analysis. There were no other exclusion criteria. This resulted in 64 patients being included in this report. The number of patients at each time point is shown in Table 1. Lysholmh There was significant difference in the scores (P < 0.001) and post-hoc tests revealed that the pre-operative Lysholm score was significantly different from all post- operative scores, as depicted in Fig. 2. There were no significant differences noted between any of the post- operative scores. patellar chondral lesion. The outcome variables, VAS pain, KOOS subscales and Lysholm, were evaluated via a factorial analysis of variance (ANOVA) across all time points. Exploratory analyses were conducted to test for relationships between the change in scores from baseline and the patients’ BMI, age and defect size. The a priori alpha level was set at p = 0.05. Statis- tical analyses were performed using MedCalc, version 19.4 (MedCalc Software, Ostend, Belgium). patellar chondral lesion. The outcome variables, VAS pain, KOOS subscales and Lysholm, were evaluated via a factorial analysis of variance (ANOVA) across all time points. Exploratory analyses were conducted to test for relationships between the change in scores from baseline and the patients’ BMI, age and defect size. The a priori alpha level was set at p = 0.05. Statis- tical analyses were performed using MedCalc, version 19.4 (MedCalc Software, Ostend, Belgium). KOOS Scores According to recommendations for use of the KOOS score, [23] we did not take into account the total KOOS Fig. 2 The scores for the Lysholm test across all time points Fig. 2 The scores for the Lysholm test across all time points Fig. 2 The scores for the Lysholm test across all time points Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Gille et al. BMC Musculoskeletal Disorders Page 5 of 9 difference between the pre-operative mean of 5.7 ± 1.8 and all post-operative scores (p < 0.001). It should be noted that there were no significant differences between the pain score at any of the post-operative time points. in our results but separately evaluated the 5 domains (symptoms, pain, quality of life, sport and leisure and activities of daily life). We noted a significant improve- ment in all the domains of the KOOS. The KOOS score for symptoms improved from a mean of 48.7 ± 23 to a mean of 87.5 ± 12.5 (p < 0.001) (Fig. 3) The pain sub- scale improved from a mean of 52.4 ± 16.3 to 89.8 ± 13.2 (p < 0.001), while quality of life (QoL) more than doubled, going from 26.8 ± 178 pre-op to 83.3 ± 14.6 post-oper- atively (p < 0.001) (Fig. 4) and sports and leisure activi- ties showed a similarly large improvement, rising from 21.7 ± 14.4 pre-operatively to 85 ± 12.6 post-operatively (p < 0.001) while the KOOS domain for activities of daily living (ADL) improved from 55.2 ± 21.2 pre-operatively to 92.6 ± 8.8 post-operatively (p < 0.001). in our results but separately evaluated the 5 domains (symptoms, pain, quality of life, sport and leisure and activities of daily life). We noted a significant improve- ment in all the domains of the KOOS. The KOOS score for symptoms improved from a mean of 48.7 ± 23 to a mean of 87.5 ± 12.5 (p < 0.001) (Fig. 3) The pain sub- scale improved from a mean of 52.4 ± 16.3 to 89.8 ± 13.2 (p < 0.001), while quality of life (QoL) more than doubled, going from 26.8 ± 178 pre-op to 83.3 ± 14.6 post-oper- atively (p < 0.001) (Fig. Regressionsh The exploratory analysis examined relationships between demographic variables and the Lysholm score at the last follow-up. While neither age nor BMI showed a relation- ship with the Lysholm score at the last follow-up, there was a trend (p = 0.073) towards a relation between defect size and improvement in Lysholm score, as shown in Fig. 6. Although not significant, the data suggests that patients with larger defects tended to exhibit greater changes in Lysholm score at their last follow-up visit. KOOS Scores 4) and sports and leisure activi- ties showed a similarly large improvement, rising from 21.7 ± 14.4 pre-operatively to 85 ± 12.6 post-operatively (p < 0.001) while the KOOS domain for activities of daily living (ADL) improved from 55.2 ± 21.2 pre-operatively to 92.6 ± 8.8 post-operatively (p < 0.001). Discussionh The results of this study, showing a sustained improve- ment in patient outcomes for several years after surgery, supports the use of AMIC Chondro-Gide® as a surgical treatment of chondral lesions of the patella. We observed a significant improvement between the pre-operative and post-operative scores related to function (Lysholm and Pain Similar to the previous outcomes that have been listed, the VAS score for pain (Fig. 5) showed a significant In evaluating clinical data, we also considered the min- imal clinically important difference (MCID) in the Fig. 3 The scores for the KOOS subscale for symptoms, at each time point in the follow-up period Fig. 4 QoL subscale over the follow-up period Fig. 3 The scores for the KOOS subscale for symptoms, at each time point in the follow-up period Fig. 3 The scores for the KOOS subscale for symptoms, at each time point in the follow-up period Fig. 3 The scores for the KOOS subscale for symptoms, at each time point in the follow-up period Fig. 4 QoL subscale over the follow-up period Fig. 4 QoL subscale over the follow-up period Fig. 4 QoL subscale over the follow-up period Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 6 of 9 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 outcomes. This has been reported to be 10.1 points for a review of the patient records at each site did Fig. 5 The change in VAS over time Fig. 6 The regression plot that shows the change in Lysholm score relative to the patient’s defect size Fig. 5 The change in VAS over time Fig. 5 The change in VAS over time Fig. 6 The regression plot that shows the change in Lysholm score relative to the patient’s defect size Fig. 6 The regression plot that shows the change in Lysholm score relative to the patient’s defect size a review of the patient records at each site did not reveal complications in this cohort, the loss to follow-up may have been influenced by reoperations or failures. outcomes. This has been reported to be 10.1 points for Lysholm [24] while the KOOS Sports and Recreation sub- scale has been reported to be 30 points, [25]. Therefore, among the patients who exceeded the MCID for Lysholm at their last follow-up visit, we noted a responder rate of 0.83 for the Lysholm tests. For the sport and recreation subscale of the KOOS, the responder rate was 0.84. Complications and failures When the registry was initiated in 2002 it was not designed to capture complications and failures. Although Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 7 of 9 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 rate of 0.83, which is somewhat better than the responder rate of 0.7 that was recently reported for ACI [31]. Considering that a majority of patients presented with injuries resulting from sports activities, we also felt it would be worthwhile to examine the responder rate with regard to the KOOS sport and recreation sub- scale, with an MCID calculated as 30 points [25]. Our patients cohort showed a responder rate of 0.84 for this subscale. KOOS) as well as a significant reduction in pain (VAS). Among our patients, the improvements in all KOOS subscales, well above the MCID and with a correspond- ingly high responder rate, indicates that the treatment is an effective means of treating patellar chondral lesions. Since there were no significant differences noted between any of the post-operative time points it seems appar- ent that the improvements in the patients’ outcomes are maintained throughout the follow-up period of this study.h Recent data has noted that concomitant corrective surgery for patellar instability results in low failure rate with satisfactory clinical outcomes at mid-term follow- up [14]. Among the cohort in our study, corrective tib- ial osteotomies for some patients had been recorded, but the numbers do not allow for a meaningful compar- ison of outcomes data with regard to this aspect of the procedure. The necessity of physiological knee func- tion, as a component of chondral repair, was clearly emphasized with regard to proper alignment being a critical factor in long-term success of cartilage repair [32]. Indeed, it has recently been stated that cartilage repair without respecting alignment is fruitless [33]. These results are consistent with our own experience in the knee, in which we noted positive outcomes up to 7 years postoperatively [18]. In that dataset, we had recorded outcomes after treatment of lesions on all artic- ulating surfaces of the knee. The results from our earlier paper and our current patellofemoral data are consistent with studies that have reported positive longer-term out- comes in the hip [26] and in the ankle [27] when AMIC with Chondro-Gide was used in the repair of focal chon- dral lesions. Complications and failures In addition to the results we have reported here, as well as our previous data [18] other publications have detailed positive patient outcomes following AMIC in the knee. A case series of patients treated with AMIC for large chon- dral knee defects has shown consistent improvements in patient outcomes over 7 years [28]. An RCT, which investigated the use of biological adjuvants in the AMIC procedure, had reported sustained benefits up to 9 years status post [29]. Specific to the patellofemoral joint, a small cohort study with 5 years follow-up had stated that AMIC is considered to be a reliable one-step alterna- tive for the treatment of large, isolated patellar cartilage defects [16]. p p g g Certainly, our study is not without limitations. While a registry collects real-world data based on all patients, the follow-up can be problematic. In contrast to an RCT with dedicated time points, the follow-up in a registry simply follows standard of care. As an exam- ple, if a patient is unsatisfied with their treatment and they seek treatment elsewhere, then they will be lost to follow-up, thus reinterventions or revision surgery will not be captured. In the recent study the number of patients who had data at each post-operative time point decreased as time progressed from the baseline evalu- ation, which is a limitation of the registry and one that would have certainly decreased the power of the statis- tical analysis. With regard to the statistical analysis, in a clinical study in which the patients are seen at sev- eral pre-established timepoints, a repeated-measures analysis of variance is typical. However, this registry had collected post-operative data on patients when they were seen in routine visits that followed standard of care. Thus the lack of planned visits, set to a time schedule in the study protocol, precluded a repeated- measures ANOVA. Therefore, the factorial analysis, which may limit the power of the study, was a realistic method of statistical analysis. Furthermore, we did not collect radiographs outside of the normal standard of care. While an assessment of such images can provide research utility, all of the surgeons would have used imaging as part of the normal treatment and would have detected any anomalies in the course of treatment. For these reasons, as well as ethics and cost, there were no additional radiographs. Complications and failures While MFx is often considered a first line treatment for focal, chondral defects, due to its simplicity and low- cost, the data on its durability, in terms of pain relief and improved function, may make other options more attrac- tive. A randomized controlled trial (RCT) that com- pared microfracture (MFx) to AMIC reported that while AMIC showed a sustained benefit, the outcomes for MFx patients started to worsen between the 2-year and 5-year follow-ups [19]. Similarly, it was seen in a case–control study with 4-year follow-up that the AMIC procedure for the treatment of patellar chondral defects results in better IKDC and Lysholm scores along with a significant reduction of the VAS score [30]. Additionally, the Tegner scale demonstrated that patients exhibited a higher level of activity after AMIC, relative to MFx, while the AMIC group evidenced a lower rate of failure [30]. Additionally, we took into consideration the MCID in the assessment of outcomes. The MCID for Lysholm and VAS has been reported to be 10.1 and 2.7, respec- tively [24] and among this cohort, we had a responder Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Page 8 of 9 4. Wheatley MGA, Rainbow MJ, Clouthier AL. Patellofemoral Mechanics: a Review of Pathomechanics and Research Approaches. Curr Rev Musculo- skelet Med. 2020;13(3):326–37. 4. Wheatley MGA, Rainbow MJ, Clouthier AL. Patellofemoral Mechanics: a Review of Pathomechanics and Research Approaches. Curr Rev Musculo- skelet Med. 2020;13(3):326–37. Declarations 14. Waltenspül M, Suter C, Ackermann J, Kühne N, Fucentese SF. Autologous Matrix-Induced Chondrogenesis (AMIC) for Isolated Retropatellar Carti- lage Lesions: Outcome after a Follow-Up of Minimum 2 Years. Cartilage. 2021;13(1_suppl):1280S–90S. Funding O A Open Access funding enabled and organized by Projekt DEAL. This study was not supported by external research funding. The authors received no funding for this study. 12. Benthien JP, Behrens P. The treatment of chondral and osteochondral defects of the knee with autologous matrix-induced chondrogenesis (AMIC): method description and recent developments. Knee Surg Sports Traumatol Arthrosc. 2011;19(8):1316–9. Availability of data and materials Datasets generated and analysed during current study are not publicly avail- able due to controlled personal data agreement and data security. 13. Zhang C, Cai YZ, Lin XJ. One-step cartilage repair technique as a next generation of cell therapy for cartilage defects: biological characteristics, preclinical application, surgical techniques, and clinical developments. Arthroscopy. 2016;32(7):1444–50. Acknowledgements None. Acknowledgements None. 9. Widuchowski W, Lukasik P, Kwiatkowski G, Faltus R, Szyluk K, Widuch- owski J, et al. Isolated full thickness chondral injuries. Prevalance and outcome of treatment. A retrospective study of 5233 knee arthrosco- pies. Acta chirurgiae orthopaedicae et traumatologiae Cechoslovaca. 2008;75(5):382–6. Consent for publication 17. Gille J, Behrens P, Volpi P, De Girolamo L, Reiss E, Zoch W, et al. Outcome of Autologous Matrix Induced Chondrogenesis (AMIC) in cartilage knee surgery: Data of the AMIC Registry. Arch Orthop Trauma Surg. 2013;133(1):87–93. Author details 1 1 Universitätsklinikum Schleswig-Holstein - Campus Lübeck, Lübeck, Germany. 2 OrthoPraxis, Zofingen, Switzerland. 3 ORTHODOK, Tonndorfer Hauptstraße 71, 22045 Hamburg, Germany. 4 Orthopaedic Department Kantonsspital Fribourg, University of Berne, Bern, Switzerland. 5 Department of Spine Disorders and Pediatric Orthopedics, University of Medical Sciences, Poznan, Poland. 19. Volz M, Schaumburger J, Frick H, Grifka J, Anders S. A randomized controlled trial demonstrating sustained benefit of Autologous Matrix- Induced Chondrogenesis over microfracture at five years. Int Orthop. 2017;41(4):797–804. Received: 4 August 2022 Accepted: 25 September 2023 Received: 4 August 2022 Accepted: 25 September 2023 20. Piontek T, Ciemniewska-Gorzela K, Szulc A, Naczk J, Slomczykowski M. All- arthroscopic AMIC procedure for repair of cartilage defects of the knee. Knee Surg Sports Traumatol Arthrosc. 2012;20(5):922–5. 21. Briggs KK, Lysholm J, Tegner Y, Rodkey WG, Kocher MS, Steadman JR. The reliability, validity, and responsiveness of the Lysholm score and Tegner activity scale for anterior cruciate ligament injuries of the knee: 25 years later. Am J Sports Med. 2009;37(5):890–7. Authors’ contributions JG, ER, PB, RJ, TP performed the surgical procedures on the patients in this study. JG wrote the original draft of the manuscript. ER, PB, RJ, and TP reviewed and edited the manuscript. All authors read and approved the final manuscript. 10. Strauss EJ, Galos DK. The evaluation and management of cartilage lesions affecting the patellofemoral joint. Curr Rev Musculoskelet Med. 2013;6(2):141–9. 10. Strauss EJ, Galos DK. The evaluation and management of cartilage lesions affecting the patellofemoral joint. Curr Rev Musculoskelet Med. 2013;6(2):141–9. 11. Migliorini F, Eschweiler J, Schenker H, Baroncini A, Tingart M, Maffulli N. Surgical management of focal chondral defects of the knee: a Bayesian network meta-analysis. J Orthop Surg Res. 2021;16(1):543. 11. Migliorini F, Eschweiler J, Schenker H, Baroncini A, Tingart M, Maffulli N. Surgical management of focal chondral defects of the knee: a Bayesian network meta-analysis. J Orthop Surg Res. 2021;16(1):543. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 18. Gille J, Reiss E, Freitag M, Schagemann J, Steinwachs M, Piontek T, et al. Autologous Matrix-Induced Chondrogenesis for Treatment of Focal Cartilage Defects in the Knee: A Follow-up Study. Orthop J Sports Med. 2021;9(2):2325967120981872. Ethics approval and consent to participate The study was performed in compliance with the regulations of the ethi- cal review boards of all participating institutions. All participants provided informed consent prior to their participation in the registry. Ethical approval for this study was obtained from the ethics review board of the University of Lübeck (file No. 19–178). The conduct of the study, the acquisition, provision and analyses of data in this study are in agreement with the European General Data Protection Regulations and in accordance with the Declaration of Helsinki. 15. Sadlik B, Puszkarz M, Kosmalska L, Wiewiorski M. All-arthroscopic autolo- gous matrix-induced chondrogenesis-aided repair of a patellar cartilage defect using dry arthroscopy and a retraction system. J Knee Surg. 2017;30(9):925–9. 16. Tradati D, De Luca P, Maione A, Uboldi FM, Volpi P, de Girolamo L, et al. AMIC-Autologous Matrix-Induced Chondrogenesis Technique in Patellar Cartilage Defects Treatment: A Retrospective Study with a Mid-Term Follow-Up. J Clin Med. 2020;9(4):1184. 2. Arokoski JP, Jurvelin JS, Vaatainen U, Helminen HJ. Normal and pathologi- cal adaptations of articular cartilage to joint loading. Scand J Med Sci Sports. 2000;10(4):186–98. 3. Camarero-Espinosa S, Rothen-Rutishauser B, Foster EJ, Weder C. Articular cartilage: from formation to tissue engineering. Biomater Sci. 2016;4(5):734–67. Conclusionh The complexity and mechanical demands of the patel- lofemoral joint create a challenging environment for the repair of focal chondral defects. The results of our ongoing registry study, in which patients reported sig- nificant, sustained improvements in KOOS, Lysholm and pain, support the use of the AMIC procedure as an effective treatment for the repair of chondral lesions on the retropatellar surface. 5. Cleather DJ. The patella: a mechanical determinant of coordination dur- ing vertical jumping. J Theor Biol. 2018;446:205–11. 5. Cleather DJ. The patella: a mechanical determinant of coordination dur- ing vertical jumping. J Theor Biol. 2018;446:205–11. 6. Roos PE, Barton N, van Deursen RW. Patellofemoral joint com- pression forces in backward and forward running. J Biomech. 2012;45(9):1656–60. 7. Scott SH, Winter DA. Internal forces of chronic running injury sites. Med Sci Sports Exerc. 1990;22(3):357–69. 7. Scott SH, Winter DA. Internal forces of chronic running injury sites. Med Sci Sports Exerc. 1990;22(3):357–69. 8. Flanigan DC, Harris JD, Trinh TQ, Siston RA, Brophy RH. Prevalence of chondral defects in athletes’ knees: a systematic review. Med Sci Sports Exerc. 2010;42(10):1795–801. 8. Flanigan DC, Harris JD, Trinh TQ, Siston RA, Brophy RH. Prevalence of chondral defects in athletes’ knees: a systematic review. Med Sci Sports Exerc. 2010;42(10):1795–801. References 1. Lin W, Klein J. Recent Progress in Cartilage Lubrication. Adv Mater. 2021;33(18):e2005513. 2. Arokoski JP, Jurvelin JS, Vaatainen U, Helminen HJ. Normal and pathologi- cal adaptations of articular cartilage to joint loading. Scand J Med Sci Sports. 2000;10(4):186–98. 3. Camarero-Espinosa S, Rothen-Rutishauser B, Foster EJ, Weder C. Articular cartilage: from formation to tissue engineering. Biomater Sci. 2016;4(5):734–67. 22. Roos EM, Lohmander LS. The Knee injury and Osteoarthritis Outcome Score (KOOS): from joint injury to osteoarthritis. Health Qual Life Out- comes. 2003;1:64. 23. KOOS user’s guide, Updated August 2012 [updated August 2012. Avail- able from: http://www.koos.nu. 3. Camarero-Espinosa S, Rothen-Rutishauser B, Foster EJ, Weder C. Articular cartilage: from formation to tissue engineering. Biomater Sci. 2016;4(5):734–67. 24. Jones KJ, Kelley BV, Arshi A, McAllister DR, Fabricant PD. Comparative effectiveness of cartilage repair with respect to the minimal clinically important difference. Am J Sports Med. 2019;47(13):3284–93. 24. Jones KJ, Kelley BV, Arshi A, McAllister DR, Fabricant PD. Comparative effectiveness of cartilage repair with respect to the minimal clinically important difference. Am J Sports Med. 2019;47(13):3284–93. Page 9 of 9 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 Gille et al. BMC Musculoskeletal Disorders (2023) 24:964 25. Chahal J, Lansdown DA, Davey A, Davis AM, Cole BJ. The clinically impor- tant difference and patient acceptable symptomatic state for commonly used patient-reported outcomes after knee cartilage repair. Am J Sports Med. 2021;49(1):193–9. 26. de Girolamo L, Jannelli E, Fioruzzi A, Fontana A. Acetabular Chondral Lesions Associated With Femoroacetabular Impingement Treated by Autologous Matrix-Induced Chondrogenesis or Microfracture: A Com- parative Study at 8-Year Follow-Up. Arthroscopy. 2018;34(11):3012–23. 27. Richter M, Zech S, Meissner S, Naef I. Autologous matrix induced chon- drogenesis plus peripheral blood concentrate (AMIC+PBC) in chondral lesions at the ankle as part of a complex surgical approach - 5-year follow-up. Foot Ankle Surg. 2022;28(8):1321–6. 28. Schiavone Panni A, Del Regno C, Mazzitelli G, D’Apolito R, Corona K, Vasso M. Good clinical results with autologous matrix-induced chondrogen- esis (AMIC) technique in large knee chondral defects. Knee Surg Sports Traumatol Arthrosc. 2018;26(4):1130–6. 29. de Girolamo L, Schonhuber H, Vigano M, Bait C, Quaglia A, Thiebat G, et al. Autologous Matrix-Induced Chondrogenesis (AMIC) and AMIC Enhanced by Autologous Concentrated Bone Marrow Aspirate (BMAC) Allow for Stable Clinical and Functional Improvements at up to 9 Years Follow-Up: Results from a Randomized Controlled Study. J Clin Med. 2019;8(3):392. 30. References Migliorini F, Eschweiler J, Maffulli N, Driessen A, Rath B, Tingart M, et al. Management of Patellar Chondral Defects with Autologous Matrix Induced Chondrogenesis (AMIC) Compared to Microfractures: A Four Years Follow-Up Clinical Trial. Life (Basel, Switzerland). 2021;11(2):141. 31. Niethammer TR, Altmann D, Holzgruber M, Gulecyuz MF, Notohami- prodjo S, Baur-Melnyk A, et al. Patient-Reported and Magnetic Resonance Imaging Outcomes of Third-Generation Autologous Chondrocyte Implantation After 10 Years. Arthroscopy. 2020;36(7):1928–38. 32. Kaiser N, Jakob RP, Pagenstert G, Tannast M, Petek D. Stable clinical long term results after AMIC in the aligned knee. Arch Orthop Trauma Surg. 2021;141(11):1845–54. 33. Martin R, Jakob RP. Are Cartilage Repair and Restoration Procedures in the Knee without Respecting Alignment Fruitless? A comprehensive review. J Cartil Joint Preserv. 2022:100074. https://www.sciencedirect.com/scien ce/article/pii/S2667254522000373. Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W2242151821	https://revistas.ucr.ac.cr/index.php/rbt/article/download/18223/22976	English	null	Aspergillus and Penicillium (Eurotiales: Trichocomaceae) in soils of the Brazilian tropical dry forest: diversity in an area of environmental preservation	Revista de Biología Tropical	2,016	cc-by	6,185	Received 27-II-2015. Abstract: Soil is a complex biological system that plays a key role for plants and animals, especially in dry forests such as the Caatinga. Fungi from soils, such as Aspergillus and Penicillium, can be used as bioindica tors for biodiversity conservation. The aim of this study was to isolate and identify species of Aspergillus and Penicillium in soil, from the municipalities of Tupanatinga and Ibimirim, with dry forests, in the Catimbau National Park. Five collections were performed in each area during the drought season of 2012, totaling 25 soil samples per area. Fungi were isolated by suspending soil samples in sterile distilled water and plating on Sabouraud Agar media plus Chloramphenicol and Rose Bengal, and Glycerol Dicloran Agar. Isolates were identified by morphological taxonomy in the Culture Collection Laboratory and confirmed by sequencing of the Internal Transcribed Spacer of rDNA. A total of 42 species were identified, of which 22 belong to the genus Aspergillus and 20 to Penicillium. Penicillium isolates showed uniform distribution from the collecting area in Tupanatinga, and the evenness indices found were 0.92 and 0.88 in Tupanatinga and Ibimirim, respectively. Among isolates of Aspergillus evenness, the value found in Tupanatinga (0.85) was very close to that found in Ibimirim (0.86). High diversity and low dominance of fungi in soil samples was observed. These results con tributed to the estimation of fungal diversity in dry environments of the Caatinga, where diversity is decreasing in soils that have undergone disturbance. Rev. Biol. Trop. 64 (1): 45-53. Epub 2016 March 01. Key words: Catimbau, Caatinga, fungi, richness, taxonomy. Knowledge of tropical forests plays a key role in developing global strategies for biodi versity conservation. In Brazil, the tropical dry forest is known as Caatinga (Tupi language; caa = forest and tinga = white) owing to the predominant type of vegetation (Santos et al., 2012). The Caatinga is a unique ecosystem in Brazil, and despite its socioeconomic importan ce to the country, it is subject to intense natural resource exploitation and has been little studied and protected. According to the literature, only 14 % of tropical forest studies are related to the biodiversity of the Caatinga, and 86 % are conducted in humid regions (Sánchez-Azofeifa et al., 2005). Soil is a dynamic and complex biological system, in which different organisms play key roles in the maintenance and survival of plant and animal communities. Neiva Tinti Oliveira¹ 1. Departamento de Micologia, Programa de Pós-Graduação em Biologia de Fungos, Universidade Federal de Pernambuco, Av. Nelson Chaves s/n, CEP 50760-420, Recife, PE; renan.rnb@gmail.com, jadsondpb@hotmail.com, phelipeoller@yahoo.com.br, netinti@hotmail.com 1. Departamento de Micologia, Programa de Pós-Graduação em Biologia de Fungos, Universidade Federal de Pernambuco, Av. Nelson Chaves s/n, CEP 50760-420, Recife, PE; renan.rnb@gmail.com, jadsondpb@hotmail.com, phelipeoller@yahoo.com.br, netinti@hotmail.com 2. Programa de Pós-Graduação em Ciências Biológicas-UFPE; nelsonradar2005@hotmail.com 2. Programa de Pós-Graduação em Ciências Biológicas-UFPE; nelsonradar2005@hotmail.co g ç g ; @ 3. Micoteca URM-UFPE; ivanaggalvao@hotmail.com, anthonyalves1@hotmail.com, mariajf2005@hotmail.com, cristina.motta@ufpe.br 3. Micoteca URM-UFPE; ivanaggalvao@hotmail.com, anthonyalves1@hotmail.com, mariajf2005@hotmail.com, cristina.motta@ufpe.br Aspergillus and Penicillium (Eurotiales: Trichocomaceae) in soils of the Brazilian tropical dry forest: diversity in an area of environmental preservation Renan do Nascimento Barbosa¹, Jadson Diogo Pereira Bezerra¹, Phelipe Manoel Oller Costa¹, Nelson Correia de Lima-Júnior², Ivana Roberta Gomes Alves de Souza Galvão³, Anthony Alves dos Santos-Júnior³, Maria José Fernandes³, Cristina Maria de Souza-Motta¹,³ & Neiva Tinti Oliveira¹ Renan do Nascimento Barbosa¹, Jadson Diogo Pereira Bezerra¹, Phelipe Manoel Oller Costa¹, Nelson Correia de Lima-Júnior², Ivana Roberta Gomes Alves de Souza Galvão³, Anthony Nelson Correia de Lima-Júnior², Ivana Roberta Gomes Alves de Souza Galvão³, Antho MATERIALS AND METHODS Study site: Soil samples were collected in areas from the Catimbau National Park in Pernambuco, Brazil in the municipalities of Ibimirim (08°27’ S - 37°19’66’ ‘W) and Tupa natinga (08°29’ S - 037°19’58 W) during the drought season (July-November) of 2012. The vegetation of the park exhibits very different floristic and structural patterns, which com prise five distinct Caatinga vegetation types: shrub-arboreal; shrub with savannah elements; shrub with elements of rock fields; evergreen forest vegetation and shrub sub-evergreen (Rodal, Andrade, Sales & Gomes, 1998). The municipality of Ibimirim shows traces of the practice of withdrawal of native vegetation for fuel and grazing activities, and includes homes of small farmers who make constant use of this soil. In the municipality of Tupanatinga, there is a greater amount of plant species typical of Caatinga, such as cacti Cereus jamacaru DC., Pilosocereus gounellei (FAC Weber) (Byles & GD Rowley) and Melocactus zehntneri (Britton & Rose) Luetzelb., and no grazing activities or housing. The soil sampled in the two collection areas was chemically analyzed in the Physics and Soil Fertility Laboratory of the Federal Rural University of Pernambuco (Table 1). The characterization of the mycodiversity of soil will not only contribute to preservation strategies but also can be used for develo ping bioindicators of environmental changes associated with disturbances caused by mana gement practices and pollutants (Lambais, Cury, Maluche-Baretta, & Büll, 2005). The prospecting of fungi can lead to increased understanding and preservation of the sources of genetic diversity, increased understanding of the distribution patterns of microorganisms and the involvement of biodiversity in the functioning and sustainability of ecosystems (Øvreås, 2000). Among the areas of importance in the Caa tinga, is the Catimbau National Park (PARNA Catimbau), created in 2002. The PARNA Catimbau is distributed among the area munici palities of Buíque (12 438 ha), Tupanatinga (23 540 ha) and Ibimirim (24 809 ha) in the State of Pernambuco, Brazil. Its landscape consists of walls of colorful sandstone formations, and unique vegetation. It attracts large numbers of visitors each year (SNE, 2002), and this area has also been used by small farmers for extrac ting wood from the forest areas for subsistence or for sale to local businesses as fuel. Soil sampling and processing: Five collections were performed in each area, and each collection was randomly assigned five 25 m2 quadrants (5×5 m) with a minimum distan ce of 10 m between them. Received 27-II-2015. Fungi are the predominant soil microorganism in terms of Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 45 biomass and a number of different species can be found in soil including the genera Aspergillus and Penicillium (Cruz, Santos, Lima, Moreira & Souza-Motta, 2013; Oliveira, Cavalcanti, Fernandes, & Lima, 2013). Those genera are widespread, occurring frequently in soils, and have important roles in the proces ses of biodeterioration, as well as significant importance in the medical field, because some of them are pathogenic to humans and other animals (Houbraken & Samson, 2011; Pitt & Hocking, 2009). yet surveyed. Therefore the aim of this study was to gather information on the diversity and distribution of Aspergillus and Penicillium spe cies in soils of the Catimbau National Park in Pernambuco, Brazil. TABLE 1 TABLE 1 Chemical properties of soils collected in the municipalities of Tupanatinga and Ibimirim in Brazilian tropical dry forest in the National Park Catimbau-Pernambuco, Brazil, in 2012 Zone Chemical properties mg dm3 cmolc dm3 % Fe Cu Zn Mn P pH K Na Al Ca Mg MO Tupanatinga 107.40 0.40 0.50 2.20 7 4.2 0.1 0.43 0.8 0.80 0.30 3.84 Ibimirim 57.40 0.20 3.80 24.30 8 6.2 0.54 0.05 0.0 5.80 0.30 1.84 Iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), phosphorus (P), potassium (K), aluminum (Al), calcium (Ca), magnesium (Mg), organic matter (OM). Iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), phosphorus (P), potassium (K), aluminum (Al), calcium (Ca), magnesium (Mg), organic matter (OM). The polymerase chain reaction (PCR) ampli fication for region ITS1-5.8S-ITS2 of rDNA was carried out with the primers ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) according to White, Bruns, Lee and Taylor (1990). All amplification reactions were carried out in volumes of 25 µL with 19 µL PCR buffer (Tris-HCl 20 mM pH 8.4; NH4), 2.0 mM MgCl2, 0.20 mM dNTP mix, 0.40 lM of each primer, 0.04 U/µl Taq DNA polymerase (Fer mentas Life Sciences), 25 ng DNA and sterile ultra-pure water up to 30 µl. Amplification was carried out in a thermocycler programmed for 8 min at 95 °C initial denaturation followed by 35 cycles of 30 s at 95 °C, 45 s at 47 °C and 3 min at 72 °C, and 10 min final exten sion at 72 °C. University of Pernambuco (UFPE), Pernambu co, Brazil. The soil collections were authorized by the Ministério do Meio Ambiente (MMA)/ Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio); permission number: 33358-1/authentication code 39911643 issued on 22th march, 2012. Fungal isolation: Fungi isolation from soil samples was carried out using a modified version of a serial dilution technique described by Clark (1965). In summary, 25 g of each soil sample was diluted in 225 mL of sterile distilled water. One milliliter of the 10-3 dilu tion was spread onto Sabouraud Agar with chloramphenicol (0.17 g/L), Bengal Rose (0.05 g/L) and Dicloran Glycerol (DG-18). The Petri dishes were incubated at 28 ± 2°C for 3 days. After incubation, the colonies were subcultured to Malt Agar and chloramphenicol (0.17 g/L) was added until single colonies were obtained. MATERIALS AND METHODS In each quadrant, six equidistant subsamples were collected at a depth of 0-20 cm and combined, consisting of a total of five mixed samples in each collection area. The samples were stored in labeled plastic bags, and manipulated in the Phytopathoge nic Fungi Laboratory, Mycology Department, Center of Biological Sciences (CCB), Federal There has been some study on the fungi in PARNA Catimbau (Cruz et al., 2013; Oli veira et al., 2013); however, soil samples have covered only a small part of the Park in the municipality of Buíque. Thus, taking into account the rapid loss of biodiversity and the role of fungi in the balance of an ecosystem, it is necessary to extend the knowledge of fungi, as well as their distribution in areas not Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 46 TABLE 1 The PCR products were checked in 1 % agarose gel electrophoresis for 30-35 min at 100 V. The amplification product obtained was purified with GenJET PCR Purification Kit – Fermentas®. All PCR products were sent to Plataform of Sequencing-LABCEN/CCB (UFPE) to be sequenced. The sequences obtai ned were analysed in GenBank with BLASTn to search for similarity with the sequences deposited. Molecular operational taxonomic units (MOTUs) were defined using a 98 % ITS region identity threshold. Fungal identification: The fungi were identified in the research laboratory of the Cul ture Collection (URM), Mycology Department, Universidade Federal de Pernambuco, Brazil, using specific methodology and literature des cribed in Jurjević et al. (2012), Klich (2002), Pitt (1991), Samson and Frisvad (2004), Samson and Houbraken (2011). Isolates with unclear mor phological identification were selected for molecular identification. The genomic DNA extraction was performed according Góes- Neto, Loguercio-Leite and Guerrero (2005). Ecological analyses: After the identifica tion of the filamentous fungal species from the different soils, ecological indices were applied: species richness consisted of the total number Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 47 are very similar morphologically. Thus, spe cies with dubious identification were extracted DNA and amplified ITS region and β-tubulin. Fifteen Molecular Operational Taxonomic Units (MOTUs) were identified based on the sequencing of the ITS region of rRNA with nucleotide differences from the other fungal sequences deposited in GenBank (National Center for Biotechnology Information, Bethes da, Maryland, USA) ranging from 1 to 2 %. The numbers of accesses in the GenBank are: A. tamarii (KM613134); A. westerdij kiae (KM613135); A. caelatus (KM613136); A. niveus (KM613137); A. aculeatus (KM613138); A. niger (KM613139); A. caela tus (KM613140); A. versicolor (KM613141); A. lentulus (KM613142); A. viride-nutans (KM613143); A. aureolus (KM613144); A. terreus (KM613145); P.simplicissimum (KM613146); P. adametzii (KM613147) and P.citrinum (KM613148). of species (S) in a unique sample. The distri bution of each fungal species was calculated for each sampling using the following formula: Di = (Ni/N) * 100, where Di = distribution of the species; Ni = number of colony forming units (CFU) of the species; N = total number of CFU. According to this formula, the species frequencies can be classified as: < 0.5 % = rare, ≥ 0.5 < 1.5 % = occasional, ≥ 1.5 < 3.0 % = common, ≥ 3.0 % = abundant (Schnittler & Stephenson, 2000). RESULTS A total of 3 139×106 colony forming units per gram of soil (CFU/g) were found in the municipalities studied. Isolates numbering 2 531 originated from Tupanatinga and 608 from Ibimirim, distributed across 42 species: 22 belonging to the genus Aspergillus and 20 to the genus Penicillium. The species, number of CFUs and the relative abundance (%) are shown in Table 2. The species A. aureoterreus, A. lentulus, A. niveus, A. pulvinus, A. ruber, A. westerdijkiae, P. implicatum, P. janczewskii, P. restrictum, P. vinaceum occurred only in Tupa natinga soils while A. recurvatus was isolated only in soil samples from Ibimirim. Conside ring the total obtained in the two areas in rela tion to the classification of relative abundance according Schnittler & Stephenson (2000), 14 % of the isolates were classified as rare (Di: < 0.5 %), 26 % occasional (Di ≥ 0.5 < 1.5 %), 29 % common (Di ≥ 1.5 < 3.0 % ) and 31 % were abundant (Di ≥ 3.0 %). Analyzing each collec tion area separately, 21 % of the species were rare, 17 % occasional, 26 % common and 33 % abundant in Tupanatinga and 02 % were consi dered rare, 29 % occasional, 19 % common and 26 % abundant in Ibimirim (Table 2). Comparing the two collection areas, it was possible to obtain a large number of Colony Forming Units x 103 g solo-1 (CFUs) of Asper gillus (1 143) and Penicillium (1 388) in Tupa natinga, and small number of CFUs Aspergillus (235) and Penicillium (373) in Ibimirim, (F = 6.236, p = 0.001 and F = 5.149, p = 0.001 respectively); however, it was not possible to observe a significant difference in density bet ween the genera in the same area by Tukey test at 5 % probability. The ecological indices indicated that the values of diversity and richness of Aspergillus and Penicillium species are close between the studied areas. The distribution of Aspergillus species showed greater uniformity in Ibimirim with an evenness index 0.86. However, Peni cillium to the soil showed greater evenness in Tupanatinga with a value of 0.92 (Table 3). This result may be related to the density found for each collection site. TABLE 1 Means were compared by Tukey’s test at 5 % significance level using the ASSISTAT 7.7. Species Richness, Shannon- Wiener’s Diversity, Pielou’s Equitability and Berger-Parker’s Dominance were calculated using the PAST 1.7 software (Hammer, Harper, & Ryan, 2001). DISCUSSION Most species found in this study are already known to occur in soils in Caatinga; however, A. aureolus, A. aureoterreus, A. caelatus, A. Aspergillus and Penicillium genera con tain a high number of species, many of which Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 48 TABLE 2 Colony Forming Units, absolute frequency (fa) and relative abundance (%) of species of Aspergillus and Penicillium isolated from soils in the municipalities of Tupanatinga (A) and Ibimirim (B) in Brazilian tropical dry forest in the National Park Catimbau-Pernambuco, Brazil, in 2012 Colony Forming Units, absolute frequency (fa) and relative abundance (%) of species of Aspergillus and Penicillium isolated from soils in the municipalities of Tupanatinga (A) and Ibimirim (B) in Brazilian tropical dry forest in the National Park Catimbau-Pernambuco, Brazil, in 2012 Táxon 1st occurrence for Caatinga’s soil Tupanatinga Ibimirim Overall analysis A B fa % Clas. fa % Clas. fa % Clas. Aspergillus aculeatus Iizuka 130 5.14 A 23 3.78 A 153 4.87 A A. allahabadii B.S. Mehrotra & Agnihotri 63 2.49 C 11 1.81 C 74 2.36 C A. aureolus Fennell & Raper X X 14 0.55 O 7 1.15 O 21 0.67 O A. aureoterreus Thom & Raper X 25 0.99 O 0 0.00 - 25 0.80 O A. caelatus BW Horn X X 7 0.28 R 10 1.64 C 17 0.54 O A. flavus Link 154 6.08 A 4 0.66 O 158 5.03 A A. fumigatus Fresen 55 2.17 C 14 2.30 C 69 2.20 C A. insuetus (Bainier) Thom & Church 5 0.20 R 5 0.82 O 10 0.32 R A. lentilus Balajee & K.A. Marr X 21 0.83 O 0 0.00 - 21 0.67 O A. nidulans Fennell & Raper X X 9 0.36 R 4 0.66 O 13 0.41 R A. niger Tiegh 160 6.32 A 43 7.07 A 203 6.47 A A. niveus Blochwitz 1 0.04 R 0 0.00 - 1 0.03 R A. pulvinus Kwon-Chung & Fennell 53 2.09 C 0 0.00 - 53 1.69 C A. recurvatus Raper & Fennell X 0 0.00 - 6 0.99 O 6 0.19 R A. ruber Thom & Church X 4 0.16 R 0 0.00 - 4 0.13 R A. sydowii (Bainier & Sartory) Thom & Church 6 0.24 R 2 0.33 R 8 0.25 R A. tamarii Kita 155 6.12 A 19 3.13 A 174 5.54 A A. TABLE 3 Species richness, Shannon-Wiener’s Diversity (H’), Pielou’s Equitability (J’) and Berger-Parker’s Dominance (d) of Aspergillus (A) and Penicillium (P) isolated from soils of the counties of Tupanatinga and Ibimirim in Catimbau National Park during the dry season of 2012 Species richness, Shannon-Wiener’s Diversity (H’), Pielou’s Equitability (J’) and Berger-Parker’s Dominance (d) of Aspergillus (A) and Penicillium (P) isolated from soils of the counties of Tupanatinga and Ibimirim in Catimbau National Park during the dry season of 2012 Zone Richness Diversity Equitability Dominance A P A P A P A P Tupanatinga 21 20 2.6 2.7 0.85 0.92 0.14 0.11 Ibimirim 16 16 2.4 2.4 0.86 0.88 0.22 0.17 of fungi. The isolates that were selected for sequencing in this study that had their iden tification confirmed by analysis of molecular data, showed a maximum 2 % divergence from any sequence in the GenBank. Nilsson, Kristiansson, Ryberg, Hallenberg and Larsson (2008) showed that 2 % is an acceptable mar gin for intraspecific differences in Ascomycota sequences for the ITS rDNA region. lentulus, A. nidulans, A. recurvatus, A. ruber, A. viridinutans, A. westerdijkiae, P. miczynskii and P. sclerotiorum are being referenced for the first time to this soil ecosystem. In the natural environment of Northeastern Brasil, several studies have shown the presence of species of the genera Aspergillus and Penicillium and others Acomycota, Basidiomycota e Muco romycotina (Cavalcanti, Oliveira, Fernandes, & Lima, 2006; Silva, Tiago, Mattos, Paiva, & Souza-Motta, 2011). q g Aspergillus species of the Niger group were also well represented with A. niger being considered abundant in both areas. This species is widely distributed and is often found in dry regions. Its distribution is related to the cli mate, vegetation and soil. Klich (2002), when studying the biogeography of Aspergillus in samples of soil and leaf litter, noted that this fungi occurs more frequently in desert envi ronments and that A. niger has been isolated more frequently in areas of cultivated soils and tropical forests. According to the same author, members of the Nidulantes section are rare in soils that have experienced anthropogenic influence, which corroborates with the results obtained in this study, which was isolated only one species of the Nidulantes section. , ) Oliveira et al. (2013) researched filamen tous fungi in soils of the Catimbau National Park (Buíque-PE, Brazil) and found a total of 85 taxa; 71 species of anamorphic fungi, eight Zygomycetes and six Ascomycota. DISCUSSION terreus Thom 71 2.81 C 22 3.62 A 93 2.96 C A. versicolor (Vuill.)Tirab. 11 0.43 R 6 0.99 O 17 0.54 O A. violaceofuscus Gasperini 73 2.88 C 52 8.55 A 125 3.98 A A. viridinutans Ducker & Thrower X X 77 3.04 A 7 1.15 O 84 2.68 C A. westerdijkiae Frisvad & Samson X 49 1.94 C 0 0.00 - 49 1.56 C Penicillium adametzii K.M. Zalessky 89 3.52 A 53 8.72 A 142 4.52 A P. aurantiogriseum Dierckx 119 4.70 A 5 0.82 O 124 3.95 A P. bilaiae Chalab 32 1.26 O 6 0.99 O 38 1.21 O P. citreonigrum Dierckx 153 6.05 A 19 3.13 A 172 5.48 A P. citrinum Sopp 159 6.28 A 44 7.24 A 203 6.47 A P. corylophilum Dierckx 138 5.45 A 14 2.30 C 152 4.84 A P. decumbens Thom 20 0.79 O 12 1.97 C 32 1.02 O P. griseofulvum Dierckx 95 3.75 A 51 8.39 A 146 4.65 A P. implicatum Dierckx 41 1.62 C 0 0.00 - 41 1.31 O P. janczewskii Zaleski 83 3.28 A 0 0.00 - 83 2.64 C P. janthinellum Biourge 77 3.04 A 4 0.66 O 81 2.58 C P. miczynskii K.M. Zalessky Stolk & D.B. Scott X X 40 1.58 C 9 1.48 O 49 1.56 C P. minioluteum Dierckx 57 2.25 C 62 10.20 A 119 3.79 A P. restrictium J.C. Gilman & E.V. Abbott 99 3.91 A 0 0.00 - 99 3.15 A P. sclerotiorum Beyma X X 66 2.61 C 9 1.48 O 75 2.39 C P.simplicissimum (Oudem.)Thom 10 0.40 R 11 1.81 C 21 0.67 O P. spinulosum Thom 26 1.03 O 48 7.89 A 74 2.36 C P. verruculosum Peyronel 55 2.17 C 16 2.63 C 71 2.26 C P. vinaceum J.C. Gilman & E.V. Abbott 23 0.91 O 0 0.00 - 23 0.73 O P. waksmanii K.M. Zalessky 6 0.24 R 10 1.64 C 16 0.51 O TOTAL 2531 100 608 100 3139 100 Classification of species occurrence: rare (R), occasional (O), common (C) and abundant (A). Classification of species occurrence: rare (R), occasional (O), common (C) and abundant (A) Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 49 TABLE 3 TABLE 3 TABLE 3 If the stress level continues to increase, the competition decreases, resulting in an increase in diver sity. However, when the disturbance reaches high levels, elimination occurs between species and the diversity decreases again (Clarke & Warwick, 1994). The distribution of isolates among the identified species had become more uniform among isolates of Penicillium in Tupanatinga with evenness indices equal to 0.92. However, among isolates of Aspergillus the evenness found in Tupanatinga was very close to that found in Ibimirim. This is probably due to the characteristics of the genus as well as the density of fungal isolates. Additionally, com munities with high evenness tend to have low dominance among species, since the evenness measure is related to the distribution of sam pled individuals with the number of species (Cullen, Rudran, & Valladares-Padua, 2004). In the presente study, those species were classified as abundant (34 %), common (26 %), rare (14 %) and ocasional (26 %). Oliveita et al. (2013) found a total density of 1 150×10³ CFU/g, with Aspergillus (18 species) and Peni cillium (28) species classified as abundant. De Fede, Panaccione and Sextone (2001) and Grayston, Grifftih, Mawdesley, Campe bell, and Bardgett (2001) found that in soils with high organic matter content, the microbial population tends to remain more stable over a period, probably owing to the wealth of ecolo gical niches and variety of carbon sources. In the present study, the highest content of organic matter in the soil was found in Tupanatinga, and the pH of the soils was acidic (Table 1), differing from Moreira and Siqueira (2006) who found that the soils of dry or semi-arid regions are usually alkaline. ( ) p Penicillium commune was not isolated in any of the soil samples and P. restrictum was only obtained from Tupanatinga. This is interesting because in both collection sites, the native vegetation remains in good condition. Studies have shown that fungi are found with densities ranging from 104 to 106 organisms per gram of soil (Blackwell, 2011). According Bononi (1998), in environments with high density populations of fungi, different types of ecological relationships among populations of fungi and/or other bodies are established. In the present study, high diversity and low dominance of fungi was observed in soil from Ibimirim, confirming the affirmation of Brower and Zar (1984) that a community with high diversity will have low dominance. TABLE 3 The abundant genera were Aspergillus (28 taxa) and Penicillium (18). Cruz et al. (2013) also studied the diversity of Penicillium in soils of the Caatinga in Catimbau National Park, but only in one area and in soils of Atlantic Forest. They obtained a total of 370 isolates from soils of the National Park, with P. aurantiogriseum, P. glabrum and P. lividum being the most dominant species. The authors also noted that 14 species occurred only in soils of the Caa tinga, including P. adametzii, P. citrinum, P. implicatum, P. janczewskii, P. minioluteum, P. spinulosum, P. verruculosum and P. waksma nii. All these fungi species also occurred in the present study, indicating their ability to thrive in dry environments. Penicillium species belonging to the sec tion Citrine were also well represented in this survey. According to Monteiro (2012) the Citri ne section commonly occurs in soils and is also good producers of secondary metabolites that may confer benefit by providing a competitive advantage when colonizing a new substrate. We used a culture-based approach and the sequencing of ITS region of the rRNA gene clarifications regarding the morphology taxo nomy. The ITS region was chosen because this region has the highest probability of allowing the successful identification of a broad range The least abundant species found for the genus Aspergillus was A. niveus (0.03 %) which were isolated only in Tupanatinga. For the genus Penicillium, the lowest abundan ce was of P. waksmanii (0.51 %) in the two Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 50 collection areas. Several studies have shown that different plant species have the ability to select certain fungal communities (De Bellis, Kernaghan, & Widden, 2007). De Bellis et al. (2007) studied the relationship between groups of soil fungi and plant communities in a boreal forest in Québec (Canada) and noted that the species of fungi were related to plant species and not to the chemical properties of the soil. However, it is noteworthy that the type of vegetation also influences the physicochemi cal properties and composition of soil organic matter. There are reports that plants can cause changes in soil properties that lead to complex interactions with vegetation and may influence the soil fungal community (Nielsen, Osler, Campbell, Burslem, & van Der Wal, 2010). is minimal, diversity is reduced owing to com petitive exclusion between species. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 TABLE 3 The diver sity and dominance of the fungal populations in soils depend on the occurrence of specific habitats for certain species. The decrease in the diversity, richness and evenness and increasing dominance has been linked to levels of environ mental stress. However, it is possible that there may be an increase in diversity with increasing stress level. At one point where the stress level In Brazil, most of the work has been carried out in humid environments, so the results of this research contribute to the pro vision of information on the diversity of fungi in soils of dry environments as well as on the mycodiversity of Caatinga soils. The microor ganisms, including fungi, are the first orga nisms to demonstrate the effects of changes in an environment on large populations and on ease of dispersion, beyond the short generation time. The results suggest that the diversity of the fungal community in the soil may become reduced in soils that have undergone some Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (1): 45-53, March 2016 51 sort of disturbance such as removal of native vegetation. Thus, the preservation of some impacted areas is essential for the resilience of the ecosystem. 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Chemical and microbiological properties (pp. 1460-1466). USA: American Society of Agronomy, Soil Science Society of America. RESUMEN Aspergillus y Penicillium (Eurotiales: Trichocoma ceae) en suelos de un bosque seco tropical brasileño: diversidad en un área protegida. El suelo es un sistema biológico complejo, que desempeña un papel fundamental en las plantas y los animales, especialmente en los bosques secos como la Caatinga. Los hongos del suelo, tales como Aspergillus y Penicillium, pueden ser utilizados como bioindicadores para la conservación de la biodiversidad. El objetivo de este estudio fue aislar e identificar las especies de Aspergillus y Penicillium del suelo, en los municipios de Ibimirim y Tupanatinga en el Parque Nacional Catimbau. Cinco colecciones se llevaron a cabo en cada área durante la estación seca de 2012, un total de 25 muestras de suelos por área. 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https://openalex.org/W4313396449	http://journals.rta.lv/index.php/EID/article/download/6945/5784	English	null	EXAMINING INCLUSION AND EXCLUSION IN HIGHER EDUCATION: A CASE STUDY OF BLIND STUDENTS AT A RURAL BASED SOUTH AFRICAN UNIVERSITY	Education. Innovation. Diversity	2,022	cc-by	11,183	EXAMINING INCLUSION AND EXCLUSION IN HIGHER EDUCATION: A CASE STUDY OF BLIND STUDENTS AT A RURAL BASED SOUTH AFRICAN UNIVERSITY Clever Ndebele1, Milton Gadisi2 1Walter Sisulu University, Directorate of Learning and Teaching Mthatha Campus, South Africa 2University of Venda, Centre for Higher Education Teaching and Learning South Africa Clever Ndebele1, Milton Gadisi2 Clever Ndebele1, Milton Gadisi2 Walter Sisulu University, Directorate of Learning and Teaching Mthatha Campus, South Africa University of Venda, Centre for Higher Education Teaching and Learning South Africa Abstract. This study sought to examine barriers to learning for blind students at a rural based South African University. Premised on the qualitative design, data were collected through semi-structured individual interviews with 5 blind students, two academic support staff and four lecturers who were identified through purposive sampling. Emerging themes from the data were identified through content analysis of the verbatim responses. The study found that reasonable accommodation was not provided for blind students in the lecture halls at the university under study. The study further found that, study materials and computers in the university library and mainstream computer laboratories were not adapted for blind students. Third, lecturers were not trained to teach blind students with some lecturers using PowerPoint presentations while teaching when blind students could not access the screens. The study recommends universal design for all learning facilities, thorough training for all staff teaching students with disabilities and the development of a disability policy in the university. y Keywords: blind, braille, reasonable accommodation, disability, universal design. words: blind, braille, reasonable accommodation, disa To cite this article: Ndebele, C. & Gadisi, M. (2022). Examining Inclusion and Exclusion in Higher Education: A Case Study of Blind Students at a Rural Based South African University Education. Innovation. Diversity, 2(5), 21-36. DOI: https://doi.org/10.17770/eid2022.2.6945 Introduction The recognition of the rights of people with disability has been on the United Nations agenda for a while (UN Convention on the Rights of the Child (UNCRC, hereafter) 1989; UN Standard Rules on the Equalisation of Opportunities for Persons with Disabilities (UNSREOPD, hereafter) 1993; UN Convention on the Rights of Persons with Disabilities (UNCRPD, hereafter) 2006). Several countries, including South Africa, have ratified some of these conventions and started putting in place legislation to accommodate learners with disability. Legislation in the UK requires all public authorities, including higher education institutions, to actively promote equality of opportunity for people with disabilities (Goode, 2007; Richardson, 2015; Vickerman and Blundell 2010). In Brazil, the educational integration of persons with disabilities (visual, auditory, physical and intellectual) has increased (De Camargo et al., 2013). Australia, the United States of America and Israel, have legislation concerning the integration of students with disabilities into higher education (Fuller, Bradley and Healey 2004). Recently, the United Arab Emirates in the Middle East made efforts to promote the rights of people with disabilities (Alhammadi, 2016). In Australia, policy initiatives and legislation ensure that students with disability are presented with an equitable experience in, and access into, higher education (Dryer et al., 2016). In Zimbabwe, disability policies are in place although outdated (Chikukwa & Chimbwanda, 2013). Findings from the literature indicate that despite a growth of interest in widening access, participation and inclusive higher education, the voices of disabled students themselves have hardly been heard (Fuller, Bradley, & Healey, 2004; Vaccaro, Kimball, Ostiguy& Wells, 2015; Moriña, 2019). Furthermore, while legislation has been enacted the 20 world over as shown in the preceding paragraphs to enable access for students with disabilities, little research seems to have been conducted on the category of blind students and their experiences of the higher education context. Studies conducted seem to either focus on students with disabilities in general or where research focus is on visually impaired students, blind students and partially sighted students are lumped together. (see for example, Fuller, Bradley, & Healey, 2004; Whitburn, 2014; Seyama. Morris, & Stilwell, 2014; Cunnah, 2015; Kendall, 2016; Patterson & Loomis 2016; Moriña, 2019; Kim & Kutscher, 2020). It is incumbent upon universities to guarantee the necessary conditions and opportunities to ensure that all students can engage and learn. In this regard, it is useful to listen to the voices of the students (Moriña, 2019). Introduction This will help institutions of higher education better understand how they can support collegiate success among students with disabilities (Kim & Kutscher, 2020). Sachs and Schreuer (2011) aver that the opportunity that legislative changes present for the inclusion of students with disabilities in higher education institutions, and the resources dedicated to that purpose, call for an in-depth examination of the results to determine how these students participate in academic and student life at university. The massification of higher education in South Africa following the advent of democracy meant that populations previously disadvantaged under apartheid now had access to education. In South Africa, the Ministerial Statement on the Implementation of the University Capacity Development Grant program 2021-2023 (DHET, 2020) concludes that the system has not yet been able to transform sufficiently to effectively and equitably support the success of previously marginalised groups. Disaggregation of the student data in the Ministerial Statement is only by race and gender. No statistics are given on the retention, throughput and graduation rates of students with disabilities. It is against this background that this study, grounded in the qualitative paradigm, used individual semi-structured interviews conducted with five blind students who were studying at the rural based university to examine the issue of inclusion and exclusion of students with disabilities in higher education. The population of this study consisted of all registered students with disclosed disabilities, all academic support staff in the Disability Unit and all lecturers teaching blind students. Purposive sampling was used to identify the blind students through available university records. Interpretative phenomenological approach (IPA) was used to engage participants to understand their lived experiences from their perspectives. The study seeks to contribute to the debate on the plight of students with disabilities with a specific focus on blind students in rural disadvantaged contexts. Review of Literature Conceptualising Disability The South African White Paper on the Rights of Persons with Disabilities (WPRPD, 2015) conceptualises disability as a complex and evolving concept and argues that defining it must take into account that, “current definitions of disability have evolved over time, and reflect a more progressive view of disability than was the case in the past” (p.17). Among the various attempts to conceptualise disability, two dominant models have emerged, the medical model and the social model of disability. The medical model focuses attention on the nature of the person’s impairment and the degree to which this impairment may or may not prevent the person from carrying out various tasks or participate in activities in ways regarded as normal (Howell, 2005). This model sees disability as inherent in the individual, rather than as a social condition vested in the social milieu (Ndlovu & Watson, 2016). The model focuses on individual deficit or impairment, and attributes any restriction that the individual confronts in his or her everyday life as the 21 inevitable and tragic consequence of that impairment (Hammell, 2006; Ohajunwa, Mckenzie, & Lorenzo, 2015). The emphasis is on the impairment rather than the abilities the person might possess. The disability rights movement rose against the medical model arguing that the circumstances of people with disabilities and the discrimination they face are socially created and have little to do with the impairments of people with disabilities (Howell, 2005). This gave rise to the alternative social model of disability. Rather than focusing on individual impairment, this model focuses on the physical and social barriers which exclude people with disabilities and renders them powerless and voiceless (Watson, 2004). As Armstrong, Armstrong and Spandagou (2011) show, a person’s impairment is not the cause of disability, but rather disability is the result of the way society is organised, which disadvantages and excludes people with impairments. The solution to the problems of disabled people in this paradigm therefore lies in restructuring society in order to accommodate them. South African legislation, for example, the White Paper on the Rights of Persons with Disability (WPRPD, 2015) is aligned to the social model of disability. Visual Impairment Visual impairment relates to those students who are categorized as legally blind, having a visual acuity of 6/60 or less in the better eye, and/or a visual field of less than 10 degrees (Permvattana, Armstrong, & Murray, 2013). According to Shepherd (2001), the visual system can be considered as the dominant sensory modality in humans as almost half the brain is devoted to sight, and about 70% of the total capacity of the brain devoted to processing sensory information is devoted to handling visual information. Chikukwa and Chimbwanda (2013) define visual impairment or low vision as, “a severe reduction in vision that cannot be corrected with standard glasses or contact lenses and reduces a person's ability to function at certain or all tasks” (p.4). The level of visual impairment ranges from severe short-sightedness to blindness (Ghafri, 2015). Most learning typically occurs visually. The challenge facing visually impaired students is that the enormous amount of learning that normally takes place via vision must now be achieved using other senses and methods. The focus of this paper is on the challenges faced by blind students at a rural based South African university. Legislative framework on education for people with disabilities in South Africa Following the demise of apartheid, several pieces of legislation have been promulgated to advance the rights of people with disabilities in South Africa. These include the Constitution of the Republic of South Africa (CRSA, hereafter) (1996), the White paper on an Integrated National Disability Strategy (INDS, hereafter) (1997), the National Plan for Higher Education (NPHE, hereafter) (2001), White Paper 6 on Special Needs Education: Building an Inclusive Education and Training System (SNEBIETS, hereafter) (2001), the White Paper for Post-School Education (PSET, hereafter) (2013) and the White Paper on the Rights of People with Disability (WPRPD, hereafter) (2015). The South African legislation as explained in the paragraphs that follow, steers people away from the medical conception of disability discussed in a previous subsection, which focuses on individual impairment or deficit and instead focuses on the elimination of physical and social barriers that exclude people with disabilities and renders them powerless and voiceless - a tenet of the social model of disability. The CRSA (1996) declares all people as equal and outlaws discrimination on any basis and guarantees the right to quality services for persons with disabilities. The 22 entrenched Bill of Rights (BR) of the CRSA (1996) Subsection 3, states that no person may unfairly discriminate directly or indirectly against anyone. The INDS (1997) acknowledges that the majority of people with disabilities in South Africa have been excluded from the mainstream of society and have thus, been prevented from accessing fundamental social, political and economic rights. The strategy cites several factors as having contributed to the neglect of people with disabilities, among them the political and economic inequalities of the apartheid system; social attitudes, which have perpetuated stereotypes of people with disabilities as dependent and in need of care; and a discriminatory and weak legislative framework that has sanctioned and reinforced exclusionary barriers. y Another piece of legislation, NPHE (2001) advocates for an increase in the number of non–traditional students entering higher education particularly women and people with disabilities. The plan requires universities to indicate in their three-year plans, strategies, time-frames and targets to increase the enrolment of students with disabilities. The year 2001 saw the publication of the Education White Paper 6: SNEBIETS which further outlined measures to include students with disabilities in the education system. The PSET (2013), like the other pieces of legislation refers to the issue of inclusive education for all. Legislative framework on education for people with disabilities in South Africa The White Paper argues that the achievement of greater social justice is closely dependent on equitable access by all sections of the population to quality education and points out that, “the post-school system must respond to the special education and training needs of various social groups such as the youth, the disabled…” (p.10) The most recent legislation on the issue of disability is the WPRPD (2015). The vision of the WPRPD is the creation of a free and just society inclusive of all persons with disabilities as equal citizens. This is aligned to the social model of disability, which argues that the solution to the problems of people with disabilities lies in restructuring society in order to accommodate them. In this regard, the WPRPD (2015) commits duty bearers to realising the rights of persons with disabilities by:  Accelerating implementation of existing legislation that advocates equality for persons with disabilities;  Accelerating implementation of existing legislation that advocates equality for persons with disabilities;  Taking calculated action to ensure that their rights as equal persons are upheld;  Removing discriminatory barriers to access and participation;  Ensuring that universal design informs access and participation in the planning,  Ensuring that universal design informs access and participati  Ensuring that universal design informs access and participation in the planning, budgeting and service delivery value chain of all programmes (p.11). budgeting and service delivery value chain of all programmes (p.11). It can be seen from the above discussion, though not necessarily the case for all 26 universities, that the South African higher education system provides support for students with disabilities within the diversity rights framework (Matshedisho, 2007) guided by national legislation and underpinned by the principle of fundamental human rights for all. Most public universities in South Africa have an office responsible for students with disabilities commonly referred to as a Disability Unit (DU). The philosophy behind establishing the DUs according to Tugli et al. (2013) is to promote the equal participation of people with disabilities in all spheres of university life and to eliminate unlawful disability discrimination, including disability related harassment. These units, however, although in existence, do not always result in quality support for the students with disabilities. Legislative framework on education for people with disabilities in South Africa Results from a study by Naidoo (2010) on factors affecting the academic development of students with disabilities at the University of KwaZulu-Natal show that the lack of staff in the Disability Unit and the disproportionate ratio of staff to students; lack of resources and lack of funding from the University negatively affected the efforts of the Disability Unit to offer support to students with disabilities. Similarly, a study by Tugli et al. (2013) revealed that the Disability Unit at the University of Venda was understaffed (only two staff members) and the 23 staff felt overworked and overwhelmed. Concern about Disability Units programmes being isolated or disability issues not being integrated into core areas of the institution’s functioning emerged in the findings of a study by Howell (2005). Similarly, in a pilot study on challenges faced by students with disabilities at four universities in the Western Cape Province in South Africa, the Department of Social Development (DSD, hereafter) (2015) reports that students complained of the long time it took to get braille material due to staffing constraints in the Disability Units. Thirty-two percent of their participants indicated that they encountered barriers in accessing learning materials in accessible formats. The report noted: g g p “These barriers include absence of Braille and large font material; delays in getting material transcribed or adapted into accessible formats; test scripts and old examination papers only available in small print; reference material in libraries needed for assignments and other projects not available in accessible formats.” (p. 47). g g p “These barriers include absence of Braille and large font material; delays in getting material transcribed or adapted into accessible formats; test scripts and old examination papers only available in small print; reference material in libraries needed for assignments and other projects not available in accessible formats.” (p. 47). In the same vein, the Foundation of Tertiary Institutions of the Northern Metropolis (FOTIM, hereafter) (2011) identified under-funding as a key constraint in the funding of several Disability Units. With inadequate budgets, such units cannot adequately cater for disabled students. Another major challenge facing visually impaired students in South Africa is stigmatisation and alienation faced if students disclose their condition. According to findings of the DSD (2015), participants who chose not to declare disability gave reasons relating to societal negative perceptions including labeling, alienation, attitudinal problems and stereotyping. Legislative framework on education for people with disabilities in South Africa Some of these reasons for non-disclosure as the DSD (2015) shows, are indicative of the prejudicial nature of society towards persons with disability. Study Context and Objectives This study sought to examine barriers to learning for blind students at a South African University. The specific objectives of the study were to: Identify challenges faced by blind students at a rural based university; examine ways in which blind students mitigate these challenges and explore ways of ensuring reasonable accommodation for blind students in higher education.The institution studied is a small to medium-sized, comprehensive university located in a remote rural area and draws most of the students from previously disadvantaged rural schools. The institution has a Disability Unit established in 2001 to integrate services for students with disabilities. The Unit has three staff members and an intern and supports students with the following disabilities: visual, hearing, physical, speech impairments, chronic illnesses (e.g. epilepsy), painful conditions (e.g. back injuries & carpal tunnel syndrome), psychological disabilities (e.g. bipolar disorder & severe anxiety/depression), learning disorders and temporary disabilities (e.g. broken limbs). According to statistics from the Disability Unit, 118 students disclosed their disabilities in 2014 and of these 52 were partially sighted while five were blind while in 2022, a total of 99 students disclosed their disabilities15 as partially sighted and one as blind. Four of the five interviewed participants became blind later in their lives while one participant was born blind. Four of the participants were in their undergraduate studies, one each in levels one, two, three and four of their studies while one of the participants was a postgraduate student. With regards to gender, four participants were male while one was female and all the participants were black. Research Methodology Grounded in the qualitative paradigm, individual semi-structured interviews were conducted with five blind students who were studying at the rural based university in South 24 Africa. The population of this study consisted of all registered students with disclosed disabilities, all academic support staff in the Disability Unit and all lecturers teaching blind students. Purposive sampling was used to select participants for the study and is based on the assumption that a researcher wants to discover and gain insight, and thus, selects a sample from which the most can be learned (Yssel, Pak, & Beilke, 2016). Records in the Disability Unit were used to identify the blind students. The two academic support staff in the Disability Unit and four lecturers who had blind students in their classes were also included in the sample for triangulation of data. In this study, the researchers wanted to gain insight into the challenges faced by blind students in navigating the higher education landscape, hence purposefully selecting those affected by the impairment (blindness). This produced rich data. p p y g y p p The interpretative phenomenological approach (IPA) was used both during the interviews and the data analysis process. IPA is a qualitative approach which aims to provide detailed examinations of personal lived experience (Smith and Osborn 2015). IPA approach argues that good research interviewing recognises that the course and content of an interview cannot be laid down in advance (Smith, Flowers, & Larkin 2009; Alase, 2017). According to Tuffour (2017), the aim of IPA is to look in detail at how someone makes sense of life experience, and to give detailed interpretation of the account to understand the experience. IPA has a commitment to understanding particular experiences in context (Rose et al. 2019). Such experiences cannot be predetermined. Rather than a rigid interview schedule that would be followed religiously therefore, a prompt sheet with a few main themes for discussion with the participants was produced to guide a loosely structured interview process (Biggerstaff and Thompson 2008). The theme statements were developed after a thorough review of literature sources that speak to the experience that is being studied, that is, blind students in higher education in rural contexts. The prompt sheet checklist ensured that while participants were given the freedom to take the lead in the conversation, the data gathered would still speak to the purpose of the research. Research Methodology The interview schedule was merely the basis for kick-starting the conversations with participants. As Jeong and Othman (2016) show, it is important that IPA researchers as a rule, utilise the open-ended question formula. During the interviews, the researchers did not only listen to what the participants described about their experiences, but also focused on the interpretation of those experiences through probing, and asking critical questions to the participants, as suggested by Mavhandu- Mudzusi (2016). In addition to field notes taken during the interview, all interviews were recorded and transcribed verbatim in order for both researchers to be able to participate in the process of analysis. Coding was first conducted by each researcher individually and then in a joint discussion leading to a decision on core themes as suggested by Berggren, Rowan, Bergbäck and Blomberg (2016). Interviews of each participant were analysed individually, to identify emerging themes. Following this, the researchers looked for common patterns across the analysed cases. This resulted in restructuring and relabeling the original individual themes into few overarching themes (Lourens & Swartz 2016a). Challenges relating to mobility and accessibility One of the themes that emerged from the data was in relation to accessibility and mobility around campus due to carelessness in infrastructure development and negligence by fellow students and academics. The following were examples of responses on the issue: g p p • Changes along the route to places of choice are intermittently changed without our knowledge. We end up falling on trenches(BSA) • Changes along the route to places of choice are intermittently changed without our knowledge. We end up falling on trenches(BSA) • I once fell into the ditch and luckily it was not so deep. I could not proceed to my class that day as I was mildly injured (BSD). • I once fell into the ditch and luckily it was not so deep. I could not proceed to my class that day as I was mildly injured (BSD). • It is not easy to move on the paths of the university because there are light poles erected on the middle of the paths (BSE). • No. there are a lot of disturbances. People drive around the campus as if they are on a freeway. People park everywhere. Security staff are not consistent in monitoring this challenge (L3) • No. it is horrible. University community parks the cars everywhere and they drive so fast on the campus roads. This is not good for those students with disabilities (AS1). • No. it is horrible. University community parks the cars everywhere and they drive so fast on the campus roads. This is not good for those students with disabilities (AS1). The results show that new infrastructure development projects around the university were not communicated to the students leading to challenges for blind students as shown in the responses above. The student concerns are corroborated by both the academic support staff (AS1) and the lecturers (L3). More stringent security measures would go a long way in helping curb careless driving and undesignated parking in the university. The issue of mobility and accessibility is also reported in the literature. Howell and Lazarus (2003) argue that barriers for students with disabilities are exacerbated by higher education institutions in South Africa that remain largely physically inaccessible to many disabled students, especially physically disabled and blind students. Ethical considerations Working with people with a range of special needs demands sensitivity and an increased awareness of the great vulnerability of many of these research participants (Magwa & Magwa 2015). Appointments were made with each of the blind students individually where the research project and its purpose was explained to them. Participants were informed that their participation in the study was entirely voluntary and that they could withdraw at any time without consequence. Further, they were informed that confidentiality and anonymity would be maintained and that their identities would not be disclosed as pseudonyms would be 25 used. Participants gave informed consent. The five students were then given alphabetic name codes; Blind Student A (BSA), Blind Student B (BSB), Blind Student C (BSC), Blind Student D (BSD) and Blind Student E (BSE). Academic support staff (AS) were coded numerically as AS1 and AS2 while lecturers (L) were coded as L1, L2, L3 and L4. Ethical clearance was sought from the Research Ethics Committee of the University following the university’s ethical clearance application procedures and was granted under reference number CHETL/11/01/E0811. Results and Discussion The results are presented and discussed according to the following emerging themes: challenges related to mobility and accessibility, instructional challenges, assessment practices, lack of knowledge, interaction with non-disabled students, academic support from the Disability Unit and academic support from the library. Samples of verbatim responses from the participants are used in the discussion of each identified theme. The choice of which participant to quote in each instance was guided by both the typicality of the response with regards to the theme identified and the need to represent all participants as much as possible in the quoted responses. Instructional challenges One issue identified by academic staff and lecturers was the issue of accessibility to learning nd teaching in the lecture halls as a result of lack of universal design in the construction of ecture rooms: One issue identified by academic staff and lecturers was the issue of accessibility to learning and teaching in the lecture halls as a result of lack of universal design in the construction of ecture rooms:  Chairs in these lecture halls are located far from the white board making it difficult for visually impaired to see what is written. Most students with visually impairment just go to classes only to listen to the lectures (ADS1)  The sitting arrangements are in a form of a stadium and it is difficult for a blind student to walk through. Chairs are built in and in a form of a row and it becomes difficult for a blind student to walk through to the chair (L4)  Some lecture halls are not conducive for teaching visually impaired students. For example, lecture halls A and E. The board is far from the seats (L2) Such infrastructure design issues identified by lecturers in the responses reveal lack of universal design planning in the furniture installation in the lecturer rooms. Ensuring that universal design informs access and participation in the planning, budgeting and service delivery value chain of all programmes (WPRPD, 2015) will ensure that all categories of students are catered for. Negative attitudes of lecturers and insensitivity to the needs of blind students during the actual learning and teaching process were major issues raised by the blind students, for example:  Some lecturers are arrogant. They do not provide pamphlets and they tell us to consult with other students. I followed one lecturer the other day and he told me; ‘you disabled students like to be treated special. He is not God. I had to quit the module’ (BSB).  As a lecturer I have never given such students special handouts, except that they write their tests and examinations in Disability Unit where they are well-catered (L2).  My students get materials like other students. I don’t differentiate them because I was not orientated or trained to care for the students with disabilities (L4).  They send materials to us and we adapt them according to the needs of students. Challenges relating to mobility and accessibility Lourens and Swartz (2016a) found that simply getting around campus was a challenging task for the students who spoke of threats of motor vehicles and obstructions in the environment such as holes in sidewalks, low-hanging branches and road works. Similarly, a study by Chikukwa and Chimbwanda (2013) in Zimbabwe, found that totally blind students faced problems of construction work (trenches dug up everywhere), parked cars and water puddles. In this regard, amendments to campus design that are not only 26 visually impaired student-friendly, but also environmentally friendly are warranted (Berggren et al. 2016). This, according to (UNCRPD) (2006) means the design of products, environments, programmes and services should be usable by all people, to the greatest extent possible, without the need for adaptation or specialised design. student however, felt accessibility was better for blind students compared to physicall dent however, felt accessibility was better for blind students compared to physically d students although he had problems with elevators as shown in this response: One student however, felt accessibility was better for blind students compared to nt however, felt accessibility was better for blind students compared to physically udents although he had problems with elevators as shown in this response: , y p p y y d students although he had problems with elevators as shown in this response: • Access into buildings is better to blind students because we can manage to go to the other floors of the building using steps. The lift in the main administration is not user friendly to the blind person as the buttons are not clearly marked in Braille and there is no voice synthesizer to help the blind user (BSB). • Access into buildings is better to blind students because we can manage to go to the other floors of the building using steps. The lift in the main administration is not user friendly to the blind person as the buttons are not clearly marked in Braille and there is no voice synthesizer to help the blind user (BSB). From the analysis of the except above, it is indicative that students with sight challenge emphasise the need for good access into buildings. However, the access in existence is not user friendly to students with sight problems because there are no braille signs and audio configuration to assist in effective usage by the blind. Instructional challenges (2016) conducted research into the knowledge, attitudes and experiences of staff within Australian universities. This research suggested that universities need to be more informed, and consistent with legislation in the area of disability as staff at times held negative or hostile attitudes towards students with disabilities. Similarly, Chikukwa and Chimbwanda (2013) in their study found that some lecturers were reluctant to modify their classroom procedures, giving students only partial accommodations. They further argue that given the significance of lecturer attitudes to the success of blind and visually impaired students, it is important to understand the significance of attitudes of not only the lecturers but also of peers and other campus administrators to the success of these students. Studies in the literture emphasise the importance of lecturer preparation programmes to provide extended experiences for future lecturers to facilitate conceptual shifts and improve attitudes about assisting students living with disabilities (Barton-Arwood, Lunsford, & Suddeth, 2016; Zongozzi, 2022) As the social model of disability argues, a person’s impairment is not the cause of disability, but rather disability is the result of the way society is organised, which disadvantages and excludes people with impairments (Armstrong, Armstrong and Spandagou 2011). Instructional challenges The challenge only pops up if a student did not divulge his/her disability to the lecturer (AS1). There was justification by those lecturers who refused to give blind students materials so that they could take them for adaptation, for example - converting material to braille arguing that this amounted to special treatment. This argument y lecturers, in our view, cannot 27 be accepted as this is a category of students that does indeed need special treatment. The significance of the lecturer as partner in the success of the blind student is also evident from the student responses. This reproduction of negative attitudes in higher learning emanates from people viewing disability negatively (Ndlovu and Walton 2016) and could be attributed to the medical model of disability, which sees disability as inherent in the individual, rather than as a social condition. Ryan (2011) as cited in Dryer et al. (2016) conducted research into the knowledge, attitudes and experiences of staff within Australian universities. This research suggested that universities need to be more informed, and consistent with legislation in the area of disability as staff at times held negative or hostile attitudes towards students with disabilities. Similarly, Chikukwa and Chimbwanda (2013) in their study found that some lecturers were reluctant to modify their classroom procedures, giving students only partial accommodations. They further argue that given the significance of lecturer attitudes to the success of blind and visually impaired students, it is important to understand the significance of attitudes of not only the lecturers but also of peers and other campus administrators to the success of these students. Studies in the literture emphasise the importance of lecturer preparation programmes to provide extended experiences for future lecturers to facilitate conceptual shifts and improve attitudes about assisting students living with disabilities (Barton-Arwood, Lunsford, & Suddeth, 2016; Zongozzi, 2022) be accepted as this is a category of students that does indeed need special treatment. The significance of the lecturer as partner in the success of the blind student is also evident from the student responses. This reproduction of negative attitudes in higher learning emanates from people viewing disability negatively (Ndlovu and Walton 2016) and could be attributed to the medical model of disability, which sees disability as inherent in the individual, rather than as a social condition. Ryan (2011) as cited in Dryer et al. Lack of knowledge The need for training of staff who work with disabled students came out strongly in the findings of this study. This appeared in responses from both students, academic support staff and lecturers as shown in these responses:  Lecturers do not really know how to assist a blind student. They demonstrate, project their PowerPoint presentations and write on the board forgetting that a blind student cannot see and needs special attention (BSC).  In the class, the lecturer writes a lot on the board and narrates so little of what they have written. Sometimes they will just point at facts on the board saying this and that, when you combine this and that you get this (BSA).  New staff should be inducted on how to interact with disabled students.  New staff should be inducted on how to interact with disabled students. Unfortunately, we have a backlog due to the pandemic (AS1). Unfortunately, we have a backlog due to the pandemic (AS1).  Lecturers should be trained to take care of students with disabilities (L4). With regards to the lecturers’ capacity to help blind students, the results from all the categories of participants (students, lecturers and academic support staff) indicate that lecturers were not conversant with how to deal with these students. The way they presented their materials in class did not accommodate the needs of blind students. There was, however, one student who was positive and who stated that her lecturers were friendly and understanding as shown in this response: My lecturers are friendly. They never ask questions that demand me to draw. I request all the slides from the lecturer immediately after lesson. Sometimes they give me hard copies which I take to the disability unit for scanning, editing and brailing (BSE). As shown in the responses, projecting power point presentations on the screens and writing on the whiteboards did not accommodate blind students if this was not accompanied by verbal reading of what was on the slides. Whatever is projected or written on whiteboards should also be read out aloud to accommodate this category of students. The issue of lack of knowledge and training of lecturers is reported in the results from a study by Mushome and Monobe (2013) which revealed that lecturers found teaching visually impaired students a problem as they had never been trained to teach this category of students. Assessment practices Another major finding under instructional challenges related to assessment with regard to whether or not the assessment practices used accommodated the condition of blind students. All the five students were positive although they alleged that there was poor planning at times; • Blind students write their tests and examinations in the adapted exam lab in the Disability Unit. Test papers are either Brailed or given in an electronic format. With the aid of screen reading software, I find it very easy to read and write (BSE). • I had to write a little bit late sometimes when the lecturer could not provide an electronic question paper to the exam department. The exam had to be scanned, edited and brailed while waiting in the exam room (BSC). • The University staff seem to believe that students with disabilities should only be assisted in the disability unit. Even lecturers, when they have challenges with disabled students, they send them to the disability unit (AS1). The Disability Unit should be lauded for ensuring assessment accommodation for the blind students. The Unit seems to be aligned to the social model of disability, which focuses on removing the physical and social barriers which exclude people with disabilities (Watson, 2004). Adapting the assessment instruments for students with disabilities also resonates with one of the requirements of the WPRPD (2015) which legislates for the removal of discriminatory barriers to access and participation. Academic support staff in the Disability Unit, however, expressed concern at the fact that academics were abrogating their responsibilities and dumping interventions related to students with disabilities on the Disability Unit rather than co-owning the student support. It is significant to note that the findings of this study contradict those of Vickerman and Blundell (2010) where 11.1% of disabled students indicated that their assessments did not cater for their needs, compared with 28 3.6% of their non-disabled peers. The results further contradict those of Dryer at el. (2016) which showed that students with disability were often challenged by assessment practices. The issue raised in the last response above however, about examinations and tests being adapted while the students were already waiting in the examination room to write, points to poor coordination between the lecturers, the examinations department and the Disability Unit. Assessment practices The Disability Unit, the lecturer who sets the examinations paper and the examination department need to liaise with each other to ensure that all examinations have been adapted for disabled students before the examination date. Interaction with non-disabled students One of the obstacles faced by people with disabilities in general and blind students in particular as shown in the results of this study is stigmatisation and isolation by abled people. The blind students in this study felt that their abled counterparts rejected them and did not want to associate with them. This rejection and isolation by non-disabled students was expressed as follows: • Some abled students do not like to form a group with a disabled student. They have negative perceptions that maybe we shall be a burden to them (BSD). • The abled students do not easily fuse with disabled students. When lecturers require us to form groups, they isolate us (BSB). • They don't want to help them. They say there are getting marks from something that they didn’t work for (L3). • All disabled students are side-lined by the able students. The able students do not want to create friendship with the disabled students (L1). • The university is not good for the formation of diversity groups since students are not trained for this kind of formation (AS1). The rejection and isolation by abled students was expressed in cases where abled students did not want to form groups with students with disabilities when group work tasks were assigned. The sentiments from the blind students are echoed by the academic support staff (AS1) and the lecturers (L1 and L3). The argument for student training in issues of diversity suggested by one of the lecturers is indeed laudable. Not all non-disabled students were labelled as having negative attitudes towards blind students. One blind student had not experienced any problems interacting with non-disabled students while one lecturer had witnessed cases of abled students assisting a blind student in her class: • I make friendships with my classmates. I do not have problems when it comes to formation of groups in the class (BSE). • I make friendships with my classmates. I do not have problems when it comes to formation of groups in the class (BSE). formation of groups in the class (BSE). • Yes, my students always assist the visually the impaired student, they always assist her with finding venues, with recording presentations for her and in case she doesn’t come to class they always update her (L4). Lack of knowledge Chikukwa and Chimbwanda (2013) also found in their study that most lecturers lacked special training in handling students with disabilities even though they had first degree qualifications and even postgraduate degrees in Special Needs Education. If academics are to respond effectively to the needs of the visually impaired student, they will need to invest time in relevant staff development (Shepherd, 2001). As Vickerman and Blundell (2010) show, “Whilst equality legislation is an important part of the jigsaw, it is vital that this is matched by the education and training of higher education (HE) staff to respond proactively to the diverse needs of the disabled students they support” (p.28). On the issue of policies, citing the University of South Africa (UNISA), Zongozzi, (2022 argues that, “Although UNISA appears to have good disability policies in place, the problems mentioned so far seem to stem from poor 29 implementation of these policies” (p.1653). It is noteworthy however, that one of the five blind students interviewed was positive and stated that her lecturers were friendly and understanding. Academic Support from the Disability Unit Results show that the Disability Unit was seen as a valuable resource centre for the blind students. Two students spoke positively about the Disability Unit. Factors external to the disability unit that negatively affected efforts to support blind students were identified by academic support staff: pp • The Disability Unit is the only accessible building in the campus. The staff is very h l f l b t th lib t ff t t t h t i t (BSD) pp • The Disability Unit is the only accessible building in the campus. The staff is very helpful but the library staff are not competent enough to assist us (BSD). pp • The Disability Unit is the only accessible building in the campus. The staff is very helpful but the library staff are not competent enough to assist us (BSD). y y g p ff y helpful but the library staff are not competent enough to assist us (BSD). • The Disability Unit staff are trying their level best but the staff is inadequate to cater for us effectively (BSB). • As a unit we provide them with assistive devices on loan as soon as a challenge is identified. The problem is only their personal adapted devices that are purchased by their bursaries. You will find that the unit initiate the procurement process of assistive devices this year and they are only to be purchased two years later. Sometimes the devices are purchased when the student has dropped out or completed the degree (AS1). • When the unit has secured some funds from Department of Higher Education for the improvement of its services, it becomes rocket science to release such funds. Something should be done to alleviate such delays (AS2) • When the unit has secured some funds from Department of Higher Education for the improvement of its services, it becomes rocket science to release such funds. Something should be done to alleviate such delays (AS2). Something should be done to alleviate such delays (AS2). Timely provision of learning and teaching resources is critical for all students and even more critical for students with special needs such as blind students who do not have alternative access to learning without such resources. Procurement delays as identified by the academic support staff have to be rectified to promote equity of outcomes for blind students. Interaction with non-disabled students Such social stigmatisation, discrimination, isolation and stereotyping of disability can also contribute to depression and withdrawal of some students with disabilities (Tugli et al. 213). Fvazza et al. (2016) aver that, children with disabilities are among the world’s most stigmatised and excluded population because of limited understanding and knowledge about persons with disabilities. Such stigmatisation and labelling, as the literature shows, might result in students concealing their disabilities where these are not easily visible. Yssel, Pak and Beilke (2016) found that one barrier was reluctance on the part of students to disclose their disabilities and be labelled. Citing Jacoby and Austin (2007), Vickerman and Blundell (2010) suggested that having a disability can increase the perception that they are devalued and stigmatised, and as such, this may be why some students were concerned about disclosure in case it results in negativity and lack of access. A study by Lourens and Swartz (2016b) found that some partially sighted students, whose impairment was less obvious, went to great lengths to conceal their visual impairment in order to gain acceptance and inclusion into non-disabled peer groups. The fact that not all 30 abled students were labelled as having negative attitudes towards blind students shows that there are some students who are accommodating.  There is no internet access in the residence (BSA). Academic Support from the Library Two of the students, as shown in the verbatim quotes below, felt the library was not conducive enough for them citing lack of resources and moody unapproachable staff: • There are no electronic or Braille books in the library. As from 2013, an adapted lab was established but still running short of important tools such as Pearl reading cameras, IPAL solo standalone reading device and many more (BSA). • There are no electronic or Braille books in the library. As from 2013, an adapted lab was established but still running short of important tools such as Pearl reading cameras, IPAL solo standalone reading device and many more (BSA). • In the library, they have a school representative who assists me whenever I am looking for references but the staff members are often moody and unapproachable. There are no electronic books (BSE). pp ( ) • The library is very good inside because there is a ground floor where the blind students can walk freely and have access of computers and internet (L3). f y f p ( ) • The lab for disabled students in the library is too small with little resources (AS1). • The lab for disabled students in the library is too small with little resources (AS1). • Blind students need a dedicated human support. Blind students need someone to direct them in the library (AS2). • Blind students need a dedicated human support. Blind students need someone t direct them in the library (AS2). While lecturers viewed the availability of an adapted computer laboratory section in the library as adequate, academic support staff felt the adapted section could be enlarged. In unison with students, as shown in the responses, academic support staff felt resources for blind students in the library were inadequate. Close collaboration between the library and the Disability Unit could be one way in which adapted resources could be incorporated into the library budget. Students with disabilities, including blind students, like any students need access to library resources to complete assigned tasks. Although an adapted lab had been built in the library, it had not been equipped with requisite resources. It should be noted that there was dearth of literature relating specifically to blind students. Studies reviewed referred generically to visually impaired students who would include partially sighted students. This study therefore, contributes to the debate by foregrounding the plight of totally blind students. Academic Support from the Disability Unit All lecturers were not sure of the provision of devices because this responsibility was vested in the Disability Unit. One student had issues with the operating hours of the Disability Unit. Unlike the university library that closed in the evening, the Disability Unit closed early leaving these students without access to adapted resources as shown in this response: • The main challenge happens after the Disability Unit is locked when the admin staff go home because we will not have access to the adapted lab. There are no internet cables in the hostels. If installed, we will be able to access the network licensed Job Access with Speech (JAWS), the screen reading software (BSE). This lack of after-working-hours access could be because of the reported shortage of staff in the unit. Such staff shortages are not peculiar only to this institution. Naidoo (2010) also reported that lack of permanent staff at the DU at the University of KwaZulu-Natal resulted in delays in students receiving study and examination related materials. While according to Tugli et al. (2013) the philosophy behind establishing the DUs is to promote the equal participation of people with disabilities in all spheres of university life, it appears from the results that at the university under study, blind students had no access once Disability Unit staff left at the end of their normal working day. Lack of ‘after-working hours’ access seems novel and peculiar to this study as such a concern could not be found in the literature reviewed. Blind students received disability grants, which they used to procure laptops for their studies. The students, however, lamented the lack of internet services in the residences, which rendered their devices useless as shown in these responses: 31  From 2010 we were given laptops that cannot access internet as there is no WIFI in the campus (BSC).  From 2010 we were given laptops that cannot access internet as there is no WIFI in the campus (BSC).  From 2010 we were given laptops that cannot access internet as there is no WIFI in the campus (BSC).  AS1 No. there are no adapted labs in the residence. Visually impaired students have to travel to the library to study in the evening. The university should build labs that are adapted. This will help students not to travel long distances at night.  AS1 No. Academic Support from the Disability Unit there are no adapted labs in the residence. Visually impaired students have to travel to the library to study in the evening. The university should build labs that are adapted. This will help students not to travel long distances at night. This calls for the need for planning for an after-hours service for these blind students. A shift system could be introduced or alternatively arrangement could be made to have a security officer man the Disability Unit adapted lab after working hours. The university could also consider installing WIFI in residences as the students lamented the lack of internet services in the residences, which rendered their laptop devices useless in accessing information. Alternatively, accessible computers should be made available in the usual computer rooms that the general student population uses so that students with disabilities can access these after the disability unit has closed. It was worrying to note that three out of the four lecturers had not bothered to check what conditions were like in the residences for blind students as they either were not sure or confessed to never having been to student residences. Conclusions and Recommendations It can be concluded from the results of this study that considerable strides have been made towards accommodating the needs of students with disabilities in line with the social model of disability-both in relation to inclusive policy legislation in South Africa and efforts by the Disability Unit at this university. Tenets of the medical model can however still be seen in the lack of adequate institutional arrangements around universal design and in the way lecturers and students without disabilities treat students with disabilities. The study found that reasonable accommodation was not provided for blind students in the lecture halls at the university under study as study materials in the library were not properly adapted, computers in the university library and mainstream computer laboratories were not accessible to blind students and lecturers were not trained to teach blind students. Some lecturers used PowerPoint presentations while teaching when blind students could not access the screens. The study recommends universal design for all learning facilities which, according to UNCRPD (2006) means the design of products, environments, programmes and services to be usable by all people, to the greatest extent possible, without the need for adaptation or specialised design. Adapted computers should be made available in the usual computer rooms that the general student population uses so that students with disabilities can access these after the disability unit has closed for the day. The study further recommends thorough training for all staff on teaching students with various disabilities. A short term recommendation offered while the university is still looking for resources to hire more staff is that a shift system be introduced or alternatively, arrangements be made to have a security officer stationed at the adapted lab after working hours. The university should also consider installing WIFI in the residences. In addition, it is recommended that the Disability Unit, the lecturers and the examinations department need to liaise with each other to ensure that all examinations have been adapted for disabled students before the examination date. The study further recommends improvement in the university’s procurement processes to ensure that all students in general and blind students in particular receive the requisite resources needed for learning and teaching on time. Academic Support from the Library A study exploring the use of the library by visually impaired students (Sehić & Faletar, 2014) found that in most cases, these students visited libraries only after all other options had been exhausted because their experience had taught them that their academic libraries did not possess adequate technology and resources needed for their studies. In this regard, Eskay and Chima (2013) assert that the education system in developing countries not fully embraced or adopted the technology associated with special library services for the visually impaired 32 students. This is evidenced in the lack of production and distribution facilities for reading materials for these students. Ekwelem (2013) advises that as more people with disabilities attend higher education institutions, it is incumbent upon library management to provide the same level of service to them as is provided to users without disabilities. In the current case study, close collaboration between the library and the Disability Unit could be one way in which adapted resources can be incorporated into the library budget. 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Accessible Quality Higher Education for Students with Disabilities in a South African Open Distance and e-Learning Institution: Challenges, International Journal of Disability, Development and Education, 69(5), 1645-1657, DOI: https://doi.org/10.1080/1034912X.2020.1822518 36
https://openalex.org/W2803643444	https://www.matec-conferences.org/articles/matecconf/pdf/2018/24/matecconf_fatigue2018_02011.pdf	English	null	The role of pores and microstructural heterogeneity on the tooth root fatigue strength of sintered spur gears	MATEC web of conferences	2,018	cc-by	4,705	1 INTRODUCTION sintered density, the damage usually occurs very early, located at the necks between powder particles, which are the weakest point of the microstructure. A widespread damage occurs and the final failure is often caused by the coalescence of numerous defects previously initiated [11]. By increasing the sintered density to high values, even up to near-full density, the triggering of the damage is delayed and the pores can be regarded as local defects of different criticality, able to concentrate the stresses and to act as internal notches or, in certain situations, even as cracks [5, 12 to 15]. In this case the fatigue response is dictated by the most critical defect, which is generally an irregularly shaped pore located in the regions of maximum stress. The structural parts produced by powder metallurgy (P/M) represent a competitive solution, alternative to traditional production, in many industrial sectors, including the automotive industry that employs a considerable portion of the total production of P/M parts. The progressive improvement of the production processes has led to the design and fabrication of structural parts able to withstand high stress levels. The gears represent one of these applications, where maximum advantages in terms of competitiveness and quality can be achieved by using a near net shape technique such as P/M [1-5]. The structural parts produced by powder metallurgy (P/M) represent a competitive solution, alternative to traditional production, in many industrial sectors, including the automotive industry that employs a considerable portion of the total production of P/M parts. The progressive improvement of the production processes has led to the design and fabrication of structural parts able to withstand high stress levels. The gears represent one of these applications, where maximum advantages in terms of competitiveness and quality can be achieved by using a near net shape technique such as P/M [1-5]. The research efforts have always been devoted to setting up processes able to increase the relative density of the sintered parts up to 90% and more; in addition, the formulation of new high strength pre-alloyed powders and the development of surface modification techniques have strongly improved the ‘in service’ performances as well as the quality of the final product. Despite this positive trend, some crucial aspects still remain open. One of these is the comprehension of the synergistic effect of porosity and microstructure in the damage process of the material when subjected to ‘in service’ loads [6-10]. The role of pores and microstructural heterogeneity on the tooth root fatigue strength of sintered spur gears Matteo Benedetti1, Vigilio Fontanari1, Alberto Molinari1, Pietro Valcozzena1, and Wolfgang Pahl2 1Department of Industrial Engineering, University of Trento, 38122 via Sommarive 9, Trento, Italy 2GKN Si M l 39031 i d ll F bb i h 5 B i (BZ) I l 1Department of Industrial Engineering, University of Trento, 38122 via Sommarive 9, Trento, Italy 2GKN Sinter Metals, 39031 via delle Fabbriche 5, Brunico (BZ), Italy Abstract. The automotive industry employs a considerable amount of sintered parts, mainly as transmission and engine components. Gears are the parts that mostly benefit, in terms of cost saving, from the near net shape P/M technology. However, the porosity along with the heterogeneous microstructure can detrimentally affect the mechanical behaviour, especially the fatigue strength. The possibility of increasing sintered density up to 90% and more, the use of high strength alloys, as well as post sintering treatments have been extensively investigated obtaining consistent increases in the fatigue strength. The present study focuses on the effects of porosity and microstructure on tooth root bending fatigue of small module spur gears. The aim is to investigate the synergistic contribution of pore morphology and microstructure heterogeneity to the initiation of fatigue cracks and to the following crack paths. High density parts produced by high strength pre-alloyed powders were studied. Part of the specimens was case-hardened to obtain a martensitic/bainitic microstructure in the surface layer. Bending fatigue tests up to a fatigue endurance of three million cycles were performed. A careful fractographic analysis was conducted. The obtained results were discussed using the fracture mechanics approach of Murakami, considering the pores as pre-existing defects, whose propagation strongly depends on the microstructural heterogeneity. © 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: vigilio.fontanari@unitn.it © 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/). MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 * Corresponding author: vigilio.fontanari@unitn.it d by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution ommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Cre License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2.1 Microstructure and porosity The density was measured using the Archimedes method according to ASTM B311, which permits a quantitative evaluation of the total and open porosity. The porosity was also studied by image analysis using an optical microscope, in order to identify the pores morphology and also their spatial distribution in the gear body. In particular, local measurements were carried out at the tooth root, as well as inside the tooth and in the gear body. Table 2 lists the mean porosity values calculated according to ASTM B311, while table 3 reports the results of the image analysis measurements. It can be observed that porosity has a non-homogeneous distribution into the gear volume. 1 INTRODUCTION A critical aspect for the application of the Murakami model concerns the definition of the critical pore and the identification of the hardness that has to be included in the formula. The gears were produced by GKN Sinter Metals. For A85Mo05 a conventional sintering cycle was conducted in a continuous furnace at a temperature of 1120 °C for 30 min in an atmosphere of endogas (A85Mo05); DDH and DHP underwent to a sinter-hardening cycle that provides forced cooling from the temperature of 1120 ° C down to 300 °C, followed by stress relieving in a separate oven at 180 ° C for 1 h. The A85Mo05 gears were case hardened (austenitizing at 860°C with oil quenching) after sintering to improve the mechanical behaviour of the surface layer. In the context of this research topic, the present work is aimed at investigating the tooth root fatigue behaviour of small module spur gears produced by P/M technology, starting from different pre-alloyed powders, compacted up to high green density and consolidated adopting different sintering conditions. The principal objective is to explore the synergic effect of pore structure and microstructure on the fatigue behaviour. For this reason, the fatigue response is discussed starting from an in-depth fractographic analysis aimed at identifying the role of porosity and microstructure in the initiation and propagation of cracks. The Murakami model is applied for distinguishing the synergistic role of porosity and heterogeneous microstructure in defining the fatigue limit of the material. 1 INTRODUCTION This aspect turns out to be particularly critical for structural pieces that work under cyclic loading conditions and are therefore prone to fatigue damage. It can be observed that, for relatively low The microstructural heterogeneity can play a very important role in the damage localization and in the definition of a preferential crack path [5,8]. Particularly critical are the soft phases that usually envelop the critical pores and represent the microstructure of first propagation of the fatigue crack. In some papers [6-8], the pores are treated as pre- existing cracks and a fracture mechanics approach is adopted to predict the propagation conditions of crack- like defects. The Murakami criterion [16], initially proposed for determining the fatigue limit of steels containing inclusions, can be very promising among the fracture mechanics based methods. In particular, in its most general formulation this approach envisages estimating the fatigue limit of the material with the MATEC Web of Conferences 165, 02011 (2018) MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 Fig. 1. Geometry of the P/M spur gears. following equation, that includes also the influence of the load ratio R. 𝜎𝜎𝑙𝑙𝑙𝑙𝑙𝑙= 𝐶𝐶𝐶 𝐻𝐻𝐻𝐻+120 ൫√𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎൯1/6 ∙ቀ 1−𝑅𝑅 2 ቁ 𝛼𝛼 (1) (1) The Vickers hardness HV accounts for the effect of the microstructure, whereas the term 'area' contains the information on the defect geometry, i.e. the dimension of the defect projected on the critical plane of crack propagation, characterized by the maximum normal stress (opening mode I). Finally, C and α are empirical parameters related respectively to the position of the pore with respect to the surface and to the material microstructure. In particular C = 1.41 for surface defects, C = 1.56 for internal defects, while the exponent α = 0.226+HV.10-4. Fig. 1. Geometry of the P/M spur gears. Fig. 1. Geometry of the P/M spur gears. Table 1. Nominal compositions of the powders Powder label Composition (%wt) A85Mo05 0.85 Mo, Fe bal. pre-alloyed DHP 1.5 Mo, 2.0 Cu, 4.0 Ni, Fe bal diffusion bonded DDH 1.5 Mo, 2.0 Cu, Fe bal diffusion bonded Table 1. Nominal compositions of the powders Bergmark [6] used the Murakami model for sintered products by introducing a further corrective factor that indirectly takes into account the effect of porosity in terms of variation of the elastic modulus with respect to that of the fully dense material. 2 EXPERIMENTAL ACTIVITY The experimental activity was carried out on the P/M spur gears shown in Fig. 1, whose main constructive parameters are: number of teeth N= 20, module m = 0.71 mm and pressure angle θ =20°. The tooth root bending fatigue strength for three types of material is investigated. The reference standard is ISO 6336 [17] for the calculation of the load capacity of spur and helical gears. Table 2. mean density, total porosity and open porosity following ASTM B311. A85Mo05 DHP DDH Density (g.cm-3) 7.55 7.35 7.41 Total porosity(%) 4.09 7.56 6.36 Open porosity(%) 0.60 1.39 0.96 Table 2. mean density, total porosity and open porosity following ASTM B311. Table 2. mean density, total porosity and open porosity following ASTM B311. The gears are produced using pre-alloyed as well as diffusion bondend powders, whose nominal compositions are summarized in Table 1. In the blending phase, carbon in the form of graphite is added in a quantity equal to 0.25% wt for A85Mo05, and 0.15% wt both for DHP and DDH. 2 2 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 Fig. 3. Microstructure of DHP Table 3. mean porosity values in different regions of the gear measured using image analysis on metallographic sections. Table 3. mean porosity values in different regions of the gear measured using image analysis on metallographic sections. 2.1 Fatigue testing Specimens surviving the 3.106 cycles are considered as runouts. The choice of 3.106 cycles is dictated by the ISO6336 standard. In fact, for this fatigue live, an endurance factor equal to one is assumed in the design formula. The fatigue tests were conducted with a resonant Rumul Mikroton 20 kN testing machine operating at frequencies of about 150 Hz. The testing configuration shown in fig. 6 provides for the simultaneous gripping of two teethwith intespaciong of five teeth. The fatigue curve in the finite life regime was evaluated using the Basquin law reported by Eqn. 2. A least-square regression of the results of 15-20 tests, distributed on different stress levels was carried out: 𝜎𝑎𝑁𝑙𝐶 Unfortunately, due to the very small module of the teeth, this condition cannot be precisely reproduced by the testing configuration. For this reason, the stress state in the tooth root region is evaluated with a finite element analysis, that allows to model the effective testing configuration: In this way stress at the tooth root can be correctly calculated. 20-node brick element were used to build up the FE model. The sub-modelling technique was adopted to obtain a refined evaluation of the local stress. The FEM sub-model developed for the analysis is shown in fig. 7. The FE convergence analysis was performed by reproducing the ISO 6336 loading configuration and comparing the FE tooth root bending stress with that calculated using the ISO6336 formula. 𝜎𝜎𝑎𝑎∙𝑁𝑁1/𝑙𝑙= 𝐶𝐶 (2) (2) In this formula σa represents the stress amplitude and N is the number of cycles to failure. The dispersion of the results is calculated using the central standard deviation of the data, assuming uniform scatter for different fatigue lives. A measure of the results scattering is expressed by the ratio between the stress amplitude corresponding to 90% and 10% probability of failure: 𝑇𝜎𝜎𝑎 𝑇𝑇𝜎𝜎,10:90 = 𝜎𝜎𝑎𝑎,90 𝜎𝜎𝑎𝑎,10 (3) (3) Fig. 6. Tooth bending fatigue testing configuration Fig. 7. FEM Sub-model of the loaded tooth: the applied loads are represented by the red arrows. A load ratio R of 0.1 was adopted. A fatigue life interval between 3.104 and 3.106 cycles was explored. The fatigue strength at 3.106 cycles was determined using the staircase method performing at least 15 tests. Table 3. mean porosity values in different regions of the gear measured using image analysis on metallographic sections. Porosity % A85Mo05 DHP DDH Tooth body 7.13 8.85 7.63 Tooth root 6.29 7.99 6.20 gear body 3.04 7.15 4.17 The A85Mo05 specimens contains irregularly shaped pores, not uniformly distributed in the gear volume: the remarkable difference in pores content between the tooth and the gear body is evident (table 3). While A85Mo05 exhibits the lowest total porosity among the three variants, its pore content in the critical tooth root region is comparable with other materials. Concerning the microstructure (fig. 2), a surface layer with martensitic microstructure and limited presence of bainite and retained austenite can be observed. The bulk is instead characterized by lower bainite and martensite. Fig. 4. Microstructure of DDH Fig. 2. Microstructure of A85Mo05 Fig. 4. Microstructure of DDH Due to the post sintering treatments, all the materials are characterized by a surface layer with higher hardness compared to the bulk one. The A85Mo05 alloy was modified by case-hardening, while the other two material variants are produced by sinter-hardening, undergoing a controlled cooling from the sintering temperature down to 300 °C, whose effects are more evident in the surface layer. The microhardness profiles (HV0.05) are plotted in figure 5: the surface hardening extends to a depth of nearly 0.5 mm. Fig. 2. Microstructure of A85Mo05 Fig. 2. Microstructure of A85Mo05 The DHP steel is characterized by a more homogeneous pore distribution in the gear volume. The microstructure is heterogeneous (fig. 3) with austenite (white areas), lower bainite (light gray areas) and martensite (dark gray areas). The distribution of the alloy elements is inhomogeneous in the diffusion bonded powders and during sintering there is no complete diffusion of Ni and Cu into the base powders (1.5% Mo Prealloyed iron). The Ni-rich austenite is predominantly located around the pores [18]. The martensite is richer in Cu, while the base powder, poor in Ni and Cu, is predominantly bainitic. Fig. 5. Microhardness profiles in the surface layer The DDH steel shows a non-uniform pore distribution, comparable to A85Mo05. The microstructure (fig. 4) is predominantly martensitic in the surface layer with some evidences of lower bainite, while the upper bainite prevails in the bulk. Also in this case the pre-alloyed powders do not presents a homogeneous distribution of Cu, consequently areas richer in Cu are still present after sintering and assume a martensitic microstructure upon cooling. Fig. 5. Table 3. mean porosity values in different regions of the gear measured using image analysis on metallographic sections. Microhardness profiles in the surface layer 3 3 https://doi.org/10.1051/matecconf/201816502011 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 2.1 Fatigue testing ) Fig. 11. A85Mo05: a) crack path, b) initiation site ) Fig. 11. A85Mo05: a) crack path, b) initiation site ) Fig. 11. A85Mo05: a) crack path, b) initiation site a) 2.1 Fatigue testing The evidence of a knee in the fatigue curve at fatigue lives lower than about 2.106 cycles may be indicative of the presence of a fatigue limit, the confirmation thereof would however require investigating the response to longer lifetimes. In the figures 8-10 the results of the bending fatigue tests and the P10, P50 and P90 fatigue curves are plotted. Fig. 6. Tooth bending fatigue testing configuration Fig. 8. Results of bending fatigue tests on A85Mo05 Fig. 8. Results of bending fatigue tests on A85Mo05 Fig. 6. Tooth bending fatigue testing configuration Fig. 6. Tooth bending fatigue testing configuration Fig. 7. FEM Sub-model of the loaded tooth: the applied loads are represented by the red arrows. Fig. 8. Results of bending fatigue tests on A85Mo05 Fig. 7. FEM Sub-model of the loaded tooth: the applied loads are represented by the red arrows. A load ratio R of 0.1 was adopted. A fatigue life interval between 3.104 and 3.106 cycles was explored. The fatigue strength at 3.106 cycles was determined using the staircase method performing at least 15 tests. 4 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 Fig. 9. Results of bending fatigue tests on DHP Fig. 10. Results of bending fatigue tests on DDH Fig. 9. Results of bending fatigue tests on DHP The fracture path for DHP (Fig. 12) and for DHH (Fig. 13) is much more influenced by the presence of pores that define preferential propagation directions and determined a very serrated path Fig. 9. Results of bending fatigue tests on DHP pores that define preferential propagation directions and determined a very serrated path a) b) Fig. 11. A85Mo05: a) crack path, b) initiation site a) a) ) Fig. 10. Results of bending fatigue tests on DDH The principal parameters that describe the fatigue behaviour for the three materials are collected in table 4. Table 4. Characteristic parameters of the fatigue curves.𝜎𝑎𝑇𝜎 Table 4. Characteristic parameters of the fatigue curves. 𝜎𝜎𝑎𝑎,3∙106 (P50) 𝑇𝑇𝜎𝜎,10:90 C m A85Mo05 321 (MPa) 1.42 3163 (MPa) 5.90 DHP 307 1.26 2858 5.71 DDH 262 1.59 3846 5.00 b) The obtained results are compared with results published in [3-5] for similar microstructures with different porosity contents. In general, the literature data show a progressive but not particularly marked decrease in the fatigue strength with density for relative densities higher than 90%, whereas a very marked worsening occurs at lower densities. 2.2 Fractographic analysis The fractographic study was carried out both by metallographic observations and by electron scanning microscopy (SEM) analysis. In the first case, the main objective is to study the preferential crack path, whereby the tooth is sectioned with a plane orthogonal to the gear axis, polished and prepared with the conventional metallographic procedure. The SEM analysis, on the other hand, is aimed at identifying the crack initiation sites and at evaluating the size of the critical pores. a) The representative images for the three material variants are shown in the following figures. The crack path for A85Mo05 (Fig. 11a) is not very serrated: a limited evidence of deviations due to the presence of porosity can be observed. In fig. 11b a surface pore is highlighted which has probably played the role of crack initiation site. In the same region some evidences of intergranular fracture have been observed, that is probably produced by grain boundary embrittlement due to cementite precipitation after carbon enrichment during case hardening. 5 5 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 b) Fig. 12. DHP: a) crack path, b) initiation site a) b) Fig. 13. DDH: a) crack path, b) initiation site ATEC Web of Conferences 165, 02011 (2018) ATIGUE 2018 b) Fig. 12. DHP: a) crack path, b) initiation site a) b) Fig. 13. DDH: a) crack path, b) initiation site For both material variants the SEM analysis allows to identify pores with very articulated morphology close to the surface, which can be reasonably considered as initiation sites for the fatigue crack. Some evidences of multiple crack initiation followed by crack coalescence have been observed. that shown in figure 14. This observation agrees with the crack propagation behaviour reported in the technical literature [5,8,12,13]. Fig. 14. DDH: non propagating cracks An extensive analysis of the most critical pores on the fracture surface was carried out to estimate the characteristic size of the initial defect to be used in the Murakami formula. To calculate the area of the critical pores the maximum Feret diameter is used, the evaluation of which is schematically described in figure 15. The area to be inserted in equation 1 is that of the circle defined by the maximum Feret diameter, that inscribes the pore. b) that shown in figure 14. This observation agrees with the crack propagation behaviour reported in the technical literature [5,8,12,13]. b) a) Fig. 14. 3 Conclusions The tooth root bending fatigue behaviours for three P/M material variants characterized by high density and high strength microstructures were studied. The main conclusions can be listed as follows: 1) the fatigue curves have a marked knee that can suggest to consider the fatigue strength at 3.106 cycles as a reasonable estimate of the fatigue limit. However, the scatter of the fatigue results is quite large and therefore additional tests will be needed to explore longer fatigue lives. 2) by studying the porosity distribution and morphology as well as the inhomogeneous microstructure after sintering characterized by evident differences in hardness, the factors that most affect the fatigue response can be evaluated. Table 5. Fatigue strength estimated with the Murakami model considering the average microstructural hardness 𝑎𝑎𝑎𝑎 Table 5. Fatigue strength estimated with the Murakami model considering the average microstructural hardness 𝑎𝑎𝑎𝑎 g g considering the average microstructural hardness HV mean √𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (µm) σl, Mur (MPa) σl, exp (MPa) ∆(%) A85Mo05 873 111 524 321 +63 DHP 744 152 437 307 +42 DDH 758 136 452 262 +73 3) The SEM analysis of the fracture surfaces allows to identify the critical pores and to advance the hypothesis that the main crack is formed as a result of the coalescence of multiple cracks nucleated in the most stressed region. 4) For the evaluation of the critical pore it is necessary to conduct the SEM fractographic analysis, as the metallographic analysis does not allow to clearly identify the pores criticality. The pore area identified by SEM observation on the fracture surface approaches that of the crack surface projected on the critical plane, as required by the Murakami model Table 6. Fatigue strength estimated with the Murakami model considering the local hardness. 𝑎𝑎𝑎𝑎 Table 6. Fatigue strength estimated with the Murakami model considering the local hardness. 𝑎𝑎𝑎𝑎 HV local √𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (µm) σl, Mur (MPa) σl, exp (MPa) ∆% A85Mo05 515 111 345 321 +7 DHP 325 152 233 307 -24 DDH 378 136 264 262 +1 5) The representative parameter of the pore criticality can be the maximum Feret diameter, which identifies the circumference inscribing the serrated perimeter of the pore A consistent improvement in the estimation of the fatigue strength is obtained using the local hardness in the Murakami formula. 2.2 Fractographic analysis Scheme for the evaluation of the characteristic dimension of the pore, defined by the Feret diameter Fig. 15. Scheme for the evaluation of the characteristic dimension of the pore, defined by the Feret diameter For the evaluation of the fatigue strength using the Murakami model it is necessary to provide the hardness value representative of the material microstructure. The microstructural heterogeneity produced by the non- uniform distribution of the alloying elements can play an important role. The significant differences between the bulk microstructure and the phase surrounding the pores were previously highlighted (fig. 2). To shed light on the role of the local microstructure, the estimate of the fatigue strength using the Murakami model has been realized both considering the average hardness of the material and the local hardness taken in the surroundings of the pores, which characterizes the microstructural phase in which the crack initiation occurs. The results are collected in tables 5 and 6 and compared with the experimental values. 2.2 Fractographic analysis DDH: non propagating cracks Fig. 14. DDH: non propagating cracks a) b) Fig. 13. DDH: a) crack path, b) initiation site b) Fig. 13. DDH: a) crack path, b) initiation site Fig. 14. DDH: non propagating cracks An extensive analysis of the most critical pores on the fracture surface was carried out to estimate the characteristic size of the initial defect to be used in the Murakami formula. To calculate the area of the critical pores the maximum Feret diameter is used, the evaluation of which is schematically described in figure 15. The area to be inserted in equation 1 is that of the circle defined by the maximum Feret diameter, that inscribes the pore. b) b) Fig. 13. DDH: a) crack path, b) initiation site For both material variants the SEM analysis allows to identify pores with very articulated morphology close to the surface, which can be reasonably considered as initiation sites for the fatigue crack. Some evidences of multiple crack initiation followed by crack coalescence have been observed. Both for DDH and DHP, small cracks nucleated at pores proximal to the tooth surface (Fig. 14) have been observed. This suggests the occurrence of a first stage of diffused damage with multiple crack nucleation, followed by the onset of a main crack that can connect several cracks in its propagation path. The cracks not involved in this process of coalescence stop growing as 6 6 https://doi.org/10.1051/matecconf/201816502011 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 MATEC Web of Conferences 165, 02011 (2018) cracks propagating trough the microstructural phases enveloping the pores, takes a large part of the entire fatigue life. Moreover, a plausible explanation of the different errors obtained for the three variants can be given. As far as A85Mo05 is concerned, the overestimation may be due to the fact that the grain boundary embrittlement occurring in the case hardened layer is not taken into account by the Murakami model. On the contrary, for the sinter-hardened DHP material, characterized by soft austenite surrounding the critical pores, an underestimation of the fatigue strength is obtained. This can be due by the partial or total transformation of the retained austenite into martensite with consequent local increase in volume, that can produce crack closure slowing propagation speed or even leading to arrest the fatigue crack. These hypotheses need to be confirmed with a more in-depth study. Fig. 15. 3 Conclusions This result confirms, even considering the approximations introduced for the evaluation of the critical defect size, the considerable role played by the microstructure in the neighbourhood of the critical pores. From another point of view, it is worth noticing that the initiation phase, spent by the 6) The application of the Murakami method allows to highlight the remarkable importance of the microstructure, which acts synergistically with the pore in the damage localization 7 7 MATEC Web of Conferences 165, 02011 (2018) FATIGUE 2018 https://doi.org/10.1051/matecconf/201816502011 References 1. C.M. Sonsino CM., Powder Metall., 33(3), 235–245, (1990) 2. P.K Jones, K. Buckley-Golder, R. Lawcock et al., Int J Powd Metall., 33(3), 37–44, (1997). 3. L. Forden, S. Bengtsson, M. Bergström,. Powder Metall., 48(1):10–12, (2005) 4. S. Dizdar, P. Johansson, Proceedings of EURO PM2007, Toulouse, France; 1-7, (2007). 5. M. Benedetti, C. Menapace, Powder Metallurgy, 60 (2), 149-156, (2017) 6. A. Bergmark, Powder Metallurgy Progress, 5(3), 131-137, (2005) 7. M. Andersson, Powder Metallurgy Progress, 11 (1- 2), 21-3,1 (2011) 8. G. Straffelini, V. Fontanari, A. Hafez, M. 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https://openalex.org/W4307549822	https://www.mdpi.com/1996-1944/15/21/7513/pdf?version=1667974155	English	null	Influence of Replacing Cement with Waste Glass on Mechanical Properties of Concrete	Materials	2,022	cc-by	12,957	1 Department of Civil Engineering, Faculty of Engineering, Mugla Sitki Kocman University, Mugla 48000 Turkey 2 Department of Civil Engineering, Faculty of Engineering, Necmettin Erbakan University, Konya 42000, Tu 3 2 Department of Civil Engineering, Faculty of Engineering, Necmettin Erbakan University, Konya 42000, Turkey 3 Department of Civil Engineering, Faculty of Engineering, Zonguldak Bulent Ecevit University, 4 Department of Construction, Tomarza Mustafa Akincioglu Vocational School, Kayseri University, Kayseri 38940, Turkey 5 Department of Civil Engineering, College of Engineering, University of Duhok, Duhok 42001, Iraq 6 Department of Civil Engineering, College of Engineering, Nawroz University, Duhok 42001, Iraq 7 Department of Civil Engineering, Military College of Engineering (NUST), Risalpur 24080, Pakistan 8 Faculty of Materials Science and Engineering, Gheorghe Asachi Technical University of Iasi, 700050 Iasi Romania 5 Department of Civil Engineering, College of Engineering, University of Duhok, Duhok 42001, Iraq 6 Department of Civil Engineering, College of Engineering, Nawroz University, Duhok 42001, Iraq Department of Civil Engineering, College of Engineering, University of Duhok, Duhok 42001, Iraq 6 Department of Civil Engineering, College of Engineering, Nawroz University, Duhok 42001, Iraq 7 Department of Civil Engineering, Military College of Engineering (NUST), Risalpur 24080, Pakistan p g g g g g y q 7 Department of Civil Engineering, Military College of Engineering (NUST), Risalpur 24080, Pakistan 8 Faculty of Materials Science and Engineering, Gheorghe Asachi Technical University of Iasi, 700050 Iasi Romania * Correspondence: yozkilic@erbakan.edu.tr (Y.O.Ö.); doru.burduhos@tuiasi.ro (D.D.B Abstract: In this study, the effect of waste glass on the mechanical properties of concrete was exam- ined by conducting a series of compressive strength, splitting tensile strength and ﬂexural strength tests. According to this aim, waste glass powder (WGP) was ﬁrst used as a partial replacement for cement and six different ratios of WGP were utilized in concrete production: 0%, 10%, 20%, 30%, 40%, and 50%. To examine the combined effect of different ratios of WGP on concrete per- formance, mixed samples (10%, 20%, 30%) were then prepared by replacing cement, and ﬁne and coarse aggregates with both WGP and crashed glass particles. Workability and slump values of concrete produced with different amounts of waste glass were determined on the fresh state of concrete, and these properties were compared with those of plain concrete. 1 Department of Civil Engineering, Faculty of Engineering, Mugla Sitki Kocman University, Mugla 48000 Turkey For the hardened concrete, 150 mm × 150 mm × 150 mm cubic specimens and cylindrical specimens with a diameter of 100 mm and a height of 200 mm were tested to identify the compressive strength and splitting tensile strength of the concrete produced with waste glass. Next, a three-point bending test was carried out on samples with dimensions of 100 × 100 × 400 mm, and a span length of 300 mm to obtain the ﬂexure behavior of different mixtures. According to the results obtained, a 20% substitution of WGP as cement can be considered the optimum dose. On the other hand, for concrete produced with combined WGP and crashed glass particles, mechanical properties increased up to a certain limit and then decreased owing to poor workability. Thus, 10% can be considered the optimum replacement level, as combined waste glass shows considerably higher strength and better workability properties. Furthermore, scanning electron microscope (SEM) analysis was performed to investigate the microstructure of the composition. Good adhesion was observed between the waste glass and cementitious concrete. Lastly, practical empirical equations have been developed to determine the compressive strength, splitting tensile strength, and ﬂexure strength of concrete with different amounts of waste glass. Instead of conducting an experiment, these strength values of the concrete produced with glass powder can be easily estimated at the design stage with the help of proposed expressions. materials materials Article Inﬂuence of Replacing Cement with Waste Glass on Mechanical Properties of Concrete Özer Zeybek 1, Yasin Onuralp Özkılıç 2,* , Memduh Karalar 3 , Ali ˙Ihsan Çelik 4 , Shaker Qaidi 5,6 , Jawad Ahmad 7 , Dumitru Doru Burduhos-Nergis 8,* and Diana Petronela Burduhos-Nergis 8 1 Department of Civil Engineering, Faculty of Engineering, Mugla Sitki Kocman University, Mugla 48000, Turkey 1. Introduction The use of ordinary Portland cement (OPC) by replacing it with recycled cement in certain proportions is an interesting issue for sustainable environmental awareness [1,2]. During cement production, a high amount of energy is consumed, and a high amount of carbon dioxide (CaO2) is released into the atmosphere [3]. Therefore, cement additives are of interest to reduce cement production [4,5]. Waste is generally deﬁned as residues from industrial production processes, residues arising from the transfer, or product residues that have completed their economic life [6–14]. With the development of modern cities, the decrease in natural resources, climate change, and increasing awareness of environmental protection, there is an urgent need to develop building materials that will reduce greenhouse gas emissions [2,15]. Waste glass powder (WGP), which is increasing with the effect of industrialization and the increase in urban transformation, attracts the attention of researchers as a concrete additive material due to its economic and mechanical performance effects [4,5,16–21]. Since there is no regular storage area for waste glass, it increases the risk of soil and water pollution due to its oxidation effect. Therefore, the use of recycled glass in concrete production will provide signiﬁcant contributions to reducing environmental problems [2,3,22,23]. p According to ASTM C618-19, 2019, since waste glass consists of a large amount of calcium and an amorphous structure, it can be ground into powder to obtain pozzolanic material or cement additive [24–26]. Therefore, WGP can be used in concrete production by replacing it with cement in certain proportions [2]. Recent studies show that replacing 15–25% glass powder with cement increases the mechanical properties of concrete [6,15,21]. Aliabdo et al. used 25% substitution with cement to investigate the mechanical effect of WGP in concrete as a cement substitute. According to the results obtained, it was observed that the void ratio and density of the samples decreased, while the tensile and compressive strengths increased [27]. Al Saffar et al. in their study to test the interaction of WGP, added cement mortar with other materials and found that the compressive strength of the samples increased as the glass powder addition rate increased. They obtained the highest strength with the addition of 25% glass powder [15]. Elaqra et al. (2019) added 4% by weight to the mix to investigate the effect of WGP on fresh and hardened concrete. Citation: Zeybek, Ö.; Özkılıç, Y.O.; Karalar, M.; Çelik, A.˙I.; Qaidi, S.; Ahmad, J.; Burduhos-Nergis, D.D.; Burduhos-Nergis, D.P. Inﬂuence of Replacing Cement with Waste Glass on Mechanical Properties of Concrete. Materials 2022, 15, 7513. https:// doi.org/10.3390/ma15217513 Academic Editor: F. Pacheco Torgal Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 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/). Keywords: eco-friendly concrete; waste glass; workability; compressive strength; splitting tensile strength; ﬂexural strength https://www.mdpi.com/journal/materials Materials 2022, 15, 7513. https://doi.org/10.3390/ma15217513 Materials 2022, 15, 7513 2 of 16 1. Introduction found that the addition of 20% WGP increased the compressive strength by 19% when <75 µm [38]. They state that while the strength decreases as a result of the 7-day compressive strength test, the compressive strength does not change as a result of the 28- and 91-day tests <75 µm. In addition to strength performance, the failure processes of brittleness materials are also important. Some researchers have studied the failure processes of brittleness materials [39,40]. Since the risk of fracture will increase under sudden loads, it is beneﬁcial to take measures to increase elastic behavior. Dust and particles from soda glass were mostly used in previous studies. However, its usability in concrete was investigated by replacing ﬁne aggregate in self-compacting concrete with waste particles obtained from cathode ray tubes. The results had a positive effect on the durability properties of concrete [41]. Past studies have shown that ground glass powder can increase the pozzolanic reactivity of secondary cementitious materi- als. Therefore, the granule size of the ground glass powder has important effects [42,43]. Ahmet et al. examined different methods of using waste glass in concrete and stated that particle size, substitution ratio, and chemical composition have important effects on the mechanical durability of concrete. For example, as the grain size decreases, workability becomes more difﬁcult but pozzolanic and strength increase [44]. The particle size of the waste glass, which must be taken into account during the mix design, may affect the active silica reaction depending on the rate of substitution. p g Solid waste management is an important issue for most developing countries [45]. Instead of storing or disposing of waste materials such as glass, plastic, and metal, reusing or recycling has become a more attractive option. Waste glass has started to be widely preferred for concrete production in civil engineering applications in recent years. Since the employing of waste glass in concrete can assist to reduce environmental pollution, protect natural resources and produce low-cost concrete, WGP can be preferred instead of either natural aggregates or cement due to its pozzolanic effect. Although many studies have been conducted on this subject, there are differences in the results obtained from the literature. Thus, there still remains a need to investigate the mechanical behavior of concrete with partial substitution of waste glass and the ideal dosage of it. Based on this motivation, an experimental study was carried out on some test specimens. 1. Introduction They found that as the amount of WGP increased, the machinability increased, and the maximum compressive strength was reached with the addition of 20% WGP at 28 days of cure [28]. It is well known that in the case of ﬁne grinding of soda–lime glass, the reactivity of pozzolanic increases as the particle size decreases [29]. Zhang et al. in their study to examine the effect of WGP particle size on the mechanical and microstructures of concrete, found that the particle size distribution of WGP has a signiﬁcant effect on the properties of WGP- based concrete [30]. Shao et al. in their experimental study to observe the effect of ﬁnely ground WGP on the compressive strength of concrete found that the pozzolanic reactivity increases as the WGP particle size decreases. They found that WGP, with a particle size of 38 µm, was replaced by 30% with cement, resulting in 4.1 MPa greater compressive strength [31]. It was found that WGP pozzolanic reactivity was replaced by 0, 15, 30, 45, and 60% of weight cement, while below 30% the concrete compressive strength did not decrease due to the pozzolanic reaction between WGP cement hydration products. In fact, with the addition of 60% WGP, the resistance to chloride ion and water penetration increased continuously, while the concrete compressive strength increased by around 85% [32]. In the study by Peril and Sangle, it was observed that when WGP was mixed with 30% cement, an increase in compressive strength between <38 µm and <75 µm was between 20% and 10% [33]. Khatib et al. found in their study that there was a 1.2% increase in concrete compressive strength with the addition of 10% WGP [34]. In a similar study, Madandoust and Ghavidel found that the compressive strength of the control sample was higher than the concrete with glass powder added at every stage, the compressive strength of both samples increased with aging [35]. In their study, Tejaswi et al. stated that the compressive strength increased by 1.5% with the addition of 10% WGP. However, they found that the compressive strengths were at equal levels when 20% was substituted [36]. Vasudevan et al. Materials 2022, 15, 7513 3 of 16 observed a 1.05% increase in concrete compressive strength when 20% WFP addition was <90 µm [37]. Schwarz et al. 1. Introduction Analytical solution proposals have been developed according to the data obtained from the experimental study. The pro- posed formulas will serve as a guide for researchers and manufacturers and will accelerate future studies to be more effective. 2. Experimental Program In this study, the main aim is to investigate the effect of glass powder when it is re- placed with cement. Furthermore, the effects of all replacements with glass are investigated. Nine mixes including the reference were designed. C represents cement replacement, MIX represents cement, ﬁne aggregate, and coarse aggregate replacement. Table 1 summarizes the sample properties. For the MIX design, each type of material was replaced with certain amounts of recycled glass. The size of ﬁne aggregates is 1 mm to 4 mm and the correspond- ing waste glass was 1.7 mm to 4 mm. Size of coarse aggregate size was selected as 5–12 mm and the corresponding waste glass was also 5–12 mm. The particle size of cement particles was between 0.02 mm to 0.1 mm and the size of glass waste powder was 0.1–0.2 mm. Figure 1 demonstrates the used aggregates and cement and also their replacements which are glass powder and glass grains. Figure 2 demonstrates the recycled glass in concrete. Materials 2022, 15, 7513 4 of 16 Table 1. Sample Properties. REF No Glass Was Used C10% 10% cement was replaced with waste glass powder C20% 20% cement was replaced with waste glass powder C30% 30% cement was replaced with waste glass powder C40% 40% cement was replaced with waste glass powder C50% 50% cement was replaced with waste glass powder MIX10% 10% cement, 10% ﬁne and 10% coarse aggregates were replaced with waste glass MIX20% 20% cement, 20% ﬁne and 20% coarse aggregates were replaced with waste glass MIX30% 30% cement, 30% ﬁne and 30% coarse aggregates were replaced with waste glass Table 1. Sample Properties. REF No Glass Was Used C10% 10% cement was replaced with waste glass powder C20% 20% cement was replaced with waste glass powder C30% 30% cement was replaced with waste glass powder C40% 40% cement was replaced with waste glass powder C50% 50% cement was replaced with waste glass powder MIX10% 10% cement, 10% fine and 10% coarse aggregates were replaced with waste glass MIX20% 20% cement, 20% fine and 20% coarse aggregates were replaced with waste glass MIX30% 30% cement, 30% fine and 30% coarse aggregates were replaced with waste glass Materials 2022, 15, x FOR PEER REVIEW 4 of 17 Table 1. Sample Properties. 2. Experimental Program REF No Glass Was Used C10% 10% cement was replaced with waste glass powder C20% 20% cement was replaced with waste glass powder C30% 30% cement was replaced with waste glass powder C40% 40% cement was replaced with waste glass powder C50% 50% cement was replaced with waste glass powder MIX10% 10% cement, 10% fine and 10% coarse aggregates were replaced with waste glass MIX20% 20% cement, 20% fine and 20% coarse aggregates were replaced with waste glass MIX30% 30% cement, 30% fine and 30% coarse aggregates were replaced with waste No Glass Was Used Figure 1. Recycled glass, cement, and aggregates. Figure 2. Recycled glass in concrete. Figure 1. Recycled glass, cement, and aggregates. MIX30% glass Figure 1. Recycled glass, cement, and aggregates. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Cement was selected as CEM I 32.5 Portland cement. The water-to-cement ratio was chosen as 0.5. Figure 3 demonstrates the slump test results. The workability of the concrete produced by adding WGP increased as the waste rate increased. Accordingly, there was an increase in slump values. It is seen that the slump value increases as the recycled glass ratio increases. On the other hand, when all ingredients were replaced with glass, the slump value signiﬁcantly decreases. Madandous and Ghavidel obtained an 80 mm slump in their study with the addition of 5–20% WGP. In this study, it is seen that the slump value is 200 mm, since the additional WCP is 50%. It can be said that the results are similar to the literature [35]. Figure 1. Recycled glass, cement, and aggregates. Figure 1. Recycled glass, cement, and aggregates. g a Figure 1. Recycled glass, cement, and aggregates. Figure 1. Recycled glass, cement, and aggregates. Figure 1. Recycled glass, cement, and aggregates. Figure 1. Recycled glass, cement, and aggregates. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. g y g Figure 2. Recycled glass in concrete. Figure 2. Recycled glass in concrete. Cement was selected as CEM I 32.5 Portland cement. The water-to-cement ratio was chosen as 0.5. Figure 3 demonstrates the slump test results. The workability of the concrete produced by adding WGP increased as the waste rate increased. 2. Experimental Program Accordingly, there was an increase in slump values. It is seen that the slump value increases as the recycled glass ratio increases. On the other hand, when all ingredients were replaced with glass, the slump value signiﬁcantly decreases. Madandous and Ghavidel obtained an 80 mm slump in their study with the addition of 5–20% WGP. In this study, it is seen that the slump value is 200 mm, since the additional WCP is 50%. It can be said that the results are similar to the literature [35]. Cement was selected as CEM I 32.5 Portland cement. The water-to-cement ratio was chosen as 0.5. Figure 3 demonstrates the slump test results. The workability of the concrete produced by adding WGP increased as the waste rate increased. Accordingly, there was an increase in slump values. It is seen that the slump value increases as the recycled glass ratio increases. On the other hand, when all ingredients were replaced with glass, the slump value signiﬁcantly decreases. Madandous and Ghavidel obtained an 80 mm slump in their study with the addition of 5–20% WGP. In this study, it is seen that the slump value is 200 mm, since the additional WCP is 50%. It can be said that the results are similar to the literature [35]. Cement was selected as CEM I 32.5 Portland cement. The water-to-cement ratio was chosen as 0.5. Figure 3 demonstrates the slump test results. The workability of the concrete produced by adding WGP increased as the waste rate increased. Accordingly, there was an increase in slump values. It is seen that the slump value increases as the recycled glass ratio increases. On the other hand, when all ingredients were replaced with glass, the slump value signiﬁcantly decreases. Madandous and Ghavidel obtained an 80 mm slump in their study with the addition of 5–20% WGP. In this study, it is seen that the slump value is 200 mm, since the additional WCP is 50%. It can be said that the results are similar to the literature [35]. Materials 2022, 15, 7513 5 of 16 Figure 3. Slump test. Three types of tests were performed in order to evaluate the performance of the con‐ crete with recycled glass. A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. 2. Experimental Program Three types of tests were performed in order to evaluate the performance of the con‐ crete with recycled glass. A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. Further‐ more, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive ca‐ pability was estimated from the critical failure strength separated by the part of the spec‐ imen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical di‐ mension. 3. Experimental Results and Discussions Three types of tests were performed in order to evaluate the performance of the concrete with recycled glass. A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. Furthermore, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive capability was estimated from the critical failure strength separated by the part of the specimen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical dimension. Three types of tests were performed in order to evaluate the performance of the con‐ crete with recycled glass. 2. Experimental Program A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. Further‐ more, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive ca‐ pability was estimated from the critical failure strength separated by the part of the spec‐ imen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical di‐ mension. 3. Experimental Results and Discussions Three types of tests were performed in order to evaluate the performance of the concrete with recycled glass. A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. Furthermore, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive capability was estimated from the critical failure strength separated by the part of the specimen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical dimension. 2. Experimental Program Further‐ more, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive ca‐ pability was estimated from the critical failure strength separated by the part of the spec‐ imen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical di‐ mension. 3. Experimental Results and Discussions 0 5 10 15 20 25 REF C10% C20% C30% C40% C50% MIX10% MIX20% MIX30% Slump (cm) Figure 3. Slump test. Three types of tests were performed in order to evaluate the performance of the concrete with recycled glass. A compression test was performed on 15 × 15 × 15 cm cubic samples while splitting tests were conducted on 10 × 20 cm cylinder samples. Furthermore, bending tests were performed using 10 × 10 × 40 cm samples. Three repetitions were completed for each mix and each test. Compressive ability is the capability of a specimen to decline load under pressure. The CST was performed according to ASTM C39/C39M (C39&C39M A, 2003). In this test, specimens have dimensions of 150 mm × 300 mm. In this experiment, concrete specimens were subjected to constraint lengthwise load at a level in the offered as concerns the specimen fractures. At that point, the compressive capability was estimated from the critical failure strength separated by the part of the specimen. To provide the tensile strength of concrete where compactor load is performed in anticipation of examples unravel caused by expansion of tensile force in concrete as ASTM (Designation, 1976). In this way, cylindrical examples are divided through the vertical dimension. Figure 3. Slump test. 0 5 10 15 20 25 REF C10% C20% C30% C40% C50% MIX10% MIX20% MIX30% Slump (cm) Figure 3. Slump test. Figure 3. Slump test. Figure 3. Slump test. 3.1. Compressive Strength (CS) In this part, to carry through the compressive stren were arranged without any adulteration. Figure 4 exp 3. Experimental Results and Discussions 3.1. Compressive Strength (CS) g y g p q days period for low w/c ratio combinations formed with 100% reference and numerous ratios of recycled aggregate substituting reference aggregates, with and lacking waste glass. The CS values of the reference concrete without waste glass series at 28 days were found as 18.9 MPa, respectively. The lowest CS values were also found to be 7.10 MPa in the concrete produced with cement including 50% waste glass for 28 days periods, respec‐ tively. As observed in Figure 4, it has been detected that the CS of concrete combinations including waste glass utilization as a fractional replacement for cement were lesser than those of the corresponding concrete mixes lacking waste glass. As observed in Figure 4, In this part, to carry through the compressive strength test (CST), concrete examples were arranged without any adulteration. Figure 4 expresses the CST consequences at 28 days period for low w/c ratio combinations formed with 100% reference and numerous ratios of recycled aggregate substituting reference aggregates, with and lacking waste glass. The CS values of the reference concrete without waste glass series at 28 days were found as 18.9 MPa, respectively. The lowest CS values were also found to be 7.10 MPa in the concrete produced with cement including 50% waste glass for 28 days periods, respectively. As observed in Figure 4, it has been detected that the CS of concrete combinations including waste glass utilization as a fractional replacement for cement were lesser than those of the corresponding concrete mixes lacking waste glass. As observed in Figure 4, statistical investigation of test values shows the noteworthy impact of waste glass (as a fractional replacement for cement) on the CS of concrete. EW 6 of 17 statistical investigation of test values shows the noteworthy impact of waste glass (as a fractional replacement for cement) on the CS of concrete. Figure 4. Results of CS. In the comparison of the cement that was changed with waste glass powder, the CS 0 5 10 15 20 25 Compressive Strength (MPa) Figure 4. Results of CS. 0 5 10 15 20 25 Compressive Strength (MPa) Figure 4. Results of CS. 0 5 10 15 20 25 Compressive Strength (MPa) Figure 4. Results of CS. Figure 4. Results of CS. Figure 4. Results of CS. 3.1. Compressive Strength (CS) In this part, to carry through the compressive stren were arranged without any adulteration. Figure 4 exp 3. Experimental Results and Discussions 3.1. Compressive Strength (CS) Materials 2022, 15, 7513 6 of 16 In the comparison of the cement that was changed with waste glass powder, the CS of C10% (10% cement replaced with waste glass) is 6% greater than that of C20%. Correspondingly, the CS of C20% (20% cement replaced with waste glass) is 60% greater than that of C30%. Comparing the CS of C30% (30% of the cement was replaced with waste glass powder) it can be observed that the value is 15% greater than that of C40%. Finally, the comparison of CS of C40% (40% of the cement was substituted with waste glass powder) is 31% greater than that of C50%. Consequently, it is observed that the CS of C10% (10% cement was replaced with waste glass powder) is similar to that of reference concrete. As shown in Figure 4, while ﬁne and coarse aggregate for 10%, 20%, and 30% (MIX10%, MIX20%, and MIX30%) were exchanged with waste glass, this trend was reversed as fractional replacement of cement with waste glass promoted the CS of concrete. On the other hand, at the comparison of CS of MIX10%, MIX20%, and MIX30%, the CST consequences for the great waste glass ratio continue trends comparable to those for the small waste glass ratio. In this situation too, the CS of MIX10% is 24% greater than the corresponding mix (MIX30%). While waste glass is used instead of cement, ﬁne and coarse aggregate, remarkable improvements in strength are observed when compared with the reference concrete. Therefore, it was observed that by replacing cement with glass powder, a signiﬁcant reduction in CS will be obtained. 3.3. Flexural Performance 3.3. Flexural Performance f Flexure strength (FS) of investigational examples was established on examples after CST. The acquired norms of strength ranged between 6.6 MPa and 3.9 MPa. The conse‐ quence of the FS of the examples is presented in Figure 7. In Figure 7, it is detected FS with variable ratios of waste glass. Related to CS, FS at the initial phase failures with the incor‐ poration of waste glass. It was detected that with the addition of waste glass at 10%, 20%, 30%, 40%, and 50% of cement weight, the decrease in FS was 6.7%, 12.5%, and 21.1%, 46.5%, and 61.5% correspondingly in proportion to the reference sample (6.3 MPa). Figure 8 demonstrates a rectilinear relationship between the tensile FS of the example and the substance of waste glass addition. On the other hand, if fine and coarse aggregates in ce‐ ment are replaced with waste glass, it was noticed that the waste glass was replaced with the cement, fine and coarse aggregate at 10%, the increase in FS was 4.7% correspondingly in proportion to the reference sample (6.3 MPa). Nevertheless, if the quantity of waste glass is occupied relative to the cement content, it may be detected that the association between FS and waste glass content is related (the slope of the curves is approximate (Fig‐ Flexure strength (FS) of investigational examples was established on examples after CST. The acquired norms of strength ranged between 6.6 MPa and 3.9 MPa. The conse- quence of the FS of the examples is presented in Figure 7. In Figure 7, it is detected FS with variable ratios of waste glass. Related to CS, FS at the initial phase failures with the incorporation of waste glass. It was detected that with the addition of waste glass at 10%, 20%, 30%, 40%, and 50% of cement weight, the decrease in FS was 6.7%, 12.5%, and 21.1%, 46.5%, and 61.5% correspondingly in proportion to the reference sample (6.3 MPa). Figure 8 demonstrates a rectilinear relationship between the tensile FS of the example and the substance of waste glass addition. On the other hand, if ﬁne and coarse aggregates in cement are replaced with waste glass, it was noticed that the waste glass was replaced with the cement, ﬁne and coarse aggregate at 10%, the increase in FS was 4.7% correspondingly in proportion to the reference sample (6.3 MPa). 3.2. Splitting Tensile Strength (STS) A comparison of relative STS of the various concrete percentage combinations formed with 100% reference and numerous ratios of recycled aggregate replacing reference ag- gregates, with and without waste glass are shown in Figure 5. As presented in Figure 5, the results of STS commonly pursue a similar tendency as the compressive strength. As observed from Figure 5, the remarkable improvement in concrete tensile strength is up to 10% with waste glass used as a fractional replacement for cement. Upon altering the amount of waste glass, consequences of tensile strength are inﬂuenced similarly to the CS. As shown in Figure 5, tensile strength optimal norms are found at 10% waste glass obligat- ing maximum divided tensile. A comparative evaluation was performed where controller concrete tensile strength was deliberate as reference strength, from which concrete of the changing ratio of waste glass is collated. At 10% substitution of waste glass, STS is only 3% lower than the reference mix. The correlation of CS in competition with STS is provided in Figure 6. As shown in Figure 6, the regression model among the compressive and STS appeared to be ﬂat. Furthermore, as observed in Figure 6, the regression stroke represents a strong relationship between CS contrasted with STS having an R2 worth of more than 94 percent. IEW 7 of 17 6, the regression stroke represents a strong relationship between CS contrasted with STS having an R2 worth of more than 94 percent. Figure 5. Results of STS. Figure 5. Results of STS. Figure 5. Results of STS. Figure 5. Results of STS. Materials 2022, 15, 7513 7 of 16 7 of 16 Figure 6. STS versus CS. 3.3. Flexural Performance Flexure strength (FS) of investigational examples was established on examples after CST. The acquired norms of strength ranged between 6.6 MPa and 3.9 MPa. The conse‐ quence of the FS of the examples is presented in Figure 7. In Figure 7, it is detected FS with variable ratios of waste glass. Related to CS, FS at the initial phase failures with the incor‐ poration of waste glass. It was detected that with the addition of waste glass at 10%, 20%, 30%, 40%, and 50% of cement weight, the decrease in FS was 6.7%, 12.5%, and 21.1%, 46.5%, and 61.5% correspondingly in proportion to the reference sample (6.3 MPa). 3.2. Splitting Tensile Strength (STS) Figure 8 demonstrates a rectilinear relationship between the tensile FS of the example and the substance of waste glass addition. On the other hand, if fine and coarse aggregates in ce‐ ment are replaced with waste glass, it was noticed that the waste glass was replaced with the cement, fine and coarse aggregate at 10%, the increase in FS was 4.7% correspondingly in proportion to the reference sample (6.3 MPa). Nevertheless, if the quantity of waste glass is occupied relative to the cement content, it may be detected that the association between FS and waste glass content is related (the slope of the curves is approximate (Fig‐ Figure 6. STS versus CS. 3.3. Flexural Performance Flexure strength (FS) of investigational examples was established on examples after CST. The acquired norms of strength ranged between 6.6 MPa and 3.9 MPa. The conse- quence of the FS of the examples is presented in Figure 7. In Figure 7, it is detected FS with variable ratios of waste glass. Related to CS, FS at the initial phase failures with the incorporation of waste glass. It was detected that with the addition of waste glass at 10%, 20%, 30%, 40%, and 50% of cement weight, the decrease in FS was 6.7%, 12.5%, and 21.1%, 46.5%, and 61.5% correspondingly in proportion to the reference sample (6.3 MPa). Figure 8 demonstrates a rectilinear relationship between the tensile FS of the example and the substance of waste glass addition. On the other hand, if ﬁne and coarse aggregates in cement are replaced with waste glass, it was noticed that the waste glass was replaced with the cement, ﬁne and coarse aggregate at 10%, the increase in FS was 4.7% correspondingly in proportion to the reference sample (6.3 MPa). Nevertheless, if the quantity of waste glass is occupied relative to the cement content, it may be detected that the association between FS and waste glass content is related (the slope of the curves is approximate (Figure 8)). EW 8 of 17 Figure 6. STS versus CS. Figure 6. STS versus CS. Figure 6. STS versus CS. Figure 6. STS versus CS. 3.3. Flexural Performance 3.3. Flexural Performance 3 4 Comparison of Findings of this Study with Other Existing Stu 3.4. Comparison of Findings of this Study with Other Existing Studies 3 4 Comparison of Findings of this Study with Other Existing St 3.4. Comparison of Findings of this Study with Other Existing Studies 3.4. Comparison of Findings of this Study with Other Existing Studies The effect of waste glass on the structural performance of concrete was inves by many research teams. Effects of the use of waste glass as cement replacement o mechanical properties such as CS, STS, and FS have been investigated. In this par study, values of CS, STS, and FS for plain concrete and concrete produced from d glass powder amounts have been collected from the research publications [46–63] measured strength values of concrete produced with waste glass were first normal plain concrete strengths These normalized strength values were plotted in Figure The effect of waste glass on the structural performance of concrete was investigated by many research teams. Effects of the use of waste glass as cement replacement on some mechanical properties such as CS, STS, and FS have been investigated. In this part of the study, values of CS, STS, and FS for plain concrete and concrete produced from different glass powder amounts have been collected from the research publications [46–63]. Then, measured strength values of concrete produced with waste glass were ﬁrst normalized by plain concrete strengths. These normalized strength values were plotted in Figures 9–11, respectively, as a function of the waste glass content. REVIEW 9 of 17 p g g p respectively, as a function of the waste glass content. Figure 9. Variation of the normalized CS of the concrete produced with waste glass. 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 0 5 10 15 20 25 30 35 40 45 50 Normalized Compressive Strength (CS) Waste glass content (%) Rahman and Uddin (2018) Patil and Sangle (2013) Elaqra et al. (2019) Ibrahim (2021) Jangid and Saoji (2014) Vidya et al. (2015) Abdulazeez et al. (2020) Kalakada et al. (2022) Khatib et al. (2012) Shao et al. (2000) Tamanna and Tuladhar (2020) Metwally (2007) Ahmad et al. (2022) Nassar and Soroushian (2016) Islam et al. (2017) Du and Tan (2014) Vasudevan and Pillay (2013) Rahma et al. (2017) Ahmad et al. (2021) Kim et al. (2017) Hussain and Chandak (2015) Ali (2018) Bhagyasri et al. (2016) Patil (2020) This Study Proposed Equation (1) Figure 9. Variation of the normalized CS of the concrete produced with waste glass. 3.3. Flexural Performance 3.3. Flexural Performance Nevertheless, if the quantity of waste glass is occupied relative to the cement content, it may be detected that the association between FS and waste glass content is related (the slope of the curves is approximate (Figure 8)). W 8 of 17 ure 8)). Figure 7. Results of FS. Figure 7. Results of FS. igure 7. Results of FS. Figure 7. Results of FS. igure 7. Results of FS. Figure 7. Results of FS. Materials 2022, 15, 7513 8 of 16 Figure 8. Results of tensile flexural strength tests of concrete examples with altered waste g stances. Figure 8. Results of tensile ﬂexural strength tests of concrete examples with altered waste glass substances. Figure 8. Results of tensile flexural strength tests of concrete examples with altered waste gl stances. Figure 8. Results of tensile ﬂexural strength tests of concrete examples with altered waste glass substances. 3 4 Comparison of Findings of this Study with Other Existing Stu 3.4. Comparison of Findings of this Study with Other Existing Studies Figure 9. Variation of the normalized CS of the concrete produced with waste glass. Figure 9. Variation of the normalized CS of the concrete produced with waste glass. Materials 2022, 15, 7513 9 of 16 Figure 9. Variation of the normalized CS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 0 5 10 15 20 25 30 35 40 45 50 Normalized Split Tensile Strength (STS) Waste glass content (%) Hussain and Verma (2016) Rahman and Uddin (2018) Vijayakumar et al. (2013) Ibrahim (2021) Abdulazeez et al. (2020) Tamanna and Tuladhar (2020) This Study Ahmad et al. 2022 Ahmad et al. (2021) Hussain and Chandak (2015) Aliabdo et al. (2016) Patil (2020) Proposed Equation (1) Figure 10. Variation of the STS of the concrete produced with waste glass. Materials 2022, 15, x FOR PEER REVIEW 10 of 17 Figure 11. Variation of the FS of the concrete produced with waste glass. As shown in these figures, with the addition of waste glass, the strength values of concrete produced with glass powder generally decrease after a certain value. The maxi‐ mum decrease in compressive strength was observed in the study by Kalakada et. al., 2022. When the cement was partially replaced with glass powder at 50%, there is a 65% reduction in compression strength of the plain concrete specimen. In the same manner, a 20% addition of the waste glass leads to a 36% reduction in the splitting tensile strength 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 0 5 10 15 20 25 30 35 40 45 50 Normalized Flexural Strength (FS) Waste glass content (%) Hussain and Verma (2016) Jangid and Saoji (2014) Elaqra et al. (2019) Abdulazeez et al. (2020) Tamanna and Tuladhar (2020) Al-Zubaid et al. (2017) Ahmad et al. (2022) Nassar and Soroushian (2016) Ali (2018) Bhagyasri et al. (2016) Patil (2020) This Study Proposed Equation (1) Figure 11. Variation of the FS of the concrete produced with waste glass. As shown in these ﬁgures, with the addition of waste glass, the strength v of concrete produced with glass powder generally decrease after a certain value maximum decrease in compressive strength was observed in the study by Kalakada 2022. 3 4 Comparison of Findings of this Study with Other Existing Stu 3.4. Comparison of Findings of this Study with Other Existing Studies When the cement was partially replaced with glass powder at 50%, there is 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 0 5 10 15 20 25 30 35 40 45 50 Normalized Split Tensile Strength (STS) Waste glass content (%) Hussain and Verma (2016) Rahman and Uddin (2018) Vijayakumar et al. (2013) Ibrahim (2021) Abdulazeez et al. (2020) Tamanna and Tuladhar (2020) This Study Ahmad et al. 2022 Ahmad et al. (2021) Hussain and Chandak (2015) Aliabdo et al. (2016) Patil (2020) Proposed Equation (1) Materials 2022, 15, x FOR PEER REVIEW 10 of 1 Figure 10. Variation of the STS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. Figure 11. Variation of the FS of the concrete produced with waste glass. 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 0 5 10 15 20 25 30 35 40 45 50 Normalized Flexural Strength (FS) Waste glass content (%) Hussain and Verma (2016) Jangid and Saoji (2014) Elaqra et al. (2019) Abdulazeez et al. (2020) Tamanna and Tuladhar (2020) Al-Zubaid et al. (2017) Ahmad et al. (2022) Nassar and Soroushian (2016) Ali (2018) Bhagyasri et al. (2016) Patil (2020) This Study Proposed Equation (1) Figure 11. Variation of the FS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. Figure 10. Variation of the STS of the concrete produced with waste glass. 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 0 5 10 15 20 25 30 35 40 45 50 Normalized Flexural Strength (FS) Waste glass content (%) Figure 11. Variation of the FS of the concrete produced with waste glass. Figure 11. Variation of the FS of the concrete produced with waste glass. As shown in these figures, with the addition of waste glass, the strength values of concrete produced with glass powder generally decrease after a certain value. The maxi‐ mum decrease in compressive strength was observed in the study by Kalakada et. al., 2022. When the cement was partially replaced with glass powder at 50%, there is a 65% reduction in compression strength of the plain concrete specimen. 3.5. Scanning Electron Microscope (SEM) Analysis Scanning electron microscope (SEM) analysis was performed from the sample pieces taken after the compressive strength test from the concrete samples produced with recycled glass waste powder. SEM analysis is carried out to show the typical morphology of the surface tissue of OPC and WGP. The particles of both OPC and WGP are composed of glassy structures and irregular shapes with sharp edges. OPC particles consist of sharper edges and shapes, while WGP particle appears on smoother surfaces, sharper prismatic edges, and denser content [2]. The best images were selected to observe the effect of glass dust and glass particles in OPC concrete production. Figure 12a–f contains images where WGP is replaced by cement. Images of the mix design samples from Figure 12g–j contain the details of the change in glass particles with cement and aggregates. In order for WGP to be processed as a ﬁller by grinding in electric mills without any treatment, 74% of the particles must be passed through a 36 µm sieve [64]. The key ﬁndings of the inner image details at 500 times magniﬁcation are shown in detail in Figure 12. Judging from the general view in Figure 12a, it can be said that glass powder provides good bonding with cement and aggregates. A good interlocking is observed in terms of surface quality, but some ﬂat zones have cracks due to fracture. In Figure 12b, the appearance of glass powder particles like sharp ﬁbers is remarkable. Although there is a homogeneous distribution, gaps are seen in some regions. Voids can be removed by better mixing, shaking, and skewering [64–66]. The image in Figure 12c shows material connections more closely, such as a ﬁnely woven bee comb. Figure 12d shows the concrete surface bonding and spacing. Figure 12e,f shows the gelled state of the concrete surface. In terms of surface quality, it can be said that the glass powder is in a good correlation, but it has some holes and gaps. In the mixed model, glass powder was used as a binder with cement, and broken glass particles were used by replacing ﬁne and coarse aggregates in certain proportions. Calcium silicate hydrate (C-S-H), an amorphous phase, is deﬁned as weak crystals [67]. The ﬁne glass particles can be seen in Figure 12g. They are seen as a honeycomb in the C-S-H phase. Since this honeycomb structure provides a good pozzolanic effect, it increases compressive strength [68,69]. 3 4 Comparison of Findings of this Study with Other Existing Stu 3.4. Comparison of Findings of this Study with Other Existing Studies In the same manner, a As shown in these ﬁgures, with the addition of waste glass, the strength values of concrete produced with glass powder generally decrease after a certain value. The maximum decrease in compressive strength was observed in the study by Kalakada et. al., 2022. When the cement was partially replaced with glass powder at 50%, there is a 65% Materials 2022, 15, 7513 10 of 16 reduction in compression strength of the plain concrete specimen. In the same manner, a 20% addition of the waste glass leads to a 36% reduction in the splitting tensile strength of the plain concrete specimen in the study of Abdulazeez et al., (2020). With the addition of 50% glass powder, a 38% reduction in ﬂexural strength was observed in our experimental study. Thus, a rational design expression was developed to consider these reductions in the strength values as follows: reduction in compression strength of the plain concrete specimen. In the same manner, a 20% addition of the waste glass leads to a 36% reduction in the splitting tensile strength of the plain concrete specimen in the study of Abdulazeez et al., (2020). With the addition of 50% glass powder, a 38% reduction in ﬂexural strength was observed in our experimental study. Thus, a rational design expression was developed to consider these reductions in the strength values as follows: f = [1 + c1 × (WGPR) + c2 × (WGPR)2] × f ′ (1) (1) where f is strength values as follows: fc: compressive strength ft: splitting tensile strength; ff: ﬂexure strength; WGPR: waste glass power ratio (0 < WGPR < 50). c1 and c2 are coefﬁcients given in Table 2; f ′ is strength values of the plain concrete. Table 2. Constants for Equation (1). Strength Values (f) c1 c2 fc 0.00033 −0.00027 ft 0.00217 −0.00018 ff 0.00017 −0.00011 Table 2. Constants for Equation (1). As shown in Equation (1), CS, STS, and FS values of concrete produced with waste glass were expressed as a function of the amount of the waste glass (WGPR). These expressions can be easily used in the design stages. Figure 12. SE 4. Conclusions and Summary 4. Conclusions and Summary Waste management has gained great importance due to the increase in the amount of waste. The reuse or recycling approach of these waste products, especially in the con‐ struction sector, has become a more attractive option. In this study, the applicability of waste glass on concrete production was investigated by considering two different condi‐ tions, i.e., replacing cement with waste glass powder and replacing cement, fine and coarse aggregate with waste glasses. A series of tests were conducted on concrete samples produced with different amounts of waste glass to determine engineering properties both in the fresh and hardened cases. In the fresh case of concrete, workability and slump prop‐ erties were explored. In the case of hardened concrete, compression strength, splitting tensile strength, and flexural strength of the produced test specimens were investigated. These properties were then compared with those of plain concrete. Then, SEM analysis was carried out to explore the interaction between glass powder, cement, and aggregates. Furthermore, practical equations were developed to identify the compressive strength, splitting tensile strength, and flexure strength of concrete produced with WGP. Based on our study, the following conclusions can be drawn: • Based on the slump test, the slump value decreases as the amount of waste glass increases. In the same manner, the workability decreases when the rate of glass pow‐ der increases. • The compressive test results indicated that the usage of WGP as a replacement of Waste management has gained great importance due to the increase in the amount of waste. The reuse or recycling approach of these waste products, especially in the construction sector, has become a more attractive option. In this study, the applicability of waste glass on concrete production was investigated by considering two different conditions, i.e., replacing cement with waste glass powder and replacing cement, ﬁne and coarse aggregate with waste glasses. A series of tests were conducted on concrete samples produced with different amounts of waste glass to determine engineering properties both in the fresh and hardened cases. In the fresh case of concrete, workability and slump properties were explored. In the case of hardened concrete, compression strength, splitting tensile strength, and ﬂexural strength of the produced test specimens were investigated. These properties were then compared with those of plain concrete. Then, SEM analysis was carried out to explore the interaction between glass powder, cement, and aggregates. Figure 12. SE 4. Conclusions and Summary Furthermore, practical equations were developed to identify the compressive strength, splitting tensile strength, and ﬂexure strength of concrete produced with WGP. Based on our study, the following conclusions can be drawn: • The compressive test results indicated that the usage of WGP as a replacement of cement adversely affects the compressive strength of concrete. Compared to the ref‐ erence sample, 10%, 20%, 30%, 40% and 50% replacement of WPG reduced the com‐ pressive strength by 3%, 6%, 37%, 13% and 23%, respectively. • On the other hand when cement fine and coarse aggregates were replaced with • Based on the slump test, the slump value decreases as the amount of waste glass increases. In the same manner, the workability decreases when the rate of glass powder increases. On the other hand, when cement, fine and coarse aggregates were replaced with waste glasses, there was an increase in the compression strength, the flexural and splitting tensile strength values up to a certain value of the amount of the waste. However, further increasing the waste glass addition led to a decrease in the com‐ pression value. • According to the results of the split tensile and bending strength tests, the strength • The compressive test results indicated that the usage of WGP as a replacement of cement adversely affects the compressive strength of concrete. Compared to the reference sample, 10%, 20%, 30%, 40% and 50% replacement of WPG reduced the compressive strength by 3%, 6%, 37%, 13% and 23%, respectively. g p g g g values decrease as the value of the substitution of cement with waste glass increases. Compared to plain concrete, 50% replacement of WPG reduced the splitting tensile strength by 34%, respectively. On the other hand, this replacement reduced flexural strength by 38%. • On the other hand, when cement, ﬁne and coarse aggregates were replaced with waste glasses, there was an increase in the compression strength, the ﬂexural and splitting tensile strength values up to a certain value of the amount of the waste. However, further increasing the waste glass addition led to a decrease in the compression value. • According to the results of the split tensile and bending strength tests, the strength values decrease as the value of the substitution of cement with waste glass increases. Compared to plain concrete, 50% replacement of WPG reduced the splitting tensile strength by 34%, respectively. 3.5. Scanning Electron Microscope (SEM) Analysis Figure 12h shows how large and small glass particles are bonded with binders in concrete. It is seen in Figure 12i with good ﬁllings of binders between large Materials 2022, 15, 7513 11 of 16 ons. Cal‐ [67]. The pieces of glass. Figure 12j shows the homogeneous distribution and connection of the glass particles in the concrete. pressive strength [68,69]. Figure 12h shows how large and small glass particles are bonded with binders in concrete. It is seen in Figure 12i with good fillings of binders between large pieces of glass. Figure 12j shows the homogeneous distribution and connection of the glass particles in the concrete pieces of glass. Figure 12j shows the homogeneous distribution and connection of the glass particles in the concrete. p g [ , ] g g g p with binders in concrete. It is seen in Figure 12i with good fillings of binders between large pieces of glass. Figure 12j shows the homogeneous distribution and connection of the glass particles in the concrete particles in the concrete. (a) (b) Hidrated of cement, glass powder, fine aggregate and coarse aggregate Glass powder particles are Hole Hydrated of Cement Crack Materials 2022, 15, x FOR PEER REVIEW 12 of 1 (c) (d) Interconnection cement, glass powder and other materials Hydrated cement and WGP Interface of WGP Mixed of concrete (e) (f) (h) Huge glass particle The fine glass particles can be seen seen as a honeycomb in the C‐S‐H phase Small glass particle As binder cement and glass powders Hole and gap (g) Figure 12. Cont. (a) (b) Hidrated of cement, glass powder, fine aggregate and coarse aggregate Glass powder particles are Hole Hydrated of Cement Crack Materials 2022, 15, x FOR PEER REVIEW 12 of (c) (d) Interconnection cement, glass powder and other materials Hydrated cement and WGP Interface of WGP Mixed of concrete (e) (f) Hole and gap (h) Huge glass particle The fine glass particles can be seen seen as a honeycomb in the C‐S‐H phase Small glass particle As binder cement and glass powders (g) Figure 12 Cont Figure 12. Cont. Materials 2022, 15, 7513 12 of 16 13 f 17 Cement binder between two glasses Combination of glass pieces and cement (j) (i) piece of glass Figure 12. SEM micrographs of the obtained samples. (i) Figure 12. SEM micrographs of the obtained samples. Figure 12. SE 4. Conclusions and Summary On the other hand, this replacement reduced ﬂexural strength by 38%. • When cement, ﬁne and coarse aggregates were replaced with waste glasses there was an increase in the ﬂexural and splitting tensile strength values up to a certain value of the amount of the waste glass. However, further increasing the waste glass addition resulted in a decrease in the strength values. Replacing the waste glass by 10% resulted in a 5% increase in splitting tensile strength while replacing the waste glass by 30% resulted in a 14% decrease in splitting tensile strength. On the other hand, a 10% substitute of waste glass resulted in a 5% increase in ﬂexural strength, while a 30% substitute of waste glass resulted in an 11% decrease in ﬂexural strength. Materials 2022, 15, 7513 13 of 16 13 of 16 • It can be concluded that when waste glass is used instead of cement, ﬁne and coarse aggregate, signiﬁcant increases in strength are observed. • It can be concluded that when waste glass is used instead of cement, ﬁne and coarse aggregate, signiﬁcant increases in strength are observed. gg g g g • SEM analysis results showed that there was a good bond between the glass powder and cementitious concrete. gg g g g • SEM analysis results showed that there was a good bond between the glass powder and cementitious concrete. • The proposed equations for the compressive strength, ﬂexural, and splitting tensile strength are quite general and they can be suitable for direct adoption into design speciﬁcations of the concretes produced with waste glass. • The proposed equations for the compressive strength, ﬂexural, and splitting tensile strength are quite general and they can be suitable for direct adoption into design speciﬁcations of the concretes produced with waste glass. In this study, it was observed that waste glass can be used as a partial cement substitute, or as cement, ﬁne and coarse aggregate in concrete. Optimum waste glass dosage has also been identiﬁed for design purposes. However, lower strength values were observed when waste glass was used as a partial replacement for cement. The current study deﬁnes waste glass as suitable, reachable in large amounts, a local eco-material, cheap, that can be selected for concrete construction, in a point of view between economically and environmentally responsive. Figure 12. SE 4. Conclusions and Summary To mitigate this shortcoming, future studies will concentrate on the use of waste glass with other recycling ﬁbers. Author Contributions: Conceptualization, Y.O.Ö. and D.D.B.-N.; methodology, Y.O.Ö. and D.D.B.-N.; data curation, Ö.Z., M.K., D.P.B.-N. and Y.O.Ö.; investigation, A.˙I.Ç., Y.O.Ö., Ö.Z., M.K.; writing— original draft preparation, A.˙I.Ç., Y.O.Ö., Ö.Z., S.Q., J.A., D.D.B.-N. and D.P.B.-N.; writing—review and editing, Y.O.Ö., Ö.Z., M.K., S.Q., J.A., D.D.B.-N. and D.P.B.-N.; funding acquisition, D.D.B.-N. and D.P.B.-N. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Gheorghe Asachi Technical University of Ia¸si—TUIASI- Romania, Scientiﬁc Research Funds, FCSU-2022. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This paper was ﬁnancially supported by the Project “Network of excellence in applied research and innovation for doctoral and postdoctoral programs”/InoHubDoc, project co-funded by the European Social Fund ﬁnancing agreement no. POCU/993/6/13/153437. This paper was also supported by “Gheorghe Asachi” Technical University from Ia¸si (TUIASI), through the Project “Performance and excellence in postdoctoral research 2022”. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Batayneh, M.; Marie, I.; Asi, I. Use of selected waste materials in concrete mixes. Waste Manag. 2007, 2 [PubMed] 2. Jiang, X.; Xiao, R.; Bai, Y.; Huang, B.; Ma, Y. Inﬂuence of waste glass powder as a supplementary cementitious material (SCM) on physical and mechanical properties of cement paste under high temperatures. J. Clean. Prod. 2022, 340, 130778. [CrossRef] p y p p p g p J [ ] 3. Bilondi, M.P.; Touﬁgh, M.M.; Touﬁgh, V. 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Aksoylu, C.; Özkılıç, Y.O.; Hadzima-Nyarko, M.; I¸sık, E.; Arslan, M.H. Investigation on Improvement in Shear Performance of Reinforced-Concrete Beams Produced with Recycled Steel Wires from Waste Tires. Sustainability 2022, 14, 13360. [CrossRef] 8. Aksoylu, C.; Özkılıç, Y.O.; Hadzima-Nyarko, M.; I¸sık, E.; Arslan, M.H. Investigation on Improvement in Shear Performance of Reinforced-Concrete Beams Produced with Recycled Steel Wires from Waste Tires. Sustainability 2022, 14, 13360. [CrossRef] 9. Ali, E.E.; Al-Tersawy, S.H. Recycled glass as a partial replacement for ﬁne aggregate in self compacting concrete. Constr. Build. 8. Aksoylu, C.; Özkılıç, Y.O.; Hadzima Nyarko, M.; I¸sık, E.; Arslan, M.H. Investigation on Improvement in Shear Performance of Reinforced-Concrete Beams Produced with Recycled Steel Wires from Waste Tires. Sustainability 2022, 14, 13360. [CrossRef] 9. Ali, E.E.; Al-Tersawy, S.H. Recycled glass as a partial replacement for ﬁne aggregate in self compacting concrete. 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https://openalex.org/W4390114585	https://www.qeios.com/read/JH6ICL/pdf	English	null	Review of: "A memory dependent analysis on permeation of non-Gaussian laser pulse through human skin"	null	2,023	cc-by	149	Qeios, CC-BY 4.0 · Review, December 22, 2023 Qeios ID: JH6ICL · https://doi.org/10.32388/JH6ICL Review of: "A memory dependent analysis on permeation of non-Gaussian laser pulse through human skin" Oluwabamise Adeleye1 1 University of KwaZulu-Natal Oluwabamise Adeleye1 1 University of KwaZulu-Natal Potential competing interests: No potential competing interests to declare. The manuscript looks OK technically, with interesting results for biomedical science. However, I advise the following: The manuscript needs to be improved in the numerical analysis section, e.g., referencing previous studies (if there are and at other wavelengths) and comparing them to the results obtained. In addition, the limitations or probable sources of errors from various approximations should be made available within the study. The conclusion can also be improved with the practical impacts or recommendations for laser treatments. The conclusion can also be improved with the practical impacts or recommendations for laser treatments. Qeios ID: JH6ICL · https://doi.org/10.32388/JH6ICL 1/1
https://openalex.org/W3082858713	https://hal.archives-ouvertes.fr/hal-03160433/file/Ramos_2020.pdf	English	null	Snapshots of ADP-ribose bound to Getah virus macro domain reveal an intriguing choreography	Scientific reports	2,020	cc-by	10,869	Snapshots of ADp-ribose bound to Getah virus macro domain reveal an intriguing choreography Ana Sofia Ferreira-Ramos, Gerlind Sulzenbacher, Bruno Canard, Bruno Coutard To cite this version: Ana Sofia Ferreira-Ramos, Gerlind Sulzenbacher, Bruno Canard, Bruno Coutard. Snapshots of ADp- ribose bound to Getah virus macro domain reveal an intriguing choreography. Scientific Reports, 2020, 10 (1), 10.1038/s41598-020-70870-w. hal-03160433 Snapshots of ADP‑ribose bound to Getah virus macro domain reveal an intriguing choreography Ana Sofia Ferreira‑Ramos1,3,4, Gerlind Sulzenbacher1,4, Bruno Canard1 & Bruno Couta , Bruno Canard1 & Bruno Coutard2* Alphaviruses are (re-)emerging arboviruses of public health concern. The nsP3 gene product is one of the key players during viral replication. NsP3 comprises three domains: a macro domain, a zinc- binding domain and a hypervariable region. The macro domain is essential at both early and late stages of the replication cycle through ADP-ribose (ADPr) binding and de-ADP-ribosylation of host proteins. However, both its specific role and the precise molecular mechanism of de-ADP-ribosylation across specific viral families remains to be elucidated. Here we investigate by X-ray crystallography the mechanism of ADPr reactivity in the active site of Getah virus macro domain, which displays a peculiar substitution of one of the conserved residues in the catalytic loop. ADPr adopts distinct poses including a covalent bond between the C′′1 of the ADPr and a conserved Togaviridae-specific cysteine. These different poses observed for ADPr may represent snapshots of the de-ADP-ribosylation mechanism, highlighting residues to be further characterised. Alphaviruses are arthropod-borne viruses among which several, such as Chikungunya virus (CHIKV) and Ven- ezuelan equine encephalitis virus (VEEV), are emerging or re-emerging viruses. According to the 7th report of the International Committee on Taxonomy of Viruses (ICTV), the genus Alphavirus, belonging to the Togaviridae family, is organised into 31 species and at least ten of them are human pathogens1. Members of the Alphavirus genus are geographically and evolutionary distinguished into “Old World” (OW) alphaviruses, represented by CHIKV, and “New World” (NW) viruses, prototyped by VEEV, as well as several species corresponding to viruses that most likely appeared from recombination events between OW and NW viruses2 or viruses of marine origin.hh y pp g Their genome is a positive-sense single-stranded RNA molecule which is usually 11 to 12 kilobases long. The genome carries two open reading frames (ORFs). The first ORF can be translated directly from the genomic RNA in P123, and P1234 polyproteins, which are sequentially processed into four non-structural proteins (nsP), named nsP1 to nsP4. The proteolysis intermediates and the final nsPs constitute the transcription/replication complex (TRC) organised in spherules at the plasma membrane. In this TRC, nsP3 has been for a long time the least understood nsP, but recent studies revealed some of its functions in viral replication3. HAL Id: hal-03160433 https://hal.science/hal-03160433v1 Submitted on 5 Mar 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. www.nature.com/scientificreports www.nature.com/scientificreports Materials and methods i d i d Protein production and purification. The DNA coding sequence of the nsP3 macro domain (amino- acids 1 to 160) of Getah virus (strain M1, GenBank: ABK32031.1, amino-acids 1,333 to 1,492) was optimized for expression in Escherichia coli and synthesized by THERMO FISCHER SCIENTIFIC. The coding sequence was cloned into a pDest14 vector using the “Gateway” cloning procedure (THERMO FISCHER SCIENTIFIC). A hexahistidine (6-His) coding sequence was added at the 3′ end to produce the GETV macro domain in fusion with a C-terminal 6-His tag. A short sequence of two codons coding for methionine and lysine were added to the 5′ end in order to allow the translation in E. coli. The bacterial growth conditions for optimal expression of GETV macro domain were obtained from an incomplete factorial expression screen17. GETV macro domain was produced in E. coli Rosetta (DE3) pLysS cells (NOVAGEN). Bacteria were grown at 37 °C with shaking (200 rpm) in Terrific Broth (TB) medium (SIGMA-ALDRICH) containing 34 µg·mL−1 chloramphenicol and 100 µg·mL−1 ampicillin. The expression of the recombinant gene was induced with 0.5 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) when the bacterial cell density reached the exponential phase at OD600nm of 0.4. After induction, the incubation temperature was dropped to 25 °C for overnight expression. Subsequently, the bacterial cells were harvested by centrifugation at 4,000 g for 15 min and pellets were resuspended in 50 mL of lysis buffer made of 50 mM TRIS–HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 0.1% Triton X-100 and one tablet of complete EDTA-free protease inhibitor cocktail (ROCHE). The resuspended pellets were stored at − 80 °C until the time of use. Pellets were thawed and 0.25 mg·mL−1 of lysozyme (SIGMA-ALDRICH), 10 µg·mL−1 of DNase I (SIGMA-ALDRICH) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (SIGMA- ALDRICH) were added. The samples were incubated at 4 °C for 30 min and then sonicated. Samples were then centrifuged at 13,000 g for 1 h. The supernatant was loaded onto a 5 mL His prep column (GE HEALTHCARE) for Immobilized Metal Affinity Chromatography (IMAC) using an ÄKTA Xpress system (GE HEALTHCARE). After loading the supernatant, the column was washed with a buffer containing 50 mM TRIS-HCl, 300 mM NaCl, 50 mM imidazole, pH 8.0. The recombinant protein was eluted with 50 mM TRIS-HCl, 300 mM NaCl, and 250 mM imidazole, pH 8.0. www.nature.com/scientificreports/ analyses together with functional characterization allowed assignment of alphavirus macro domains to a pro- tein family prototyped by human macroD27. Viral macro domains were found originally to have ADR-ribose- 1″-phosphate phosphatase (A1″Pase) activity10,13. More recently, it was shown that alphavirus macro domains are able to de-ADP-ribosylate Asp or Glu side-chains of host proteins7,14,15. Some of the residues involved in ADP-ribose binding and de-ADP-ribosylation have been characterized9. However, the molecular mechanism of de-ADP-ribosylation by alphavirus macro domain remains to be clarified. i In order to better understand this mechanism, we initiated a structure-based study of the Getah virus (GETV) macro domain, which shows a peculiar substitution for one of the most conserved residues in the catalytic loop, namely serine replacing a glycine in position 30 (Fig. 1). GETV is an alphavirus isolated for the first time in Malaysia in 1955 from Culex spp. mosquitoes. GETV is geographically distributed from Asia to the north of Australia and infects mainly horses16. We produced the GETV macro domain for crystallographic studies, alone or in complex with ADP-ribose, and documented several conformations adopted by ADP-ribose in the binding site. We show here that our structures provide snapshots that could contribute to unravel the still elusive de- ADP-ribosylation mechanism at play to de-activate innate immunity responses elicited by alphavirus infection. Taken together, this ensemble of conformations might represent several snapshots of the de-ADP-ribosylation mechanism, highlighting residues that have not been evaluated to date. Snapshots of ADP‑ribose bound to Getah virus macro domain reveal an intriguing choreography nsP3 is organised into three domains: a macro domain (MD) at the N-terminal, a zinc-binding domain (ZBD) and a C-terminal hypervariable region (HVR). The HVR plays a key role in viral replication by interacting with several partners among which Ras GTPase-activating protein-binding protein (G3BP) and SH3 containing proteins4. The N-terminal domain is structurally associated to the class of macro domains, protein modules found in all domains of life. Despite structural conservation, macro domains participate in various cellular functions including DNA repair, cell dif- ferentiation, proliferation or apoptosis. Macro domains can recognize ADP-ribose (ADPr), poly-ADP-ribose (PAR) in their free or protein-bound form, or O-acetyl-ADP-ribose (OAADPr). In addition to binding, some macro domains can also catalyze several reactions such as de-ADP-ribosylation5. Like coronavirus or hepevirus macro domains6,7, alphavirus macro domains recognise ADP-ribose and have de-ADP-ribosylation activity. This latter functions are thought to be essential in both early and late replication steps8. It is postulated that de-mono- ADP-ribosylation is a viral countermeasure to cellular innate immunity mediated by the viral macro domain9. Th f h d f l h VEEV CHIKV S db (SINV) d y y y The structures of the macro domains from alphavirus species VEEV, CHIKV, Sindbis virus (SINV), Mayaro virus (MAYV) were determined using X-ray crystallography and/or NMR10–12. Structure and seque 1Architecture et Fonction des Macromolécules Biologiques, CNRS, Aix-Marseille Université, 13288 Marseille, France. 2Unité des Virus Emergents (UVE), Aix Marseille Univ-IRD 190-INSERM 1207-IHU Méditerranée Infection, 13005 Marseille, France. 3Present address: Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany. 4These authors contributed equally: Ana Sofia Ferreira-Ramos and Gerlind Sulzenbacher. *email: gerlind.sulzenbacher@afmb.univ‑mrs.fr; bruno.coutard@univ‑amu.fr Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ Materials and methods i d i d Size exclusion chromatography was next performed on a Superdex 200 HiLoad 16/600 column (GE HEALTHCARE) equilibrated in 20 mM HEPES, 300 mM NaCl, pH 7.4. The eluted protein was concentrated up to 14 mg·mL−1 using an Ultracel regenerated cellulose membrane with a 3 kDa molecular weight cut-off (Amicon Ultra-15 Centrifugal Filter, MERCK MILLIPORE). Purity and stability assessment. The purity of the protein was assessed on SDS-PAGE gels stained with Coomassie blue. Besides, protein stabilization by ADP-ribose was additionality checked by thermal shift assay (TSA)18 in a 96-well thin-wall PCR plate (BIO-RAD) using a quantitative PCR machine ICycler IQ (BIO-RAD). Briefly, the protein at a final concentration of 0.3 mg·mL−1 was mixed with a SYPRO orange solution at concen- trations recommended by the manufacturer (THERMO FISHER SCIENTIFIC) in a final volume of 25 µL and tested with the following concentrations of ADP-ribose: 0.5 mM, 0.25 mM, 0.125 mM, 0.063 mM, 0.031 mM, 0.016 mM and 0 mM. Accumulative steps of temperature from 20 to 90 °C were applied to the samples. The increase of the fluorescence emitted by the probe that binds the exposed hydrophobic regions of the denatured protein was used to monitor the denaturation of the protein. A melting temperature (Tm) was calculated as the mid-log of the transition phase from the native to the denatured protein using a Boltzmann model (ORIGIN SOFTWARE). Crystallization, co‑crystallization and crystal soaking. Initial crystallization trials were carried out with GETV macro domain at 14 mg·mL−1 using the commercial screens WIZARD CLASSIC 1&2 HT96, STRU CTURE 1&2 HT96, and STURA FOOTPRINT Screen 48 (MOLECULAR DIMENSIONS LIMITED) in 3-well sitting-drop SWISSCI crystallization plates (TPP LABTECH). 100 nL of crystallization solutions were added to 100, 200 or 300 nL of GETV macro domain solution using a MOSQUITO Robot (TTP LABTECH). The condi- tions where crystal hits were obtained were then optimized using either 96-well SWISSCI crystallization plates or 24-well hanging drop LINBRO plates. Co-crystallization experiments were set-up with GETV macro domain complemented with (1) ADP-ribose, (2) ADP-ribose and glutamic acid or (3) ADP-ribose and aspartic acid, with final concentrations of 3 mM for ADP-ribose, 50 mM for glutamic acid and 3 mM or 30 mM for aspartic acid. In general, crystals appeared after one day and were grown for approximately two weeks. Soaking experi- https://doi.org/10.1038/s41598-020-70870-w Scientific Reports \| (2020) 10:14422 \| www.nature.com/scientificreports/ Figure 1. Sequence alignment of GETV macro domain with other viral macro domains. Materials and methods i d i d The OW group from the genus alphavirus is represented in the alignment by CHIKV, MAYV and SINV macro domains, while NW group is represented by the VEEV macro domain. The human coronavirus NL63 macro domain as well as SARS coronavirus are included. The alignment was obtained using ESPript and residues highlighted with red boxes are strictly conserved, red residues represent similarity in a group, and blue frames represent similarity across groups. Secondary structure elements derived from the GETV macro domain crystal structure are represented above the alignment. Figure 1. Sequence alignment of GETV macro domain with other viral macro domains. The OW group from the genus alphavirus is represented in the alignment by CHIKV, MAYV and SINV macro domains, while NW group is represented by the VEEV macro domain. The human coronavirus NL63 macro domain as well as SARS coronavirus are included. The alignment was obtained using ESPript and residues highlighted with red boxes are strictly conserved, red residues represent similarity in a group, and blue frames represent similarity across groups. Secondary structure elements derived from the GETV macro domain crystal structure are represented above the alignment. ments were performed with crystals of GETV macro domain co-crystallized with ADP-ribose by adding mother liquor supplemented with aspartic acid to the crystallization droplet followed by 2–3 h or overnight incubation at 20 °C. Afterwards, crystals were flash-cooled in liquid nitrogen using 20% of glycerol in the mother liquid as cryo-protectant. ments were performed with crystals of GETV macro domain co-crystallized with ADP-ribose by adding mother liquor supplemented with aspartic acid to the crystallization droplet followed by 2–3 h or overnight incubation at 20 °C. Afterwards, crystals were flash-cooled in liquid nitrogen using 20% of glycerol in the mother liquid as cryo-protectant. Data collection and structure determination. Diffraction intensities were recorded on beamlines ID23-1 and ID30A-3 at the European Synchrotron Radiation Facility (Grenoble, France) and on beamline Proxima-2 at Soleil Synchrotron (Gif-sur-Yvette, France). Indexing and integration of the different datasets were performed using MOSFLM19 or XDS20. Data were scaled and merged using the CCP421 suite of programs POINTLESS22, AIMLESS 23 and TRUNCATE24. Random sets of approximately 5% of reflections, depending on https://doi.org/10.1038/s41598-020-70870-w Scientific Reports \| (2020) 10:14422 \| www.nature.com/scientificreports/ Table 1. Data collection and refinement statistics. Values in parentheses are for the highest-resolution shell. Materials and methods i d i d The structures of GETV macro domain in complex with ligands were determined either by molec- ular replacement using the native GETV macro domain as a search model or by difference Fourier synthesis.i f Refinement was performed using REFMAC26, interspersed with cycles of manual model adjustments with COOT27. Ligands were fitted into unbiased Fo–Fc difference electron density maps calculated after 10 cycles of rigid-body refinement. Hydrogens were added in the riding position. Coordinates and restraints for ADP-ribose in the close conformation were retrieved from the CCP4 ligand dictionary and a model and restraints for ADP- ribose in the open conformation were generated with the CCP4 Monomer Library Sketcher. Model quality was assessed with internal modules of Coot and with the MOLPROBITY server28. Crystallographic models are of good quality with 99.4–100% of residues in favoured regions of the Ramachandran plot and no outliers. Data col- lection and refinement statistics are summarized in Table 1 with representative electron density depicted in Sup- plementary Fig. S1. Figures representing structural renderings were generated with the PyMOL MOLECULAR GRAPHICS SYSTEM (DeLano, W.L. The PyMOL Molecular Graphics on https://www.pymol.org/). Sequence alignments were made using Clustal Omega29 and graphical rendering of the alignments, considering structural information, were made with ESPript30. The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 6QZU (native GETV macro domain), 6R0F (GETV macro domain with ADP-ribose in close conformation pose 1), 6R0G, (GETV macro domain with ADP-ribose in close conformation pose 2), 6R0T (GETV macro domain with ADP-ribose in open ring conformation), 6R0P (GETV macro domain with ADP-ribose in double open ring conformation) and 6R0R (GETV macro domain with covalently bound ADP-ribose). f Refinement was performed using REFMAC26, interspersed with cycles of manual model adjustments with COOT27. Ligands were fitted into unbiased Fo–Fc difference electron density maps calculated after 10 cycles of rigid-body refinement. Hydrogens were added in the riding position. Coordinates and restraints for ADP-ribose in the close conformation were retrieved from the CCP4 ligand dictionary and a model and restraints for ADP- ribose in the open conformation were generated with the CCP4 Monomer Library Sketcher. Model quality was assessed with internal modules of Coot and with the MOLPROBITY server28. Crystallographic models are of good quality with 99.4–100% of residues in favoured regions of the Ramachandran plot and no outliers. Materials and methods i d i d Native Closed ribose “pose 1” Closed ribose “pose 2” Open ribose Open ribose in double conformation ADP-ribose covalently bound to Cys34 Data collection Beam line ESRF ID23-1 ESRF ID30A3 ESRF ID30A3 ESRF ID30A3 ESRF ID30A3 SOLEIL Proxima2 Space group P212121 P212121 P212121 P212121 P212121 C2 Cell dimensions a, b, c (Å) 46.55, 71.36, 94.95 46.73, 71.57, 98.69 46.88, 71.65, 98.80 46.67, 71.44, 98.97 46.70, 71.45, 99.50 64.01, 46.80, 51.00; β = 104.16 Resolution (Å) 41.80–2.00 (2.05–2.00) 42.23–2.05 (2.11–2.05) 42.35–1.70 (1.73–1.70) 40.68–1.85 (1.89–1.85) 40.83–1.60 (1.63–1.60) 37.37–1.45 (1.47–1.45) Rmerge 0.134 (0.748) 0.144 (1.328) 0.062 (1.012) 0.078 (1.221) 0.059 (1.268) 0.046 (0.826) Rpim 0.079 (0.448) 0.073 (0.660) 0.040 (0.664) 0.039 (0.591) 0.034 (0.710) 0.019 (0.484) CC(1/2) 0.989 (0.603) 0.994 (0.468) 0.999 (0.527) 0.998 (0.627) 0.998 (0.559) 0.999 (0.352) I/σI 7.3 (2.1) 8.5 (1.5) 15.6 (1.6) 13.1 (1.40) 13.1 (1.2) 20.6 (2.1) Completeness (%) 99.8 (99.8) 98.6 (99.0) 97.5 (97.4) 99.6 (99.8) 98.7 (97.5) 99.9 (100) Redundancy 4.5 (4.3) 4.8 (5.1) 5.0 (5.3) 4.8 (5.1) 4.0 (4.1) 6.6 (6.5) Wilson B (Å2) 13.3 25.50 18.9 18.34 15.36 17.39 Refinement Resolution (Å) 57.05–2.00 40.66–2.05 40.70–1.70 33.98–1.85 36.41–1.60 37.40–1.45 No. reflections 20,700 19,838 34,198 27,122 41,597 24,542 Rwork 19.39 (27.70) 19.27 (31.6) 18.11 (34.20) 16.84 (30.90) 16.94 (48.50) 16.95 (53.20) Rfree 23.11 (28.70) 23.55 (32.50) 20.75 (33.50) 19.90 (34.00) 19.35 (51.70) 19.67 (54.60) No. atoms Protein 2,386 2,386 2,388 2,388 2,388 1,210 ADP-ribose – 72 72 72 72 36 Water/ligands 190/11 184/- 231/8 190/24 279/8 185/4 B-factors (Å2) Protein 12.11 37.98 27.57 46.05 26.41 19.81 ADP-ribose – 49.11 29.20 48.32 27.06 19.61 Water/ligands 34.72/47.06 37.03/- 33.42/40.84 50.42/59.22 34.40/27.28 29.13/29.04 R.m.s. deviations Bond lengths (Å) 0.009 0.007 0.005 0.007 0.006 0.009 Bond angles (°) 1.304 1.436 1.250 1.454 1.446 1.583 Ramachandran favoured 99.68 99.68 100 99.68 99.68 99.38 PDB ID 6QZU 6R0F 6R0G 6R0T 6R0P 6R0R Data collection and refinement statistics. Values in parentheses are for the highest-resolution shell. the resolution limit, were set aside for FreeR cross-validation purposes. Where data-sets of ligand complexes were in the same space group as the native data set, the composition of cross-validation data sets was system- atically taken over from the parent data set. The structure of native GETV macro domain was determined by molecular replacement with MolRep25 using the macro domain of Chikungunya virus (CHIKV, PDB 3GPG) as a search model. Results and discussion i d i Protein production, crystallization and structure determination. The sequence corresponding to the putative macro domain on the GETV nsp1234 sequence (GenBank reference ABK32031.1) is encompassing aa 1,333 to 1,492 (positions in the polyprotein; hereafter numbered 1–160 for convenience). Its sequence iden- tity with alphavirus homologues with known structures such as CHIKV and VEEV macro domains is 69% and 57%, respectively (Fig. 1). Macro domains harbour the consensus motif G(D/E/G)GV7. A focused analysis of these residues (amino acids 30 to 33 of the nsP3 sequence) of macro domains revealed that the consensus motif is followed by a Togaviridae-specific cysteine. It can also be noticed that the GETV macro domain is an excep- tion regarding the consensus motif, as its sequence harbours a serine instead of a glycine at position 30. We thus first verified that this macro domain is a bona fide ADP-ribose binding module. The recombinant GETV macro domain was produced in E. coli and purified under non-denaturing conditions. After purification, the integrity of the protein was assessed by thermal shift assay (TSA). TSA experiments showed that the protein could be denatured by heat in a classical folded-to-denatured transition phase (data not shown) with a melting tempera- ture (Tm) of 46.5 ± 0.2 °C. Addition of 0.5 mM ADP-ribose to the protein solution led to a 10 °C increase of the Tm suggesting that the recombinant GETV macro domain indeed binds ADP-ribose (Fig. 2). Structure of the GETV macro domain and comparison with other (alphavirus) macro domains. The structure of the GETV macro domain was determined at 2.0 Å resolution by molecular replacement using the CHIKV macro domain (3GPG) as a template. The crystals of the GETV macro domain belong to the space group P212121 and two molecules are present in the asymmetric unit. All residues from Ala1 to Thr160 are well defined in the electron density and the model has excellent stereochemistry with 99.2% of side-chain rotamers in the favoured conformation and 99.7% of residues in the favoured Ramachandran plot regions (Table 1).h g The structure of GETV macro domain consists of a central twisted six-stranded β sheet (strand order β1, β6, β5, β2, β4, β3) sandwiched between one α-helix (α1) at one side and three α-helices (α2, α3 and α4) at the oppo- site site (Fig. 3A). The two molecules present in the asymmetric unit are virtually identical, with an r.m.s.d. of 0.22 Å between the two chains. Materials and methods i d i d Data col- lection and refinement statistics are summarized in Table 1 with representative electron density depicted in Sup- plementary Fig. S1. Figures representing structural renderings were generated with the PyMOL MOLECULAR GRAPHICS SYSTEM (DeLano, W.L. The PyMOL Molecular Graphics on https://www.pymol.org/). Sequence alignments were made using Clustal Omega29 and graphical rendering of the alignments, considering structural information, were made with ESPript30. The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 6QZU (native GETV macro domain), 6R0F (GETV macro domain with ADP-ribose in close conformation pose 1), 6R0G, (GETV macro domain with ADP-ribose in close conformation pose 2), 6R0T (GETV macro domain with ADP-ribose in open ring conformation), 6R0P (GETV macro domain with ADP-ribose in double open ring conformation) and 6R0R (GETV macro domain with covalently bound ADP-ribose). f Refinement was performed using REFMAC26, interspersed with cycles of manual model adjustments with COOT27. Ligands were fitted into unbiased Fo–Fc difference electron density maps calculated after 10 cycles of rigid-body refinement. Hydrogens were added in the riding position. Coordinates and restraints for ADP-ribose in the close conformation were retrieved from the CCP4 ligand dictionary and a model and restraints for ADP- ribose in the open conformation were generated with the CCP4 Monomer Library Sketcher. Model quality was assessed with internal modules of Coot and with the MOLPROBITY server28. Crystallographic models are of good quality with 99.4–100% of residues in favoured regions of the Ramachandran plot and no outliers. Data col- lection and refinement statistics are summarized in Table 1 with representative electron density depicted in Sup- plementary Fig. S1. Figures representing structural renderings were generated with the PyMOL MOLECULAR GRAPHICS SYSTEM (DeLano, W.L. The PyMOL Molecular Graphics on https://www.pymol.org/). Sequence alignments were made using Clustal Omega29 and graphical rendering of the alignments, considering structural information, were made with ESPript30. The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 6QZU (native GETV macro domain), 6R0F (GETV macro domain with ADP-ribose in close conformation pose 1), 6R0G, (GETV macro domain with ADP-ribose in close conformation pose 2), 6R0T (GETV macro domain with ADP-ribose in open ring conformation), 6R0P (GETV macro domain with ADP-ribose in double open ring conformation) and 6R0R (GETV macro domain with covalently bound ADP-ribose). Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ Figure 2. Effect of ADP-ribose concentrations on the thermostability of GETV macro domain. Materials and methods i d i d (A) Titration of ADP-ribose using the thermal denaturation shift assay on GETV macro domain. For each concentration of ADP-ribose titrated onto the GETV macro domain the melting temperature was calculated (Tm). ΔTm [(Tm at a given concentration of ADP-ribose) minus (Tm with no ADP-ribose)] was calculated to quantify the change in protein stability (B). Figure 2. Effect of ADP-ribose concentrations on the thermostability of GETV macro domain. (A) Titration of ADP-ribose using the thermal denaturation shift assay on GETV macro domain. For each concentration of ADP-ribose titrated onto the GETV macro domain the melting temperature was calculated (Tm). ΔTm [(Tm at a given concentration of ADP-ribose) minus (Tm with no ADP-ribose)] was calculated to quantify the change in protein stability (B). Results and discussion i d i ADP-ribose is represented in stick-mode, with carbon atoms coloured in grey, oxygens in red, nitrogen atoms in blue and phosphorus atoms in orange. (B) Overlap of GETV macro domain, same colour coding as in (A), with the structures of CHIKV macro domain (3GPO) in red, VEEV macro domain (3GQ) in yellow and the SARS macro domain (2FAV) in grey. The N-termini, important loops and α-helices are labelled in black and violet for the GETV macro domain and in grey for SARS macro domain. For clarity, only the first β-strand is labelled. APD-ribose as observed in the GETV macro domain ADP-ribose complex “pose 1” and colour-coded as in (A) has been added for reference. Figure 3. Overall structure of GETV macro domain. (A) Cartoon representation of GETV macro domain with APD-ribose depicted as seen in the GETV macro domain ADP-ribose complex “pose 1”. β-sheets are coloured in slate, α-helices in teal and loops in pink. Secondary structure elements and the N- and C-termini are labelled. ADP-ribose is represented in stick-mode, with carbon atoms coloured in grey, oxygens in red, nitrogen atoms in blue and phosphorus atoms in orange. (B) Overlap of GETV macro domain, same colour coding as in (A), with the structures of CHIKV macro domain (3GPO) in red, VEEV macro domain (3GQ) in yellow and the SARS macro domain (2FAV) in grey. The N-termini, important loops and α-helices are labelled in black and violet for the GETV macro domain and in grey for SARS macro domain. For clarity, only the first β-strand is labelled. APD-ribose as observed in the GETV macro domain ADP-ribose complex “pose 1” and colour-coded as in (A) has been added for reference. differences occur in the loops branching structural elements (1) α1 and β3, (2) β3 and β4 and (3) β5 and α3. The β2 α1 loop, referred to as the catalytic loop, is structurally well conserved among alphavirus macro domain structures. With respect to the closest human or coronavirus macro domain structures, chiefly consisting of a central seven-stranded β-sheet flanked on either sides by three α-helices, alphavirus macro domains lack the first β-strand, and α-helices α1 and α3 (SARS-CoV annotation) are degenerated to 310-helices or reduced to loops. Structures of the GETV macro domain in complex with ADP‑ribose. Results and discussion i d i The early evaluation of the integrity of GETV macro domain by monitoring the thermostability using thermal shift assay revealed an increase of Tm by 10 °C induced by the presence of small amounts of ADP-ribose, advocating that similar to other alphavirus macro domains, GETV macro domain can bind ADP-ribose. Therefore, we produced crys- tals of the GETV macro domain in the presence of 3 mM ADP-ribose. The study of the structure of the di- manganese mono-ADP-ribosylhydrolase DraG with a trapped amino acid-ADP-ribose reaction intermediate, Lys-ADP-ribose32, inspired us to add glutamic or aspartic acids to the crystallization trials, based on the rational that in this way we could reverse the de-ADP-ribosylation reaction mediated by alphavirus macro domain, documented to de-ADP-ribosylate carboxylic amino-acid side-chains7,14,15. Thus, we added glutamic or aspartic acids to co-crystallization or soaking solutions at equal or five to fifteen-times higher concentrations than that of ADP-ribose. All the optimal crystallization and co-crystallization solutions converged to the following buffer composition: 0.2 M imidazole/malate pH 5.9 ± 0.2 and 34 ± 4% of PEG 4 K. The crystallization and soaking procedures that led to the structures discussed in this study are summarize in Table 2, and data collection and refinement statistics are presented in Table 1. No clear electron density could be detected for aspartic or glutamic acid in any of the crystal structures. However, structural analyses revealed that the addition of the amino acids to the crystallization medium was associated with conformational changes both in the catalytic loop and of ADP- ribose. Five different conformations could be observed for ADP-ribose bound to the GETV macro domain, documented here below. Structure of the GETV macro domain in complex with ADP‑ribose presenting the distal ribose in “pose 1”. In a first instance we obtained diffraction data extending to 2.05 Å resolution for GETV macro domain in complex with ADP-ribose from a crystal obtained by co-crystallization of GETV macro domain with 3 mM ADP-ribose and subsequently soaked in mother-liquor solution supplemented with 15 mM aspartic acid. The structure was solved by difference Fourier synthesis and, as for the native structure, all residues are well defined in the electron density and the final model has excellent stereochemistry. Clear difference electron density could be observed for ADP-ribose bound to each of the two macro domain chains present in the asym- metric unit. Results and discussion i d i A structural homology search with the DALI server31 revealed closest homology with macro domains from the alphavirus family such as CHIKV (3GPG), SINV (4GUA), VEEV (3GQO) and MAYV (5IQ5) with Z-scores in the range of 32.2 to 27.8 and r.m.s.d. values of 0.7 to 1.4 Å for 158–160 pairwise aligned Cα positions. g A second group of close structural homologues, with Z-scores in the range of 21.4 to 19.0 and r.m.s.d values of 1.8 to 2.2 Å for 151–154 pairwise aligned Cα positions comprises the structures of human macro domains (histone macroH2A1.1 [1ZR3], ARTD8 macrodomains 1 and 2 [3VFQ], MacroD2 [4IQY]), and a putative phos- phatase (1SPV) and YmdB (5CB5), a O-acetyl-ADP-ribose deacetylase, from E. coli. Interestingly, lower structural homologies than for the above structures of human and bacterial macro domains are calculated by DALI for the viral macro domain structures from the coronavirus family (SARS-CoV [2FAV], Human-CoV NL63, [2VRI], Human-CoV 229E [3EJG], MERS-CoV [5DUS], feline-CoV [3ETI], Avian infectious bronchitis virus (IBV) strains Beaudette [3EKE] and M41 [3EWO]), with Z-scores in the range of 16.4 to 18.8 and r.m.s.d values of 1.8 to 2.3 Å for 136–143 pairwise aligned Cα positions. Thus structure-based phylogeny of macro domain does not cluster viral alphavirus and coronavirus together, raising questions on possible functional variations. A comparison of the overall structure of GETV macro domain with the structures of macro domains from other alphaviruses shows that the alphavirus macro domain fold is very well conserved. In particular, the Cα chains of macro domains from the three Old World alphaviruses GETV, CHIKV and SINV superimpose well, as illustrated by the low root mean square deviation (r.m.s.d.) values of approximately 0.7 Å. Only minor struc- tural changes are observed at the level of the β3β4 loop (Fig. 3B). The structural homology of the GETV macro domain (OW) with other alphavirus macro domains such as VEEV macro domain (NW) and the MAYV macro domain (OW) was slightly lower with r.m.s.d. values of 0.9 and 1.4 Å, respectively. Here the main structural Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ Figure 3. Overall structure of GETV macro domain. (A) Cartoon representation of GETV macro domain with APD-ribose depicted as seen in the GETV macro domain ADP-ribose complex “pose 1”. β-sheets are coloured in slate, α-helices in teal and loops in pink. Secondary structure elements and the N- and C-termini are labelled. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. Summary of crystallization procedures. PDB code Method/component Crystallization solution Conformation of ADP-ribose 6QZU Crystallization GETV macro domain 16 mg·mL−1 0.2 M imidazole/malate pH 6.0, 30% PEG 4 K 6R0F Co-crystallization GETV macro domain 13 mg·mL−1, 3 mM ADP-ribose Soacking 15 mM aspartic acid 0.2 M imidazole/malate pH 5.9, 34% PEG 4 K ADP-ribose with closed distal ribose, “pose 1” 6R0G Co-crystallization GETV macro domain 13 mg·mL−1, 3 mM ADP-ribose, 50 mM glutamic acid 0.2 M imidazole/malate pH 6.0, 30% PEG 4 K ADP-ribose with closed distal ribose, “pose 2” 6R0T Co-crystallization GETV macro domain 13 mg·mL−1, 3 mM ADP- ribose + 30 mM aspartic acid 0.2 M imidazole/malate pH 5.9, 34% PEG 4 K ADP-ribose with open distal ribose 6R0P Co-crystallization GETV macro domain 13 mg·mL−1, 3 mM ADP-ribose, 30 mM aspartic acid 0.2 M imidazole/malate pH 6.0, 32% PEG 4 K ADP-ribose with open distal ribose in double conforma- tion 6R0R Co-crystallization GETV macro domain 13 mg·mL−1, 3 mM ADP-ribose, 3 mM aspartic acid 0.2 M imidazole/malate pH 5.9, 38% PEG 4 K ADP-ribose with distal ribose covalently linked to Cys34 Table 2. Summary of crystallization procedures. Figure 4. Interaction network between GETV macro domain and ADP-ribose in “pose 1”. ADP-ribose and interacting GETV macro domain residues are depicted in sticks and colour-coded as in Fig. 3A. Figure 4. Interaction network between GETV macro domain and ADP-ribose in “pose 1”. ADP-ribose and interacting GETV macro domain residues are depicted in sticks and colour-coded as in Fig. 3A. of Arg144 (Figs. 4, 5A). The interaction of N-6 with aspartate residues structurally equivalent to Asp10 is well conserved in most macro domain/ADP-ribose complexes and has been shown to be crucial for ADP-ribose binding in the Archaeoglobus fulgidus macro domain33. At the other hand, Arg144 is not conserved, not even within the alphavirus macro domain family, and its side-chain is rather disordered in all the structures described in this work, indicating that the stacking interaction with the adenine ring makes probably only weak contribu- tions to the binding energy. The proximal ribose interacts with GETV macro domain through a single hydrogen bond between 3′-OH and the side-chain of Thr111. It is noteworthy that Trp148, conserved in alphavirus macro domains, protrudes into the ADP-ribose binding site and makes a steric clash with the 3′-OH. Results and discussion i d i GETV macro domain binds ADP-ribose in a deep cleft and the distal ribose ring adopts a pose as observed in most other macro domain ADP-ribose complexes, henceforth called “pose 1”. The adenine moiety binds via the N-1 atom to the main-chain of Ile11 and via the N-6 atom to the side-chain of Asp10, and makes stacking interactions at one side with the side-chains of Ile11 and Val33, and at the other side with the side-chain https://doi.org/10.1038/s41598-020-70870-w Scientific Reports \| (2020) 10:14422 \| www.nature.com/scientificreports/ (D) Complex with ADP-ribose in the single open conformation. (E) Complex with ADP-ribose covalently bound to Cys34. domain and substituted by Gly in other viral macro domains harbouring the glycine-rich motif 30G(D/E/G)GV33. The tight interactions established between the distal ribose and Ser30 point towards a role in substrate binding or catalysis for this residue. Not surprisingly, we found that the most important structural changes occurring in GETV macro domain upon ADP-ribose binding could be observed in the catalytic loop β2α1 and loop β5α3, which close-up over the ligand to establish tight binding interactions. Structure of the GETV macro domain in complex with ADP‑ribose presenting the distal ribose in an unusual conformation, “pose 2”. By adding 3 mM ADP-ribose and 50 mM glutamic acid to the crystallization medium of GETV macro domain we obtained a second crystal for a GETV macro domain/ ADP-ribose complex for which diffraction data extending to 1.7 Å resolution were collected. The structure was determined by difference Fourier synthesis and the final model has excellent stereochemistry. Thr160 in chain A could not be modelled in satisfactory fashion and in chain B electron density revealed the presence of Lys0, originating from the cloning strategy that includes an AAA triplet (Lys codon) to promote efficient transla- tion initiation34. In this second GETV macro domain/ADP-ribose structure the interactions between the GETV macro domain and the adenine moiety, the proximal ribose and the phosphate groups are virtually identically to the interactions described above. However, the distal ribose is tilted by approximately 60° with respect to the “classical” pose described above and observed in all other macro domain/ADP-ribose complexes described so far (Fig. 5B), with exception of the crystal structure of human histone macroH2A1.1 in complex with ADP-ribose in form B (3IIF)35. However, in this latter crystal structure the distal ribose is tilted in the opposite direction with respect to the distal ribose observed in the present GETV/ADP-ribose structure. In the novel and unusual pose 2 the distal ribose, here in the b-anomeric configuration, establishes hydrogen bonds with Ala22 and Asn24 main-chain atoms via 3″-OH, with the side-chains of Asn24 and Ser30 via 2″-OH and with the Ser30 side-chain and the Asp31 main-chain mediated by the 1″-OH hydroxyl group. The ribose ring lines up in perfectly parallel fashion with the side-chain of Phe114. www.nature.com/scientificreports/ In the other alphavirus macro domain structures, the side-chains of the equivalent Trp residues are shifted about 1.5 to 2 Å away from the ADP-ribose binding site with respect to the position of Trp148 in the GETV macro domain. Here, a Val residue pointing from the back towards the Trp148 indole ring, Val121, impedes an equivalent rearrange- ment of the Trp148 side-chain, which appears to be highly dynamic and could not be modelled in a satisfactory fashion in all the structures described in this work. Similarly to what had been observed in other macro domain/ADP-ribose complexes, the phosphate groups of ADP-ribose are lined by the catalytic loop β2α1 and loop β5α3 and tightly coordinated by hydrogen bonds with main-chain atoms of residues Val33, Ser110, Gly112, Thr113 and Phe114 and the side-chain of Thr113. Finally, the distal ribose, observed in the anomeric α-configuration, interacts with GETV macro domain through hydrogen bonds contracted between 3″-OH and the side-chain of Asn24, between 2″-OH and the side-chains of Asn24 and Ser30, and between 1″-OH and the side-chain Ser30 and the main-chain of Asp31, and establishes a stacking interaction with the side-chain of Phe114. The Ser30 of the catalytic β2α1 loop is unique to GETV macro Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ Figure 5. The conformational trajectory of ADP-ribose bound to GETV macro domain, triggered by the presence of aspartic or glutamic acid. The overall structure of GETV macro domain is shown in cartoon and interacting residues and ADP-ribose are depicted in sticks and colour-coded as in Fig. 3A. Hydrogen bonds are shown in dashed lines. (A) Complex with ADP-ribose in “pose 1”. (B) Complex with ADP-ribose in “pose 2”. (C) Complex with ADP-ribose in the open conformation (conformation A of complex with ADP-ribose in double open conformation). (D) Complex with ADP-ribose in the single open conformation. (E) Complex with ADP-ribose covalently bound to Cys34. Figure 5. The conformational trajectory of ADP-ribose bound to GETV macro domain, triggered by the presence of aspartic or glutamic acid. The overall structure of GETV macro domain is shown in cartoon and interacting residues and ADP-ribose are depicted in sticks and colour-coded as in Fig. 3A. Hydrogen bonds are shown in dashed lines. (A) Complex with ADP-ribose in “pose 1”. (B) Complex with ADP-ribose in “pose 2”. (C) Complex with ADP-ribose in the open conformation (conformation A of complex with ADP-ribose in double open conformation). www.nature.com/scientificreports/ However, due to the ring opening the 4′′-OH is now free to make hydrogen-bonding interactions with the side-chain of Thr113 and the main-chain of Asp31. The acetate molecule, as observed in the complex with ADP-ribose in pose 2, could not be spotted in this structure. The conformations of the distal ribose in conformation B and the one in the complex with open ADP-ribose in single conformation are virtually identical (Fig. 5D). Here the linear ribose chain underwent a ~ 100° rotation around the bond connecting carbon atoms 4″-C and 3″-C, and consequently the hydrogen-bonding pattern between ADP-ribose and the GETV macro domain changed drastically. In this new conformation the 4″-OH binds the side-chain of Thr113 and the main-chain of Asp31, 3″-OH interacts with the side-chains of Asn24 and Ser30, the 2″-OH hydroxyl group contacts the side-chains of Ser30 and Cys and 1″-OH is coordinated by the main-chain atoms of Ala22 and Cys34 and hydrogen bonds the α-phosphate. An acetate molecule, reminiscent of aspartic acid added to the crystallization solution, is isosteric to the one observed in the complex with the distal ribose in pose 2 and interacts with the 3″-OH hydroxyl of ADP-ribose and the side-chain of Ser30. Structure of the GETV macro domain/ADP‑ribose complex with the distal ribose covalently bound to Cys34. Finally, we obtained a last GETV macro domain/ADP-ribose complex by co-crystallizing the protein with 3 mM ADP-ribose and 3 mM aspartic acid and diffraction data were collected to 1.45 Å reso- lution. For no apparent reason the space group in this complex changed from P212121 (as observed in all the previously described structures) to C2 and only one molecule is present in the asymmetric unit. We verified cautiously whether the position of ADP-ribose in this novel complex could have influenced the crystal pack- ing and could not retrieve any plausible explanation for the change of space group. The structure was solved by molecular replacement, using the native GETV macro domain structure as a search model and the final model presents very good stereochemistry. The interactions between GETV macro domain and the adenine moiety, the proximal ribose and the phosphate groups of ADP-ribose are specular to the interactions observed in all the GETV macro domain/ADP-ribose complexes described so far. To our surprise, ADP-ribose in this structure is found in the open conformation and a covalent bond is established between 1″-C and Cys34 SG (Fig. 5E and Sup. Fig. www.nature.com/scientificreports/ Interestingly, residual difference electron density was observed close to the distal ribose in chain A and was modelled as an acetate molecule, assuming that it represented the ordered Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ part of glutamic acid added to the crystallization medium. This carboxylic moiety coordinates the 1″-OH and 2″-OH hydroxyl groups of ADP-ribose and is further stabilized by an H-bond interaction with Ser30. We dare to speculate that the position of the acetate molecule relative to the distal ribose ring is reminiscent of a putative ADP-ribose-glutamate conjugate, a possible substrate for GETV macro domain as inferred from the function of other alphavirus macro domains7,14,15. And yet again, Ser30 appears to play a crucial role in substrate binding. Structure of the GETV macro domain in complex with ADP‑ribose presenting the distal ribose in an open ring conformation. We further obtained two additional GETV macro domain/ADP-ribose complexes by co-crystallizing the protein in the presence of 3 mM ADP-ribose and 30 mM aspartic acid. Dif- fraction data were recorded to 1.85 and 1.6 Å, respectively. Again, in these two crystal structures the interactions between GETV macro domain and the adenine moiety, the proximal ribose and the phosphate groups are com- parable to those seen in the above-described models. Conversely, preliminary electron density maps, calculated before the incorporation of the ligand, clearly indicated that the distal ribose was present in the open conforma- tion (Supplementary Fig. S1). In the complex structure at resolution of 1.85 Å the distal ribose is present in a single conformation, whereas the best way to account for difference electron density in the 1.6 Å data set was to model the distal ADP-ribose in a double open conformation (Fig. 5C,D). Both structures were determined by difference Fourier synthesis and the final models present excellent stereochemistry. In both structures no clear electron density could be observed for Thr160 in chains A and the additional Lys0 is present in chains B. In one of the conformations of ADP-ribose in the double open conformation, conformation A (Fig. 5C), the carbon atoms of the distal ribose superpose with the carbon atoms of the distal ribose ring as seen in the complex with ADP-ribose in pose 2 and the hydrogen-bonding interactions are to a large extent comparable. www.nature.com/scientificreports/ S1). It is noteworthy that in all the above described GETV macro domain/ADP-ribose structures the residues of the catalytic loop β2α1 and of the loop β5α3 are isosteric. The only exception is represented by Cys34, which adopts a double conformation in the complexes with ADP-ribose in pose 2 and ADP-ribose in the open conformation, with one of the alternate conformations of Cys34 pointing towards the distal ribose (Fig. 5B–D). Conversely, in the structure of the ADP-ribose covalent adduct a movement of approximately 1 Å could be observed for residues Ser30-Val33 of the catalytic loop β2α1. This structural rearrangement of the catalytic loop is conceivably due to a positional shift of the distal ribose chain with respect to open ribose in the single open conformation, implying a rotation of the of about 100° around the 3″-C and 2″-C bond. In the covalent complex the hydrogen bonds between 4″-OH and the side-chain of Thr113 and the main-chain of Asp31, as well as the one between 3″-OH and the side-chain of Asn24 are still maintained. However the interaction between 3″-OH and the side-chain of Ser30 is lost, and therefore the interaction between ADP-ribose and GETV macro domain is loosened. Due to the rearrangement of the open ADP-ribose chain the 2″-OH hydroxyl group now interacts with the main-chain of Cys34 and the 1″-OH group coordinates a water molecule. www.nature.com/scientificreports/ converging on the central role of the catalytic β2α1 loop. Whereas acid-base catalysis occurring via an oxocar- benium intermediate and carried out by a glutamate residue has been proposed for Thermomonospora curvata PAR glycohydrolase PARG37, the human terminal ADP-ribose protein glycohydrolase TARG1 employs a lysine residue for nucleophilic attack at the C1′’ position of ribose attached to the acceptor protein, thereby forming a transient Schiff base intermediate further resolved by an aspartic acid38. Finally, a substrate-assisted mechanism for cleavage of the ADP-ribose/protein linkage had been proposed for human MacroD2 where the α-phosphate of the conformationally strained ADP-ribose activates an ideally positioned water molecule for nucleophilic attack on the C1′′ atom39. Seemingly the ubiquitous macro domains are built on a common globular scaffold prone to accommodate ADP-ribose. Still, subtle variations in loop structures elicit profound mechanistic diversity, which calls for further functional and structural dissection of this interesting class of molecules. g Intrigued by the substitution of a highly conserved glycine residue for serine and the presence of a Togavir- idae-specific cysteine in the catalytic loop of GETV macro domain, we set out for a comprehensive structural investigation deemed to unravel the functional role of these substitutions. Inspired by the structural study of the mono-ADP-ribosylhydrolase DraG in which an amino-acid/ADP-ribose intermediate was trapped32, we co- crystallized or soaked GETV macro domains with ADP-ribose and with increasing concentrations of aspartic or glutamic acid, which could mimic host protein ADP-ribosylated side-chains, thereby reversing the de-ADP- ribosylation reaction. In a first ADP-ribose complex, obtained from a crystal where GETV macro domain had been co-crystallized with ADP-ribose and subsequently soaked in a solution containing aspartic acid, ADP-ribose had been found in the binding pocket with the distal ribose in the low energy 2′′-endo twist conformation and in a position commonly observed in other macro domain/ADP-ribose complexes (pose 1). However, when ADP- ribose and glutamic acid were added to the co-crystallization solution, in the resulting crystal structure the distal ribose (pose 2) was tilted with respect to the one observed in the previously determined ADP-ribose complex and adopted the energetically less favourable O-4′′-endo envelop configuration. Though, this strained ribose conformation is stabilized by a tighter network of hydrogen bonds contracted with the GETV macro domain when compared to the pose 1 complex structure. www.nature.com/scientificreports/ Furthermore, in one of the two molecules in the asymmetric unit an acetate molecule, reminiscent of glutamic acid added to the crystallization solution, coordinates the 2′′-OH and 1′′-OH hydroxyl groups, evoking the position of the leaving group after the de-ADP-ribosylation reaction. Apparently, it was the crystallization method deployed, soaking versus co-crystallization with carboxylic acids, which induced the conformational change between pose 1 and pose 2. When GETV macro domain was co-crystallized with ADP-ribose and aspartic acid, the distal ribose was found in two different open conforma- tions, one of them closely resembling the closed-ring ribose of pose 2, the other resulting from a rotation around C4′′ and C3′′, which positioned the aldehyde function close to the α-phosphate, buried in a pocket surrounded by Cys34, Val33, Asn21 and Ala22. Open conformations of an integral ribose in the open conformation bound to macro domains have so far not been observed, but dehydrated open ADP-ribose adducts have been observed in the crystal structures of TARG138 and DraG32. Lastly, co-crystallization of GETV macro domain with low concentrations of ADP-ribose and aspartic acid (3 mM each) led to a structure where the distal ribose is cova- lently attached to Cys34. This covalent thio-hemiacetal adduct is reminiscent of the keto-amine transition states involving a lysine residue described for DraG32 and Targ138. It is interesting to notice that an aspartate, Asp184, proposed to be the catalytic acid/base catalyst in human MacroD140, is isosteric to Cys34, as well as Asp102 of human MacroD239 and Glu114 of T. curvata PARG41, two residues proposed to play crucial roles in catalysis. Noteworthily, DraG, Targ1, MacroD1 and MacroD2 are among the closest structural homologues of GETV macro domain. The above mentioned structural similarities advocate for Cys34 being the catalytic residue in the GETV macro domain, and maybe as well in macro domains of other alphaviruses. Cys-dependent hydrolases found in Nature are mainly represented by Cys-proteases, operating through a catalytic mechanism relying on a Cys-His-Asp catalytic triad. GETV macro domain exhibits no overall structural similarity with any known Cys-protease and the only His and Asp residues found in the vicinity of Cys34, namely His67 and Asp31, though pretty well conserved throughout alphavirus macro domains, are too far apart to form a functional catalytic triad. www.nature.com/scientificreports/ The role of the peculiar Ser30 substituting a glycine in the catalytic loop of the GETV macro domain could The role of the peculiar Ser30 substituting a glycine in the catalytic loop of the GETV macro domain could ultimately not be resolved by this study. The serine residue might contribute to the trapping of ADPr in different poses that were to date not observed in the other alphavirus macro domains having a glycine in position 30. Still, the tight interactions of this residue with the distal ribose observed in the different complex structures described herein plead for a function in substrate binding or eventually a direct role in catalysis in interplay with Cys34.if Altogether the five different conformations of ADP-ribose presented in this study could picture a confor- mational itinerary (Fig. 5) representing the second part of the de-ADP-ribosylation reaction, leading from the covalent reaction intermediate through different conformational arrangements of the open ring conformations to the final products, represented by pose 2, strained in an energetically unfavourable conformation in the presence of the leaving group, and finally collapsed into the energetically favoured 2′′-endo twist conformation (pose 1) as observed in most macro domain/ADP-ribose complexes documented so far. We cannot ascertain whether the covalent Cys34-ADP-ribose adduct portrays the true interaction intermediate, or whether it represents just an artefact standing for an abortive complex. In any case, the conservation in alphavirus macro domain sequences and the proximity of Cys34 to the catalytic centre argues for an important role of this residue in the catalytic mechanism. In this respect, mutational studies, which go beyond the purpose of this report, will be necessary to ascertain the true identity of the catalytic residue of the GETV macro domain. Discussion Macro domains, named after the non-histone domain of histone variant macroH2A1.1, in which a macro domain was originally characterized36, are found in all kingdoms of life and have been shown to bind ADP-ribose and metabolites thereof. Although all sharing a common structural scaffold, they have diverged throughout evolution and some members support divers enzymatic activities such as O-acetyl-ADP-ribose deacetylation, ADP-ribose 1′′-phosphate dephosphorylation, as well as hydrolytic removal of mono- and poly-ADP-ribose attached to proteins. Diversification of enzymatic activities and substrate specificity goes necessarily along with variations in catalytic scenarios and different catalytic mechanisms have been proposed for macro domains, all https://doi.org/10.1038/s41598-020-70870-w Scientific Reports \| (2020) 10:14422 \| www.nature.com/scientificreports/ References NMR study of non-structural proteins–part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV). Biomol. NMR Assign. 9, 191–195. https://doi.org/10.1007/s12104-014-9572-0 (2015). 13. Putics, A., Gorbalenya, A. E. & Ziebuhr, J. Identification of protease and ADP-ribose 1’’-monophosphatase activities associated g 12. Melekis, E. et al. NMR study of non-structural proteins–part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV). Biomol. NMR Assign. 9, 191–195. https://doi.org/10.1007/s12104-014-9572-0 (2015).i 13. Putics, A., Gorbalenya, A. E. & Ziebuhr, J. Identification of protease and ADP-ribose 1’’-monophosphatase activities assoc with transmissible gastroenteritis virus non-structural protein 3. J. Gen. Virol. 87, 651–656. https://doi.org/10.1099/vir.0.815 (2006). 13. Putics, A., Gorbalenya, A. E. & Ziebuhr, J. Identification of protease and ADP ribose 1 monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3. J. Gen. Virol. 87, 651–656. https://doi.org/10.1099/vir.0.81596-0 (2006). 14 M Ph R L t l ADP ib lh d l ti it f Chik i d i i iti l f i li ti d (2006). 14. McPherson, R. L. et al. ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and 4. McPherson, R. L. et al. ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc. Natl. Acad. Sci. USA 114, 1666–1671. https://doi.org/10.1073/pnas.1621485114 (2017).h p g p 15. Eckei, L. et al. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases. Sci. Rep. 7, 41746. https://doi.org/10.1038/srep41746 (2017). 5. Eckei, L. et al. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive t d RNA i f ti ADP ib lh d l S i R p 7 41746 htt //d i /10 1038/ 41746 (2017) y y p p g p 6. Fukunaga, Y., Kumanomido, T. & Kamada, M. Getah virus as an equine pathogen. Vet. Clin. N. Am. Equine Pract. 16, 605–617 (2000). ( ) 17. Berrow, N. S. et al. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Cry logr. D Biol. Crystallogr. 62, 1218–1226. https://doi.org/10.1107/S0907444906031337 (2006). l f l Th f h l ll l ll 8. Geerlof, A. et al. The impact of protein characterization in structural proteomics. Acta Crystallogr. D Biol. Crystallogr. 62, 1125– 1136. https://doi.org/10.1107/S0907444906030307 (2006).f p g 9. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. Conclusion In this study, we describe by means of crystallographic structures different poses adopted by a molecule of ADP-ribose in the binding site of GETV macro domain. In addition to the pose of ADP-ribose found in other structures of alphavirus macro domain, this work reveals original features such as the opening of the distal Scientific Reports \| (2020) 10:14422 \| https://doi.org/10.1038/s41598-020-70870-w www.nature.com/scientificreports/ ribose, and its stabilization by Ser30, representing a peculiar GETV specific substitution in the catalytic loop. In addition, we were able to identify a covalent link between ADP-ribose and a cysteine, Cys34, located in the catalytic loop, as well as several poses of ADP-ribose susceptible to provide clues about the catalytic mechanism. Since Cys34 is conserved in alphaviruses, this finding would deserve to be further explored by enzymatic assays and reverse genetics. ribose, and its stabilization by Ser30, representing a peculiar GETV specific substitution in the catalytic loop. In addition, we were able to identify a covalent link between ADP-ribose and a cysteine, Cys34, located in the catalytic loop, as well as several poses of ADP-ribose susceptible to provide clues about the catalytic mechanism. Since Cys34 is conserved in alphaviruses, this finding would deserve to be further explored by enzymatic assays and reverse genetics. Data availability Th d The atomic coordinates and structure factors for the structures of GETV macro domains and complexes thereof have been deposited in the Protein Data Bank with accession numbers 6QZU, 6R0F, 6R0G, 6R0P, 6R0R and 6R0T. All the other data are available from the corresponding authors on request. Received: 17 May 2020; Accepted: 29 July 2020 References 1. Gould, E. A. et al. Understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control. Antivir. Res. 87, 111–124. https://doi.org/10.1016/j.antiviral.2009.07.007 (2010). g j 2. Weaver, S. C. et al. 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The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature 477, 616–620. https://doi. org/10.1038/nature10404 (2011). Competing interests h p g The authors declare no competing interests. Acknowledgements g We thank the European Synchrotron Radiation Facility (ESRF) and Synchrotron Soleil for beamtime allocation, and the staff of beamlines Proxima2, ID23-1 and ID30A-3 for assistance with data collection. This work was supported by the European Union Horizon 2020 Marie Skłodowska-Curie ETN “ANTIVIRALS” (Grant Agree- ment Number 642434) and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10- INSB-05-01. We thank Dr Karine Barral, Dr Sandrine Py and Dr Nicolas Papageorgiou for helpful discussions. Author contributions G.S./B.Co. designed experiments, A.S.F.R./G.S. performed experiments, A.S.F.R./G.S./B.Ca./B.Co. analyzed data G.S./B.Co. wrote the manuscript, all the authors reviewed and agreed with the manuscript. 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Chen, D. et al. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-70870-w Correspondence and requests for materials should be addressed to G.S. or B.C. 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 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. 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https://openalex.org/W2409782737	https://www.nature.com/articles/srep19851.pdf	English	null	The Prognostic Value of Alpha-Fetoprotein Response for Advanced-Stage Hepatocellular Carcinoma Treated with Sorafenib Combined with Transarterial Chemoembolization	Scientific reports	2,016	cc-by	6,639	The Prognostic Value of Alpha- Fetoprotein Response for Advanced-Stage Hepatocellular Carcinoma Treated with Sorafenib Combined with Transarterial Chemoembolization received: 25 March 2015 accepted: 15 December 2015 Published: 02 February 2016 received: 25 March 2015 accepted: 15 December 2015 Published: 02 February 2016 Lei Liu1,, Yan Zhao1,, Jia Jia1,, Hui Chen1, Wei Bai1, Man Yang1, Zhanxin Yin1, Chuangye He1, Lei Zhang1, Wengang Guo1, Jing Niu1, Jie Yuan1, Hongwei Cai2, Jielai Xia2, Daiming Fan1,3 & Guohong Han1 This retrospective cohort study aimed to evaluate the prognostic value of the alpha-fetoprotein (AFP) response in advanced-stage hepatocellular carcinoma (HCC) patients treated with sorafenib combined with transarterial chemoembolization. From May 2008 to July 2012, 118 HCC patients with baseline AFP levels >20 ng/ml treated with combination therapy were enrolled. A receiver operating characteristic curve was used to generate a cutoff point for AFP changes for predicting survival. The AFP response was defined as an AFP decrease rate [ΔAFP(%)] greater than the cutoff point. The ΔAFP(%) was defined as the percentage of changes between the baseline and the nadir values within 2 months after therapy. The median follow-up time was 8.8 months (range 1.2–66.9). A level of 46% was chosen as the threshold value for ΔAFP (sensitivity = 53.7%, specificity = 83.3%). The median overall survival was significantly longer in the AFP response group than in the AFP non-response group (12.8 vs. 6.4 months, P = 0.001). Multivariate analysis showed that ECOG ≥ 1 (HR = 1.95; 95% CI 1.24–3.1, P = 0.004) and AFP nonresponse (HR = 1.71; 95% CI 1.15–2.55, P = 0.009) were associated with increased risk of death. In conclusion, AFP response could predict the survival of patients with advanced-stage HCC at an early time point after combination therapy. Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide with more than 800,000 newly diagnosed cases per year1. It is the second most common cause of cancer-related death in the world2. A large proportion of HCC patients are diagnosed at an intermediate or advanced stage beyond curative treatments. Based on the Barcelona Clinic Liver Cancer (BCLC) staging system, transarterial chemoembolization (TACE) and sorafenib are the standard treatments for intermediate and advanced-stage HCC, respectively3,4. Because Sorafenib may improve the efficacy of TACE therapy by decreasing post-TACE angiogenesis, sorafenib combined with TACE has been considered to be a promising therapy5–7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 25 March 2015 accepted: 15 December 2015 Published: 02 February 2016 Materials and Methods All HCC patients consecutively admitted to our department between May 2008 and June 2012 who were treated with a combination therapy of sorafenib and TACE were retrospectively considered in our study. The inclusion criteria were as follows: 1) an age ≥ 18 years old, 2) an interval between sorafenib and TACE of ≤ 60 days, 3) an Eastern Cooperative Oncology Group (ECOG) performance status score ≤ 2, 4) a Child-Pugh A or B (≤ 7), and 5) no other molecular target agents. The exclusion criteria were as follows: 1) main portal vein invasion,2) con- current malignancy,3) an absence of a repeat AFP measurement within 2 months after treatment initiation,4) a baseline AFP < 20 ng/ml, and 5) poor compliance. The diagnosis of HCC was based on the American Association for the Study of Liver Disease (AASLD) criteria16. Histology was needed only in case of diagnostic uncertainty. OS was measured from the beginning of combination therapy to the date of death or the last follow-up. The require- ment to obtain informed consent was waived. The study protocol was approved by the ethics committees of Xijing Hospital. All the methods used in this study were carried out according to the approved guidelines. Treatment and follow-up. The patients received sorafenib at an initial dose of 400 mg twice daily. Later, the dose of sorafenib was modified based on the degree of adverse events (AEs). AEs were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. In our clinical practice, patients continue sorafenib treatment if the AEs can be safely controlled. TACE was performed using 10–50 mg doxorubicin mixed with 5–20 mg lipiodol. Gelatin foam was injected until the tumor-feeding vessels were com- pletely obstructed. TACE procedures were repeated according to the radiological response5. Combined therapy was defined as an interval between sorafenib and TACE of less than 60 days, regardless of the order of the two treatments. Standard follow-up evaluations, including contrast-enhanced computer tomography (CT) scans and laboratory assessments, were performed during weeks 4 and 8 after the initiation of treatment and every 8 weeks thereafter. The end of the follow-up period was either death or December 31st 2014. AFP evaluation. The serum AFP concentration was measured at baseline (before the initiation of combined therapy) and at every follow-up visit using an electro chemiluminescence immunoassay (ElecsysCobas e601, Roche). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Currently, radiological imaging evaluation is widely used for the prognostic assessment of HCC. The Response Evaluation Criteria in Solid Tumors (RECIST) focuses on whole-tumor shrinkage8. The modified RECIST (mRE- CIST) criteria measure the change of the tumor necrotic area. However, radiological imaging evaluation has sev- eral limitations9. First, it is challenging to measure tumor size when the tumor grows in a diffuse pattern. Second, radiological imaging evaluation is a relatively subjective assessment and lacks inter-observer reproducibility10. Third, our previous studies showed that RECIST and mRECIST criteria fail to predict survival at an early time point11. Therefore, alternative methods to estimate treatment efficacy are needed. hfi Alpha-fetoprotein (AFP) is a glycoprotein that is secreted in approximately 70% of HCC12. As the most com- mon biomarker of HCC, AFP has confirmed its value in screening and diagnoses in multiple studies13. Recently, several studies unanimously suggested that the AFP response was associated with longer overall survival (OS) in HCC patients after locoregional treatment modalities or systematic chemotherapy14,15. However, the prognostic value of the AFP response in patients with advanced-stage HCC who are treated with sorafenib combined with TACE remains unclear.h The aim of this study was to evaluate the prognostic value of the AFP response in patients with advanced HCC who were undergoing treatment with sorafenib combined with TACE and to explore the correlation between the AFP response and a radiological evaluation from an early time point. The Prognostic Value of Alpha- Fetoprotein Response for Advanced-Stage Hepatocellular Carcinoma Treated with Sorafenib Combined with Transarterial Chemoembolization Although the preliminary results of the first rand- omized controlled (SPACE) trial were disappointing as the time to progression (TTP) failed to show a significant difference, we consider that the reason for the failure may lie in the study design. The other trials in this field are still underway. The answer regarding the superiority of combination therapy will be provided by the results of these trials in the future. 1Department of Liver Disease and Digestive Interventional Radiology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China. 2Department of Medical Statistics, Fourth Military Medical University, Xi’an, China. 3Xijing Hospital of Digestive Diseases & State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi’an, China. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.H. (email: guohhan@126.com) Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 1 Materials and Methods The AFP variation rate (Δ AFP) was defined as the percentage of change between the baseline and the nadir within 1–2 months after combination therapy. ( ) ∆ (%) =   − /  × % ( ) − AFP AFP AFP AFP 100 1 baseline post treatment baseline ( ) 1 The AFP response was defined as an Δ AFP(%) greater than the cutoff point (Δ AFP(%) > cutoff point), whereas AFP non-response was defined as an AFP decrease rate less than the AFP variation cutoff point (Δ AFP(%) < cutoff point). The researcher who extracted the AFP data was blinded to the survival outcome. Radiological evaluation and definitions. Radiological imaging assessments were performed with contrast-enhanced spiral computed tomography (CT) at baseline (before the initiation of combined therapy) and at every follow-up visit after combined therapy. The RECIST and mRECIST criteria were used for radiological evaluation. The treatment responses were blindly assessed by three experienced clinicians (Yan Zhao, JiaJia and Wei Bai). In cases of discrepancies, the images were jointly reviewed by all of the clinicians, and a consensus deci- sion was reached. If the patients were evaluated as having a complete response (CR) or a partial response (PR) within 2 months after combination therapy, these individuals were considered to be responders. If the patients were evaluated as having stable disease (SD) or progressive disease (PD), these individuals were considered to be non-responders17. Statistical analyses. Continuous variables were presented as median values with ranges, and categorical variables were presented as frequencies with percentages. A receiver operating characteristic (ROC) curve was used to generate a cutoff point for AFP changes that predicted survival. For the area under the curve, a cutoff point with the highest sum of sensitivity and specificity was chosen as the most discriminative value of the AFP response for predicting survival. This statistic may range from 0 to 1, and cutoff points with a c-statistic > 0.7 are generally considered useful18. A Mann-Whitney U test was used to compare continuous variables, whereas a Chi-squared test was used to compare categorical variables between the AFP response and non-response groups. Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 2 www.nature.com/scientificreports/ Figure 1. Enrollment and outcomes. Figure 1. Enrollment and outcomes. The Κ coefficient was used to measure the inter-method concordance of the radiological response and the AFP response. Materials and Methods OS time was assessed by Kaplan-Meier methods, and the survival difference between groups was estimated by the log-rank test. Patients lost to follow-up or alive at the end-of-observation date were censored. Univariate and multivariate Cox regression analyses were used to test the prognostic factors of OS. Variables with a P value < 0.1 in the univariate analysis were included in the multivariate analysis. Statistical analyses were performed using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). A two-sided P value < 0.05 was considered to be statistically significant. Results P i Patient characteristics and treatment. A total of 118 patients with unresectable HCC were included in our study (Fig. 1). The median age was 48 years (range, 23–75 years). Most patients were male (86.4%) and had hepatitis B virus infection (89%), Child-Pugh class A (90.7%), an ECOG performance status of 1–2 (74.6%), and BCLC stage C (83.9%). In total, 47 (66.9%) patients had branch portal vein tumor thrombosis (PVTT). Extrahepatic spread was observed in 45 (38.1%) patients, mainly in the abdominal lymph nodes (46.5%), the lungs (39.5%) and skeleton (16.3%) (Table 1). The median number of sessions of TACE was 2 (range, 1–12), the median time taking sorafenib was 6.6 months (range 0.3–66.9 months) and the median interval between sorafenib and TACE was 3 days (range 0–55 days). The interval was < 7 days for 108 patients (91.5%), was < 15 days for 8 patients and 15–55 days for 2 cases. The median baseline AFP level was 1821.5 ng/ml (range 20.7– 121000 ng/ml), 25 (21.2%) patients had < 200 ng/ml and 93 (78.8%) patients had ≥ 200 ng/ml. Survival analysis. The median follow-up time was 8.8 months (range, 1.2–66.9). By the end of follow-up, 111 out of 118 patients (94.1%) died and 7 (5.9%) survived. The overall median survival was 8.7 months (95% CI, 6.5–10.9) (Fig. 2A). The median OS was 11.3 months (95% CI, 6.9–15.8) in the patients with PVTT and 8.7 months (95% CI, 5.1–8.9) in the patients without PVTT (P = 0.011) (Fig. 2B). The median OS of the patients with ECOG 0 was longer than that of patients with ECOG ≥ 1 (13.7 months vs. 7.6 months, P = 0.002) (Fig. 2C). The difference in OS between patients with extrahepatic metastasis and those without metastasis was not significant (10.4 months vs. 7 months, P = 0.1) (Fig. 2D). A comparison between AFP response and non-response groups. The median time from the base- line treatment to AFP follow-up was 1.4 months (range 0.4–2.0). The area under the ROC curve (c-statistic) for predicting survival was 0.716 (Fig. 3). The most discriminative value of the Δ AFP(%) for predicting survival was 46%. This cutoff point had a sensitivity of 53.7% and a specificity of 83.3%. In this study, 49 (41.5%) patients with Δ AFP(%) > 46% were classified into the AFP response group and 69 (58.5%) patients with Δ AFP(%) < 46% were classified into the non-response group. Results P i Most baseline clinical char- acteristics were similar between the AFP response and non-response groups, but the proportion of males was higher in the AFP response group than in the non-response group (Table 1). The median OS was significantly longer in the AFP response group (12.8 months, 95% CI 10.2–15.3) than in AFP non-response group (6.4 months, 95% CI4.7–8.1) (P = 0.001) (Fig. 4A). Multivariate analysis showed that ECOG ≥ 1 (HR = 1.95; 95% CI 1.24–3.1, P = 0.004) and AFP nonresponse (HR = 1.71; 95% CI 1.15–2.55, P = 0.009) were associated with increased risk of death (Table 2). A comparison between AFP response and non-response groups. The median time from the base- line treatment to AFP follow-up was 1.4 months (range 0.4–2.0). The area under the ROC curve (c-statistic) for predicting survival was 0.716 (Fig. 3). The most discriminative value of the Δ AFP(%) for predicting survival was 46%. This cutoff point had a sensitivity of 53.7% and a specificity of 83.3%. hf p y pi y In this study, 49 (41.5%) patients with Δ AFP(%) > 46% were classified into the AFP response group and 69 (58.5%) patients with Δ AFP(%) < 46% were classified into the non-response group. Most baseline clinical char- acteristics were similar between the AFP response and non-response groups, but the proportion of males was higher in the AFP response group than in the non-response group (Table 1). The median OS was significantly longer in the AFP response group (12.8 months, 95% CI 10.2–15.3) than in AFP non-response group (6.4 months, 95% CI4.7–8.1) (P = 0.001) (Fig. 4A). Multivariate analysis showed that ECOG ≥ 1 (HR = 1.95; 95% CI 1.24–3.1, P = 0.004) and AFP nonresponse (HR = 1.71; 95% CI 1.15–2.55, P = 0.009) were associated with increased risk of death (Table 2). The correlation between AFP response and radiological evaluation. Of the 118 patients, 84 (71.2%) were properly evaluated according to both RECIST and mRECIST criteria. Survival was of insufficient time to carry out contrast-enhanced CT scans in 1 patient, 3 patients did not have a complete imaging exami- nation due to clinical deterioration, 10 patients had non-measurable diffused tumor lesions in the liver, and 20 Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Variables All patients (n = 118) AFP response (n = 49) AFP non-response (n = 69) P value Age (y) Median (Range) 48 (23–75) 49 (30–74) 47 (23–75) 0.564 Sex Male/Femal - No. (%) 102 (86.4%)/16 (13.6) 38 (77.6%)/11 (22.4%) 64 (92.8%)/5 (7.2%) 0.017 Etiology HBV/HCV/Other - No. (%) 105 (89%)/2 (1.7%)/11 (9.3%) 41 (83.7%)/2 (4.1%)/6 (12.2%) 64 (92.8%)/0 (0%)/5 (7.2%) 0.146 Child-Pugh class A/B - No. (%) 107 (90.7%)/11 (9.3%) 44 (89.8%)/5 (10.2%) 63 (91.3%)/6 (8.7%) 1 ECOG 0/1–2 - No. (%) 30 (25.4%)/88 (74.6%) 16 (32.7%)/33 (67.3%) 14 (20.3%)/55(79.7%) 0.139 BCLC stage B/C - No. (%) 19 (16.1%)/99 (83.9%) 11 (24.5%)/38 (75.5%) 8 (13%)/61 (87%) 0.135 Disease burden PVTT No/yes - No. (%) 71 (33.1%)/47 (66.9%) 33 (67.3%)/16 (32.7%) 38 (55.1%)/31 (44.9%) 0.189 Extrahepatic spread No/Yes - No. (%) 73 (61.9%)/45 (38.1%) 33 (67.3%)/16 (32.7%) 40 (58%)/29 (42%) 0.256 Baseline tumor size (cm) Median (Range) 10.8 (2.9–25.3) 10.5 (3.5–22.1) 11.7 (2.9–25.3) 0.325 No. of HCC nodules 1/≥ 2/Diffused - No. (%) 86 (72.9%)/22 (18.6%)/10 (8.5%) 39 (79.6%)/8 (16.3%)/2 (4.1%) 47 (68.1%)/14 (20.3%)/8 (11.6%) 0.264 Baseline AFP (ng/ml) <200/≥ 200 - No. (%) 25 (21.2%)/93 (78.8%) 12 (24.5%)/37 (75.5%) 13 (18.8%)/56 (81.2%) 0.459 Liver biopsy Yes/No - No. (%) 19 (16.1%)/99 (83.9%) 9 (18.4%)/40 (81.6%) 10 (14.5%)/59 (85.5%) 0.573 Ascites Yes/No - No. (%) 22 (18.6%)/96 (81.4%) 10 (20.4%)/39 (79.6%) 12 (17.4%)/57 (82.6%) 0.678 Laboratory values, mean (range) Alanine aminotransferase (U/I) 57.7 (10–395) 62.9 (10–395) 54.1 (13–236) 0.355 Aspartate aminotransferase (U/I) 77.6 (16–489) 74.1 (16–362) 80 (16–489) 0.639 Total bilirubin, mg/dl 17.9 (6.9–54) 17.5 (7.1–362) 18.2 (6.9–38.4) 0.698 Platelets/mm3 163 (31–511) 150.4 (47–362) 173 (31–511) 0.166 International normalized ratio 1.1 (0.73–1.58) 1.1 (0.91–1.4) 1.1 (0.73–1.58) 0.682 Albumin, g/dl 39.5 (29.4–75.6) 40.3 (32–75.6) 38.9 (29.4–52.9) 0.201 Order of treatments Sorafenib before TACE/TACE before sorafenib/Meanwhile - No. (%) 52 (44.1%)/62 (52.5%)/4 (3.4%) 20 (40.8%)/28 (57.1%)/1 (2.1%) 32 (46.4%)/34 (49.3%)/3 (4.3%) 0.610 Interval between TACE and sorafenib (d) Median (Range) 3 (0–55) 2 (0–40) 3 (0–55) 0.073 Table 1. Baseline demographics and clinical characteristics. HBV, hepatitis B virus; HCV, hepatitis C virus; ECOG, Eastern Cooperative Oncology Group; BCLC, Barcelona Clinc Liver Cancer; PVTT, portal vein tumor thrombosis; HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. Table 1. Baseline demographics and clinical characteristics. www.nature.com/scientificreports/ HBV, hepatitis B virus; HCV, hepatitis C virus; COG, Eastern Cooperative Oncology Group; BCLC, Barcelona Clinc Liver Cancer; PVTT, portal vein tumor hrombosis; HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. patients did not have completely preserved follow-up image data. The median time for assessing radiological imaging response was 1.2 months (range, 0.7–2.0 months). The rates of CR, PR, SD and PD were 0, 7 (8.3%), 66 (78.6%) and 11 (13.1%), respectively, according to the RECIST criteria, and 24 (28.6%), 23 (27.4%), 30 (35.7%) and 7 (8.3%), respectively, according to the mRECIST criteria. The response rates (CR and PR) and nonresponse rate (SD and PD) were 8.3% and 91.7% according to the RECIST criteria and 56% and 44% according to the mRE- CIST criteria, respectively. With RECIST criteria, the median survival value of response group was not obtained because too few patients (n = 7) were classified into this group and 4 patients were censored. However, there was no difference between the response and nonresponse groups (P = 0.132) (Fig. 4B). With mRECIST criteria, the survival difference was not statistically significant between the response and nonresponse groups [14.8 months (95% CI 10.9–18.7) vs. 10.3 months (95% CI 6.8–13.8), P = 0.075] (Fig. 4C). Multivariate analysis showed that both the RECIST (HR = 2.2; 95% CI 0.9–5.6, P = 0.094) and mRECIST (HR = 2; 95% CI 0.9–2.2, P = 0.160) criteria were not independent predictors of overall survival. The outcomes of both the radiological assessment and AFP response are shown in Table 3. The patient evaluation in every response category was markedly different between the RECIST criteria and the AFP response (Κ = 0.077), whereas the majority of patients were classified into the same response categories when assessed using the mRECIST criteria and the AFP response. However, the agreement was still weak between the mRECIST criteria and the AFP response (Κ = 0.383). Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 4 www.nature.com/scientificreports/ Figure 2. The Kaplan-Meier analysis of overall survival. (A) Overall survival. (B) A comparison of survival according to portal vein thrombosis. (C) A comparison of survival according to the ECOG score. (D) A comparison of survival times according to extrahepatic metastasis. Figure 2. The Kaplan-Meier analysis of overall survival. (A) Overall survival. (B) A comparison of survival according to portal vein thrombosis. (C) A comparison of survival according to the ECOG score. www.nature.com/scientificreports/ (D) A comparison of survival times according to extrahepatic metastasis. Of the 34 patients without radiological evaluation, 8 and 26 patients were in the AFP response and AFP non-response groups, respectively. The median OS was significantly longer in the AFP response group than in the AFP non-response group (11.3 months vs. 3.9 months, P= 0.002) (Fig. 4D). Figure 3. The ROC curve for AFP values and survival. Figure 3. The ROC curve for AFP values and survival. Of the 34 patients without radiological evaluation, 8 and 26 patients were in the AFP response and AFP non-response groups, respectively. The median OS was significantly longer in the AFP response group than in the AFP non-response group (11.3 months vs. 3.9 months, P = 0.002) (Fig. 4D). Of the 34 patients without radiological evaluation, 8 and 26 patients were in the AFP response and AFP non-response groups, respectively. The median OS was significantly longer in the AFP response group than in the AFP non-response group (11.3 months vs. 3.9 months, P = 0.002) (Fig. 4D). Discussion Because AFP assessment is a simple and reproducible method to for the evaluation of the efficacy of combination treatment, our study demonstrates the feasibility of using the dynamic trend of AFP as an early biomarker for predicting survival outcomes after combination therapy in advanced HCC patients. Discussion Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 5 www.nature.com/scientificreports/ Figure 4. The Kaplan-Meier analysis of overall survival. (A) A comparison between the AFP response and nonresponse groups in the entire cohort; (B) A comparison between RECIST response and nonresponse groups in 84 patients with radiological response; (C) A comparison between mRECIST response and nonresponse groups in 84 patients with radiological response; (D) A comparison between the AFP response and nonresponse groups in 34 patients without radiological evaluation. Figure 4. The Kaplan-Meier analysis of overall survival. (A) A comparison between the AFP response and nonresponse groups in the entire cohort; (B) A comparison between RECIST response and nonresponse groups in 84 patients with radiological response; (C) A comparison between mRECIST response and nonresponse groups in 84 patients with radiological response; (D) A comparison between the AFP response and nonresponse groups in 34 patients without radiological evaluation. AFP is a well-established tumor marker for screening and diagnosing HCC, and the AFP level appears to be associated with the prognosis of HCC patients19. Previous studies demonstrated that an elevated AFP level would decrease in HCC patients after hepatic resection and would rebound in cases of HCC recurrence20. Recently, the AFP response has been reported to be a significant prognostic factor in HCC patients treated with differ- ent locoregional modalities or systemic chemotherapy14,15,21. To our knowledge, the current analysis is the first exploration of the potential prognostic value of the AFP response in HCC patients treated with sorafenib com- bined with TACE. And our study population was mainly consisted of advanced stage HCC patients, which was different from previous report. The major findings of this study were as follows: 1) the adaptive AFP variation cutoff point to predict prognosis was a 46% reduction, 2) the AFP response (a decline of more than 46% from baseline within 2 months after the initiation of combination therapy) was associated with longer OS in patients with advanced-stage HCC who were treated with sorafenib in combination with TACE, and 3) the AFP response could predict the overall survival at an earlier time point compared to radiological assessment, particularly in circumstances in which radiological evaluation could not be performed.i g p In previous studies, the AFP response was defined as an AFP level that decreased by more than 20%, 30% or 50%14,22,23. Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 Discussion HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; PVTT, portal vein tumor thrombosis; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. To avoid effect of colinearity with variables, BCLC and ascites were not included in the model. Table 2. Univariate and multivariate analysis for overall survival. HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; PVTT, portal vein tumor thrombosis; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. To avoid effect of colinearity with variables, BCLC and ascites were not included in the model. Table 2. Univariate and multivariate analysis for overall survival. HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; PVTT, portal vein tumor thrombosis; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. To avoid effect of colinearity with variables, BCLC and ascites were not included in the model. Radiological evaluation AFP Response (n = 41) Non-response (n = 43) RECIST (n = 84) CR 0 (0%) 0 (0%) PR 5 (12.2%) 2 (4.7%) SD 34 (82.9%) 32 (74.4%) PD 2 (4.9%) 9 (20.9%) Response (CR + PR) 5 (12.2%) 2 (4.7%) Non-response (SD + PD) 36 (87.8%) 41 (95.3%) mRECIST (n = 84) CR 17 (41.5%) 7 (16.3%) PR 14 (34.1%) 9 (20.9%) SD 9 (22%) 21 (48.8%) PD 1 (2.4%) 6 (14%) Response (CR + PR) 31 (75.6%) 16 (37.2%) Non-response (SD + PD) 10 (24.4%) 27 (62.8%) Table 3. The correlation between the radiological evaluation and AFP assessment. AFP, alpha-fetoprotein; RECIST, Response Evaluation Criteria in Solid Tumor; mRECIST, Modified Response Evaluation Criteria in Solid Tumor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Radiological evaluation AFP Response (n = 41) Non-response (n = 43) RECIST (n = 84) CR 0 (0%) 0 (0%) PR 5 (12.2%) 2 (4.7%) SD 34 (82.9%) 32 (74.4%) PD 2 (4.9%) 9 (20.9%) Response (CR + PR) 5 (12.2%) 2 (4.7%) Non-response (SD + PD) 36 (87.8%) 41 (95.3%) mRECIST (n = 84) CR 17 (41.5%) 7 (16.3%) PR 14 (34.1%) 9 (20.9%) SD 9 (22%) 21 (48.8%) PD 1 (2.4%) 6 (14%) Response (CR + PR) 31 (75.6%) 16 (37.2%) Non-response (SD + PD) 10 (24.4%) 27 (62.8%) Table 3. The correlation between the radiological evaluation and AFP assessment. AFP, alpha-fetoprotein; RECIST, Response Evaluation Criteria in Solid Tumor; mRECIST, Modified Response Evaluation Criteria in Solid Tumor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Discussion However, the definition of the AFP response mostly originated from personal clinical experiences or speculation but not from statistical analyses. In contrast, we used a ROC curve to generate an adaptive AFP variation cutoff point (an AFP reduction of 46%) for the AFP response. More importantly, by using this cutoff point the AFP response group had significantly longer survival than the AFP nonresponse group, and it was an independent predictor for overall survival. Thus, the AFP level could be incorporated into the algorithm for assessing the prognosis of HCC patients. Additionally, it should be noted that patient selection in previous stud- ies was different from ours. In previous studies, patients with baseline AFP < 100 ng/ml or < 200 ng/ml were excluded to differentiate from other benign liver diseases14,21. Thus, the conclusions of these studies were suitable only for patients with a relatively high baseline AFP level. In contrast, our inclusion criteria were relatively wider, only patients with a baseline AFP < 20 ng/ml were excluded from our study because not all HCC patients have an elevated AFP level. Radiological evaluations, such as those based on RECIST and mRECIST criteria responses, have been wi used in the prognostic assessment of HCC17,24. Radiological response has also been established to correlate w Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 6 www.nature.com/scientificreports/ Variable Univariate analysis Multivariate analysis HR 95% CI P value HR 95% CI P value Age (y) 1.003 0.986–1.020 0.720 – – – Sex Female vs. male 0.920 0.541–1.564 0.758 – – – Etiology Hepatitis infection vs. Other 0.749 0.398–1.410 0.370 – – – Child-Pugh class B vs. A 0.943 0.490–1.814 0.861 – – – ECOG 2 vs. 0–1 2.208 1.294–3.180 0.002 1.952 1.239–3.076 0.004 PVTT Yes vs. No 1.658 1.124–2.447 0.011 1.398 0.934–2.093 0.103 Extrahepatic spread Yes vs. No 1.376 0.938–2.017 0.103 1.214 0.825–1.787 0.324 No. of HCC nodules ≥ 2 + Diffused vs. 1 1.072 0.735–1.565 0.718 Baseline AFP (ng/ml) ≥ 200 vs 200 1 0.669–1.494 0.999 – – – AFP change Nonresponse vs. response 1.863 1.268–2.738 0.002 1.710 1.147–2.551 0.009 Table 2. Univariate and multivariate analysis for overall survival. HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; PVTT, portal vein tumor thrombosis; AFP, alpha-fetoprotein; TACE, transarterial chemoembolization. To avoid effect of colinearity with variables, BCLC and ascites were not included in the model. Table 2. Univariate and multivariate analysis for overall survival. Discussion Table 3. The correlation between the radiological evaluation and AFP assessment. AFP, alpha-fetoprotein; RECIST, Response Evaluation Criteria in Solid Tumor; mRECIST, Modified Response Evaluation Criteria in Solid Tumor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Table 3. The correlation between the radiological evaluation and AFP assessment. AFP, alpha-fetoprotein; RECIST, Response Evaluation Criteria in Solid Tumor; mRECIST, Modified Response Evaluation Criteria in Solid Tumor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. the pathological response25,26. However, in the current study, both the RECIST and mRECIST assessment within 2 months after treatment were not independent predictors of overall survival. Additionally, the agreement between radiological assessment and the AFP response was weak regardless of whether the RECIST or mRECIST criteria were used, though a majority of patients were classified into the same response categories when assessed using the mRECIST criteria and the AFP response. These results were consisted with our previous study that showed that the earliest time to evaluate the response to combination therapy was 3 months11. This result could be explained by the reality that the baseline tumor burden in Chinese patients is higher than those reported in western coun- tries. Only one TACE session may not be efficient enough to achieve complete tumor response. Moreover, the study by Georgiades et al. showed that initial nonresponders after the first TACE session could obtain prolonged survival from further treatment27. Therefore, under these circumstances, radiological assessment could not be used as an early predictor of overall survival. Additionally, our study demonstrated that the AFP response could predict the prognosis of these patients in the absence of a radiological evaluation, especially in patients with diffuse malignant tumors that could not be evaluated by radiological criteria. Hypovascular or diffusely infil- trative tumor patterns are often present in real-world clinical settings22. Establishing a correlation between AFP Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 7 www.nature.com/scientificreports/ and treatment efficacy has the potential to help assess treatment response in clinical practice when the standard imaging findings are equivocal. Another potential advantage of the AFP assessment would be reducing the cost burden of repeat radiological scans. and treatment efficacy has the potential to help assess treatment response in clinical practice when the standard imaging findings are equivocal. Another potential advantage of the AFP assessment would be reducing the cost burden of repeat radiological scans. References A., McIntire, K. R. & Klatskin, G. alpha-fetoprotein in noneoplastic hepatic disorders. JAMA 233, 38–41 (1975). 29. Chen, C. H. et al. Hepatitis B- and C-related hepatocellular carcinomas yield different clinical features and prognosis. Eur J Cancer 42, 2524–2529 (2006). 29. Chen, C. H. et al. Hepatitis B- and C-related hepatocellular carcinomas yield different clinical features and prognosis. Eur J Cancer 42, 2524–2529 (2006). Discussion p g Several limitations of this study should be recognized. First, this was a retrospective study with a relatively small number of patients. A potential bias may exist because not all the patients had follow-up AFP assessments within 2 months after treatment and consequently only the patients with complete follow-up information were included in the analysis. Further well-design prospective studies with large sample sizes are needed to confirm the prognostic value of the AFP response. Second, the serum AFP concentration might be influenced by hepati- tis, cirrhosis and liver cell necrosis. Not all HCC patients have a significantly elevated AFP level at baseline, and patients with viral hepatitis and other benign liver diseases incidentally do have an elevated AFP level28,29. An AFP reduction might also be induced not only by treatment for HCC but also by antiviral or anti-fibrosis therapy. Unfortunately, the related data were lacking because we did not collect follow-up information about these types of therapies. Third, the 46% cut-off point was based on this study cohort that mainly consisted of advanced stage HCC patients. Its application in patients with intermediate stage HCC requires further validation. p pp p g q In conclusion, our study suggested that the AFP response could predict overall survival in advanced-stage HCC patients at an early time point after the treatment of sorafenib combined with TACE. Further prospective studies are necessary to validate the prognostic effect of a decline of 46% as an accurate AFP variation cutoff point. References 3. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol 56, 908–943 (2012). 4. Llovet, J. M. et al. Sorafenib in advanced hepatocellular carcinoma. 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J Hepatol 56 Suppl 1, S75–87 (2012). 2 Global battle against cancer won’t be won with treatment alone effective prevention measures urgently needed to prevent cance Author Contributions Conceived and designed the study: Y.Z., J.J. and G.H.; collection and analysis of data: L.L., Y.Z., J.J., H.C., W.B., M.Y., J.X. and H.C.; manuscript writing: L.L., Y.Z., J.J., H.C., W.B., M.Y., Z.Y., C.H., L.Z., W.G., J.N., J.Y., H.C. and J.X.; revision of the manuscript: Y.Z., G.H. and D.F.; L.L., Y.Z., J.J., H.C., W.B., M.Y., Z.Y., C.H., L.Z., W.G., J.N., J.Y., H.C., J.X., D.F. and G.H.H. approved the final manuscript submitted. Conceived and designed the study: Y.Z., J.J. and G.H.; collection and analysis of data: L.L., Y.Z., J.J., H.C., W.B., M.Y., J.X. and H.C.; manuscript writing: L.L., Y.Z., J.J., H.C., W.B., M.Y., Z.Y., C.H., L.Z., W.G., J.N., J.Y., H.C. and J.X.; revision of the manuscript: Y.Z., G.H. and D.F.; L.L., Y.Z., J.J., H.C., W.B., M.Y., Z.Y., C.H., L.Z., W.G., J.N., J.Y., H.C., J.X., D.F. and G.H.H. approved the final manuscript submitted. Acknowledgementsh Acknowledgements This study was supported by grants from the National Natural Science Foundation of China 81172145 and 81420108020. g This study was supported by grants from the National Natural Science Foundation of China 81172145 and 81420108020. 8 Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 www.nature.com/scientificreports/ Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Liu, L. et al. The Prognostic Value of Alpha-Fetoprotein Response for Advanced-Stage Hepatocellular Carcinoma Treated with Sorafenib Combined with Transarterial Chemoembolization. Sci. Rep. 6, 19851; doi: 10.1038/srep19851 (2016). How to cite this article: Liu, L. et al. The Prognostic Value of Alpha-Fetoprotein Response for Advanced-Stage Hepatocellular Carcinoma Treated with Sorafenib Combined with Transarterial Chemoembolization. Sci. Rep. 6, 19851; doi: 10.1038/srep19851 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:19851 \| DOI: 10.1038/srep19851 9
https://openalex.org/W2996768969	http://tp.revistas.csic.es/index.php/tp/article/download/815/838, https://diposit.ub.edu/dspace/bitstream/2445/148427/1/693592.pdf	es		Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona) casi 100 años después	Trabajos de prehistoria	2,019	cc-by	6,025	TRABAJOS DE PREHISTORIA 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona) casi 100 años después* New archaeological excavations at Cova Gran and Cova Freda de Montserrat (Collbató, Barcelona) after almost 100 years F. Xavier Omsa, Juan I. Moralesa, Artur Cebriàa, Josep Mestresb y Josep M. Fullolaa RESUMEN En este trabajo se presentan los primeros datos estratigráficos, radiocarbónicos y de cultura material de las recientes excavaciones en las Coves de Montserrat (Cova Gran y Cova Freda). Estos emblemáticos yacimientos fueron excavados el año 1922 y proporcionaron uno de los primeros conjuntos cardiales de la península ibérica. Desde entonces, no volvieron a ser intervenidos de forma oficial y quedaron abandonados. En el año 2018 se han reiniciado los trabajos con sondeos en ambas cuevas, determinando la preservación de depósitos arqueológicos estratificados en algunos de ellos. En la Cova Freda se ha reconocido la existencia de un tramo de la cavidad probablemente usado como redil durante el Neolítico Antiguo Cardial y, en la Cova Gran, se han documentado un nivel y una estructura negativa de esa misma cronología, así como un potente nivel finipaleolítico. ABSTRACT This work presents the first stratigraphic, chronological and artifactual results of the recent archaeological excavations in the Coves de Montserrat (Cova Gran and Cova Freda). These emblematic sites were excavated in 1922 and provided one of the first Cardial Ware assemblages of the Iberian Peninsula. Authorized excavations were resumed in 2018 with test pits in both caves. These showed the preservation of some stratified archaeological deposits. In Cova Freda part of the cavity probably used to stall livestock during the Early Neolithic. In Cova Gran an Early Neolithic layer and pit are preserved, as well as a layer dating to the Epipalaeolithic. Palabras clave: Cova Gran; Cova Freda; NE de la península ibérica; Neolítico Antiguo; Epipaleolítico; Dataciones C14. Key words: Cova Gran; Cova Freda; NE Iberia; Early Neolithic; Epipalaeolithic; Radiocarbon dates. 1. INTRODUCCIÓN La Cova Gran y la Cova Freda se sitúan en la vertiente sur del macizo de Montserrat, en la localidad de Collbató (Baix Llobregat, Barcelona), a escasos 30 km de Barcelona. Sus coordenadas UTM ETRS89 31N son Cova Gran= X 402502, Y 4603198; Cova Freda= X 402640, Y 46030110 (Fig. 1A). Ambas están casi a la misma altitud, 500 y 502 m s. n. m. respectivamente, abiertas en el contexto general del macizo: conglomerados con interfases de lutitas. Forma la Cova Gran una única galería de unos 35 m de largo E-NE con una boca de unos 7 m de alto por 8 m de ancho. Gradualmente va descendiendo hasta hacerse impenetrable. Al final de la cavidad, a la derecha, existe un * Las intervenciones arqueológicas en la Cova Gran y la Cova Freda se integran dentro del proyecto “Transicions culturals durant el Plistocè superior i l’Holocè al litoral-prelitoral central de Catalunya” (CLT009/18/00024, 2018-2021 de la OSIC de la Generalitat de Catalunya), en el marco del grupo de investigación SERP de la Universitat de Barcelona. Sus proyectos SGR2017-00011 y HAR2017- 86509 han sufragado las dataciones de este artículo. J. I. M goza de un contrato postdoctoral Juan de la Cierva-Incorporación (IJCI-2017-31445) del Ministerio de Ciencia, Innovación y Universidades. Agradecemos al ayuntamiento de Collbató y al Patronat de la Muntanya de Montserrat su apoyo económico y en infraestructuras. a SERP, Seminari d’Estudis i Recerques Prehistòriques. Secció de Prehistòria i Arqueologia. Universitat de Barcelona. C/ Montalegre 6-8, 08001 Barcelona. Correos e.: FXO oms@ub.edu https://orcid.org/0000-0002-1642-548X; JIM jimorales@ub.edu https://orcid.org/0000-0002-8253-414X; AC arturcebria@gmail.com https://orcid.org/0000-0002-5674-4135; JMF fullola@ub.edu https://orcid.org/0000-0001-7089-1425 b VINSEUM, Museu de les Cultures del Vi. Plaça de Jaume I 1, 08720 Vilafranca del Penedès (Barcelona). Correo-e.: josepmestres@hotmail.com https://orcid.org/0000-0002-7839-7082 Recibido 13-II-2019; aceptado 24-IV-2019. Copyright: © 2019 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License 09-Prehistoria VOL76 N2-2019.indd 335 29/11/19 10:38 336 F. Xavier Oms, Juan I. Morales, Artur Cebrià, Josep Mestres y Josep M. Fullola Fig. 1. A. Localización de las “Coves de Montserrat” (Collbató, Barcelona) en la península ibérica y en el macizo de Montserrat; B. vista desde el interior de la Cova Gran (Colomines 1925: lám. VIII) y vista de la boca de la Cova Freda (Colomines 1925: lám. XXVII) (autoría SERP, Universitat de Barcelona) (A. en color en la versión electrónica). divertículo en forma de corta galería que parece vaciada recientemente. La Cova Freda tiene una estructura más compleja. La cavidad mide 232 m de longitud con un desnivel máximo de 16 m y una orientación aproximada N-S, con una pequeña apertura original que fue cerrada con una puerta hace unas décadas. Esta puerta da paso a una sala de pequeñas dimensiones (Sala I) de unos 3,5 m de largo por 12 m de ancho, la única que recibe parcialmente luz del exterior. Un estrecho corredor da acceso a la Sala II de creciente inclinación hacia el interior. Mide unos 10 m de largo por 6 m de ancho. Está afectada por importantes procesos gravitacionales y cuenta con diferentes espacios laterales (entre ellos una galería de unos 20 m de largo, en sentido N) y coladas. A través de una rampa se accede a la Sala III (en algunas ocasiones, se ha dividido esta sala en dos), de dimensiones mayores a la anterior y con una importante presencia de grandes bloques desprendidos. Uno de ellos ha provocado que algunas topografías hayan dividido esta sala en dos. Ascendiendo por uno de los bloques se accede a la Sala IV, la última, también con una gran cantidad de bloques y formaciones estalagmíticas descalcificadas. La Cova Gran y la Cova Freda fueron uno de los primeros conjuntos excavados en el Mediterráneo occidental que proporcionaron cerámicas cardiales (Colomines 1925). Al desconocerse, por entonces, paralelos se denominaron cerámicas “Montserratines”. Sin embargo, la metodología utilizada en su excavación no permitió situarlas a nivel estratigráfico. Por ese motivo fueron datadas al final de la Cultura de las Cuevas, en el Eneolítico. Hasta ese momento, en el NE peninsular, solamente habían sido publicadas cerámicas similares en la Cova de Can Pascual (Castellví de la Marca) (Bosch Gimpera 1923), datadas en el mismo periodo. Poco después, el año 1936 se publicaron los resultados de la excavación de 1928 en la Esquerda de les Roques del Pany (Torrelles de Foix). Allí, Martí Grivé (1936) detectó una cierta estratigrafía: primero un estrato con cerámica “argárica” (por las carenas, Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 336 29/11/19 10:38 Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona)… que ahora situaríamos en la Edad del Bronce), seguido por un nivel campaniforme con inhumaciones colectivas y, en la base, un nivel con un mínimo de 12 inhumaciones asociadas a cerámica “montserratina” y numerosa industria ósea (p. ej. 4 cucharas entre enteras y fragmentadas) y restos malacológicos. La Cova Bonica de Vallirana fue excavada en junio de 1936, justo antes del inicio de la Guerra Civil. Los resultados de esa campaña no fueron publicados. Por lo que desconocemos si Josep de Calasanç Serra Ràfols intuyó también alguna secuencia en esa cavidad (Baldellou 1974). La publicación de las excavaciones de la Grotta de l’Arene Candide de Finale-Ligure (Italia) y de la Cova de la Sarsa de Bocairent fueron determinantes para empezar a delimitar la cronología y ámbito de la cerámica cardial. El yacimiento ligur localizaba las cerámicas impresas en un nivel subyacente a otros con cerámicas VBQ (Vasi a Bocca Quadrata), al mismo tiempo que se dotaba al concepto “Cardial” de una presencia pan-mediterránea (Martí 2008). Gradualmente, en el NE de la península ibérica, se fueron identificando nuevos enclaves con presencia de cerámica cardial, aunque las anotaciones estratigráficas continuaban siendo prácticamente nulas: Cova de Can Montmany en Pallejà (Colomines 1947), Cova del Bolet en Mediona (Giró 1947), Cova de la Font Major en l’Espluga de Francolí (Vilaseca 1969) y Roc d’en Sardinyà en Vilassar de Dalt (Baldellou 1972) entre otros. Finalmente, a inicios de los 1980, se empiezan a publicar las primeras estratigrafías con cerámica cardial en el NE peninsular. Destacan las de la Cova del Toll de Moià (Guilaine et al. 1981), la Cova del Frare de Matadepera (Martín et al. 1985) y de Les Guixeres de Vilobí de Sant Martí Sarroca (Baldellou y Mestres 1981; Mestres 1981-1982), casi al mismo tiempo que las primeras definidas para territorios vecinos, en la Cova de l’Or de Beniarrés (Martí 1977) y en la Cueva de Chaves (Baldellou y Castán 1983). Durante buena parte del siglo XX, las “Coves de Montserrat” fueron un referente por ser uno de los primeros lugares donde se documentó una ocupación cardial y por su numerosísima colección arqueológica (especialmente cerámica). Sin embargo, gradualmente fueron perdiendo peso en las publicaciones por su falta de estratigrafía conocida y de información útil desde una perspectiva actual. A inicios de la década de los 1980, Josep Mestres, Josep Tarrús y Ramon Ten analizaron y dibujaron todo el material neolítico de Cova Gran y Cova Freda, aunque esos resultados quedaron prácticamente inéditos (Mestres et al. 1983a, Mestres et al. 1983b; Mestres 1989). Recientemente, una parte han sido incluidos en tesis doctorales sobre industria lítica (Palomo 2012) y ce- 337 rámica (Oms 2014) y en un trabajo de final de máster inédito 1. 2. LAS EXCAVACIONES DE 1922 Un acuerdo entre los monjes de la Abadía de Montserrat y los miembros de la Secció HistòricoArqueològica del Institut d’Estudis Catalans (a partir de ahora IEC) organizó la “exploración” de las cuevas ya conocidas de la vertiente sur de la montaña, cerca de Collbató. Dirigió la intervención Josep Colomines, que había ingresado en el IEC, como técnico conservador, el año 1915. Los trabajos, discontinuos, se hicieron entre abril y octubre de 1922. El año 1925 se publicó la memoria de las excavaciones (Colomines 1925) en Cova Freda y Cova Gran y de otras más modestas: Cova del Salnitre, Coves de Santa Cecília y el sepulcro de fosa de El Bruc. La información sobre las excavaciones y la documentación gráfica era realmente escasa (Fig. 1B y 1C) en la citada monografía, dedicada en su mayor al análisis de los materiales. En referencia a Cova Gran, se afirma que buena parte de ellos apareció en una única capa superficial de unos 25 cm de potencia de sedimento oscuro y carbonoso y, al mismo tiempo, que parte del conjunto afloraba al voltear los grandes bloques existentes en la mayor parte de la cueva. Sobre la Cova Freda se proporcionaron más detalles: en la Sala I se documentaron sedimentos revueltos y muy pobres, con un grosor máximo de 40 cm. La Sala II era, según Colomines, la más intacta al conservar en algunos puntos una potencia de 2,5 m. Detalla que un primer nivel estaba bastante revuelto, aunque se documentaron dos inhumaciones (boca arriba) cubiertas de cerámica ibérica. En un nivel inferior, con un sedimento más oscuro y de aspecto grumoso, apareció mucho material revuelto de diferentes fases. En la Sala III, bajo los bloques, continuaba el segundo nivel de la sala anterior, esta vez con una potencia de 1,5 m. En la Sala IV (en realidad una extensión de la Sala III) sólo documentó materiales en una covacha ya que el resto de la sala no contenía sedimentos. En la Sala V (de hecho, la IV) solamente se citan cerámicas informes en superficie. 3. LAS NUEVAS CAMPAÑAS El mes de junio de 2018 se llevaron a cabo las primeras excavaciones reguladas en las Coves de 1 Hernández, C. Propuesta de valorización de la colección arqueològica de la Cova Freda y la Cova Gran de Montserrat (Collbató-Baix Llobregat). Trabajo de final de máster inédito, Universitat Autònoma de Barcelona. Barcelona, 66 pp. Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 337 29/11/19 10:38 338 F. Xavier Oms, Juan I. Morales, Artur Cebrià, Josep Mestres y Josep M. Fullola Montserrat desde la intervención del IEC del año 1922. Las campañas consistieron en 9 días en Cova Freda (Fig. 2) y otros 9 en Cova Gran (Fig. 3), bajo la dirección de los firmantes de este trabajo. Su objetivo era comprobar el estado de conservación de ambos conjuntos, puesto que las cuevas han sido frecuentadas por excursionistas y aficionados. Por ello, presentan un deficiente estado de conservación y ha sido necesario retirar una gran cantidad de sedimentos revueltos con restos prehistóricos y subactuales. La nomenclatura utilizada para denominar los niveles en ambos yacimientos fue diferente. En Cova Freda se utilizó la numeración romana con letras para los subniveles. En Cova Gran se recurrió a un sistema de unidades estratigráficas para determinar los paquetes sedimentarios holocenos distribuidos de forma dis- continua, siendo UE100 el removido general y, a partir de UE110, los restos de estratigrafía y estructuras neolíticas. Cuando la sedimentación tenía aspecto pleistoceno, se pasó a un criterio de unidades litoestratigráficas (siendo el nivel 200 la primera). 3.1. Cova Freda Dadas las aparentemente malas condiciones de conservación de este yacimiento se realizaron sondeos de pequeñas dimensiones para comprobar la integridad estratigráfica de los diferentes sectores de la cavidad. Los trabajos arqueológicos se han centrado en las Salas I y II (Fig. 2A). En la primera sala se han realizado dos sondeos de 1 m2 que han resultado fértiles, Fig. 2. Cova Freda (Collbató, Barcelona): A. planta general de la cavidad y de detalle de la Sala I, donde se localizan los diferentes cuadros excavados; B. vista de los trabajos en la Sala I; C. vista parcial de la estratigrafía (sección Este) del sondeo en el cuadro G8 (autoría SERP, Universitat de Barcelona) (en color en la versión electrónica). Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 338 29/11/19 10:38 Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona)… mientras que los dos sondeos de 2 m2 realizados en la Sala II carecían de estratigrafía pero contaban con numerosos restos arqueológicos. En la Sala I, el sondeo del cuadro J6 tiene una potencia total de 45 cm, estando la mayoría de los estratos revueltos. Sin embargo, en la base, en un pequeño tramo (lado SE del cuadro) con un sedimento oscuro y en parte arcilloso, aparecieron materiales cerámicos informes en posición primaria. En el futuro, la ampliación de este sondeo permitirá caracterizar de manera más completa el tipo de ocupación y su cronología, así como los niveles más antiguos. El sondeo realizado en el cuadro G8 de esta sala (Fig. 2B) proporcionó interesantes resultados a nivel estratigráfico y funcional. Después de un nivel revuelto superficial (Sup), se documentó un nivel (I) in situ de coloración oscura, parcialmente arcilloso de 5-15 cm de potencia con numerosos restos faunísticos y cerámicos. Entre ellos había tres fragmentos peinados de tradición postcardial Molinot (Fig. 4: G8-I-4, 22, 339 23). Por debajo de este nivel se sucedían otros dos (IIa y IIb), separados por uno estéril (techo IIb), compuestos por laminaciones de cenizas y carbones que se extendían con un desarrollo lateral desigual por todo el cuadro. La potencia total de los dos niveles oscilaba entre los 10 y los 18 cm (Fig. 2C). A la espera de los resultados de las muestras de micromorfología, su aspecto, composición y textura nos sugiere su interpretación preliminar como dos episodios diferenciados de estabulación de rebaños. Estos paquetes destacan por la abundancia de restos faunísticos. En cambio, la cerámica es escasa, informe y muy fragmentada. El nivel basal, IIb, se apoya sobre una capa de arcillas plásticas naranjas (nivel III), de la que se excavaron unos 10 cm de potencia, qué resultaron estériles. Todo el sedimento de los niveles IIa y IIb fue recuperado y flotado. Los numerosos restos carpológicos obtenidos están actualmente en estudio. Fig. 3. Cova Gran (Collbató, Barcelona): A. planta general de la cavidad con los sectores intervenidos en 2018: B. vista de los trabajos en los cuadros E-F13 y F11 en nivel UE200; C. distribución del material de los niveles UE110 y UE111; D. distribución del material del nivel UE200 (autoría SERP, Universitat de Barcelona) (en color en la versión electrónica). Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 339 29/11/19 10:38 340 F. Xavier Oms, Juan I. Morales, Artur Cebrià, Josep Mestres y Josep M. Fullola Fig. 4. “Coves de Montserrat” (Collbató, Barcelona): cerámica e industria lítica de la Cova Gran (tramo superior) y cerámica de la Cova Freda (tramo inferior). Campaña de 2018 (autoría SERP, Universitat de Barcelona) (en color en la versión electrónica). Los sondeos de la Sala II, sin secuencia arqueológica documentada, constataron la importante alteración del espacio por todo tipo de actuaciones furtivas. Los dos sondeos proporcionaron una amplia colección de materiales situables entre el Neolítico antiguo y la época ibérica, así como numerosos restos de fauna, restos humanos, etc. 3.2. Cova Gran Los tres sondeos (dos de 1 m2) se localizaron en los tramos inicial (E-F/10-13), medio (F23) y final (F33) de la cavidad, donde no había grandes bloques. En los que se realizaron más al interior, los niveles están revueltos. En ellos se han recuperado numerosos restos arqueológicos, entre los que destaca la cerámica cardial, la industria lítica, principalmente fragmentos de láminas y numerosos restos humanos de pequeño tamaño que no habían sido documentados en las excavaciones de 1922. El tercer sondeo, situado en el vestíbulo, se inició con 4 m2 en forma de trinchera longitudinal ampliándose hasta los 7 m2 a medida que la aparición de grandes bloques de conglomerado exigía ampliar la superficie. En este sondeo, tras retirar una gran cantidad de sedimentos revueltos, se detectó una amplia estratigrafía que no ha sido agotada. En primer lugar, un nivel (UE100), revuelto, Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 340 29/11/19 10:38 Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona)… con espesor variable dependiendo del tramo, desde los casi 10 cm en los cuadros 11 y 12 a los casi 80 cm en los cuadros 14. En este último caso, asociamos la potencia con la actuación de un grupo espeleológico que realizó en esta zona un sondeo que dejó indicado en la publicación de la planimetría del yacimiento (www.espeleoindex.com). Restos de otro nivel se adosaban a la pared de la cueva. Estaba recortado por las antiguas intervenciones y protegido parcialmente por un bloque de conglomerado (UE110) (Fig. 3C). Su matriz estaba compuesta por cenizas y gravas y un componente limitado de arcillas de una coloración grisácea. Tenía una potencia cercana a los 30 cm y una extensión menor a medio metro cuadrado. Este nivel contenía escasos restos, pero significativos (Fig. 4: E12-110-1,11,12): además de fauna (principalmente restos de ovicáprido), se hallaron un borde y un fragmento informe, ambos decorados mediante impresiones cardiales así como otro borde con un acabado raspado muy profundo. Esta decoración o acabado, aunque poco habitual, ya había sido detectado en los materiales antiguos (Mestres et al. 1983b: 41). En tercer lugar, a la altura de la cata realizada por el grupo espeleológico en el cuadro E13, se documentó una estructura negativa (UE111) (Fig. 3C) en parte destruida de la que se puedo excavar la base de unos 7 cm de potencia. Tenía una matriz oscura formada principalmente por cenizas y microcarbones. La sección de la otra mitad de la estructura (todavía no excavada de unos 30-35 cm de potencia) se conserva en buenas condiciones en el cuadro G13. En el interior de esta estructura, además de fauna y cerámica informe de igual aspecto, acabados y pastas que las de la UE110, apareció como elemento más diagnóstico un segmento de círculo con retoque a doble bisel (Fig. 3 4: F13-111-14). Todo el sedimento de las UE110 y UE111 fue recogido y posteriormente flotado, proporcionando numerosos restos carpológicos, sobre todo la UE111, actualmente en estudio. Por debajo tanto de la UE110 como de la UE111 se documentó de forma continua en toda la extensión del sondeo la presencia de un nivel rojizo formado a partir de la disgregación de los conglomerados de la cueva (nivel 200) (Fig. 4B y 4D), formado por arenas y cantos centimétricos y decimétricos. En la zona más cercana a la boca de la cueva (cuadros F10, E-F11) aparece directamente bajo una pequeña capa de sedimento superficial de unos 10-15 cm de espesor. A medida que fue seguido hacia el norte, la potencia del nivel superficial aumentaba significativamente, así como la pendiente en este mismo sentido de todo el paquete. Pese a la extensión abierta, la mayor parte de la superficie presenta grandes bloques de conglomerado que han limitado en esta primera campaña la superficie excavable del nivel 200. De cara a tener una 341 primera aproximación a su potencial, la excavación se limitó casi en exclusiva a una pequeña banqueta de unos 40 cm de superficie en los cuadros E-F13, en la que se excavaron unos 30 cm de potencia y que corresponde al corte del sondeo realizado por los espeleólogos. El material recuperado del nivel 200 es relativamente abundante, cerca de 200 coordenados, destacando la industria lítica, los restos de fauna y los carbones. En el conjunto lítico se constata la talla microlaminar, tanto por la presencia de núcleos (Fig. 4: 200-E1311,16) como de laminitas, retocadas y sin retocar, y restos de talla relacionados con el mantenimiento de los frentes de explotación. Entre el material retocado señalamos las muescas y denticulados (Fig. 4: 200E13-21,22; F11-3; F13-24), por un lado, y varias laminitas de dorso con retoque abrupto de módulo muy pequeño (Fig. 4: 200-E13-63,78; F11-8; F13-19), una en forma de segmento de círculo alargado. Una identificación preliminar de la fauna recuperada muestra restos de lepóridos con patrones de fracturación antrópica, así como de cérvidos y bóvidos pequeños, posiblemente cabra. 4. Las dataciones De esta primera campaña de 2018 hemos obtenido 5 dataciones radiocarbónicas 2, cuatro de Cova Gran y una de Cova Freda (Tab. 1). En Cova Gran ha sido posible datar todos los niveles excavados y en la segunda hemos priorizado fechar el nivel IIb, el más antiguo del cuadro G8, ante la falta de materiales diagnósticos. El resto de niveles de Cova Freda se datarán conforme avancen las excavaciones y se profundice en sus dinámicas de formación y su representatividad cultural. De la Cova Gran se han realizado cuatro dataciones: una de la UE110 sobre una semilla carbonizada de Triticum c/f dicoccum (0,01 g) recuperada de la flotación de sedimento entre cotas de -340-355; una de la UE111 sobre una semilla carbonizada de Triticum aestivum/durum (0,013 g), recuperada de la flotación de sedimento entre cotas de -410-415; y dos del nivel 200, sobre carbón de Juniperus sp. coordenados individualmente a cotas -410 y -411. De la UE110 se obtuvo un resultado de 6360 ± 30 BP (Beta-508798), 5467-5234 cal BC. De la UE111 una datación de 6380 ± 30 BP (Beta-508799), 5468-5310 cal BC. Mientras que las dos fechas del nivel 200 proporcionaron fechas muy similares pertenecientes a los últi2 Según las convenciones habituales, las calibraciones de dataciones neolíticas se presentan mediante “cal BC”, y las paleolíticas mediante “cal BP”. Los datos completos se encuentran en la tabla 1. Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 341 29/11/19 10:38 342 F. Xavier Oms, Juan I. Morales, Artur Cebrià, Josep Mestres y Josep M. Fullola Yacimiento Nivel Muestra Referencia D13 BP SD Cal BC 2σ Cal BP 2σ Cova Gran Cova Gran 110 Triticum c/f dicocum Beta-508798 -21.9 6360 30 5467-5234 7416-7247 111 Triticum aestivum/durum Beta-508799 -21.5 6380 30 5468-5310 7417-7259 Cova Gran 200 Carbón Juniperus sp. Beta-505684 -23.3 10230 40 10166-9825 12033-11847 Cova Gran 200 Carbón Juniperus sp. Beta-505685 -20.9 10160 40 10082-9681 11942-11742 Cova Freda IIb Triticum cf. aestivum/durum Beta-508800 -22.6 6410 30 5469-5327 7418-7276 Tab. 1. Dataciones radiocarbónicas de la Cova Gran y la Cova Freda de Montserrat, calibradas mediante el software OxCal v. 4.3.2, con la curva IntCal’13 (Reimer et al. 2013). Los restos han sido identificados por Ethel Allué (carbones, IPHES, Tarragona) y por Ferran Antolín (semillas, UBasel, Suiza). mos momentos del Pleistoceno, 10160 ± 40 y 10230 ± 40 BP (Beta-505684 y Beta-505685), 11842 ± 100 y 11940 ± 93 cal BP. Por último, de la Cova Freda se dató el nivel IIb a partir de una semilla carbonizada de Triticum cf. aestivum/durum (0,01 g) recuperada de la flotación de sedimento entre cotas de -220-225. Proporcionó una fecha de 6410 ± 30 BP (Beta-508800), 5469-5327 cal BC. Las dos fechas sobre carbón del nivel 200 de Cova Gran sitúan las ocupaciones documentadas dentro del Paleolítico superior final. Su total coherencia interna está confirmada por los test estadísticos T y χ² (Test T= 1.5315, χ²=3.84 a 1 grado de libertad), simplificando sus valores a una datación de 10195 ± 28 BP, 12060-11740 cal BP. Las dataciones neolíticas de Cova Gran también son homogéneas entre sí y pertenecen a una misma fase comprendida entre 6370 ± 21 BP, 5454-5307 cal BC (Test T=0.222, χ²=3.84 a 1 grado de libertad). A su vez, la datación del nivel IIb de Cova Freda es coherente con las dos anteriores (Test T=1.407407, χ²=5.99 a 2 grados de libertad), mostrando un episodio común del Neolítico Cardial en ambas cuevas entre 5465-5316 cal BC (6383 ± 17 BP). Estas tres dataciones se insertan perfectamente dentro del Neolítico Antiguo Cardial, en concreto en la primera mitad -Fase 1- de ese periodo según las últimas síntesis (Oms 2017). 5. DISCUSIÓN Y CONCLUSIONES La primera campaña en Cova Gran y Cova Freda ha permitido una primera aproximación a su funcionalidad a pesar de la escasa extensión excavada. Las ocupaciones del Paleolítico superior final de Cova Gran supone la identificación por primera vez de secuencia paleolítica en las cuevas montserratinas. Además, son uno de los escasos ejemplos documentados en el tramo prelitoral central del NE de la península ibérica, junto con la capa 21 de la Cova de Can Sadurní y el nivel III de la Cova de la Guineu (Fullola et al. 2011; Morales et al. 2013). A la espera de que el avance de las excavaciones proporcione conjuntos más significativos que permitan profundizar en el carácter de las ocupaciones y los procesos de formación, es interesante constatar que el nivel 200 se sitúa cronológicamente en los momentos finales del GS-1 o Dryas reciente. Esta fase cuenta con numerosos datos radiocarbónicos en el NE peninsular, sin embargo, los contextos no están siempre bien definidos (Aura et al. 2011). La fase del Neolítico antiguo en Cova Gran ha sido, por el momento, definida a través de un pequeño tramo de un nivel relicto y una estructura negativa de tipo fosa-silo. Las condiciones favorables de habitabilidad de esta cueva sugieren su uso como un asentamiento puntual (del que ahora mismo no es posible determinar la intensidad) y donde, además, se llevaron a cabo prácticas de almacenaje. Por otro lado, la sala I de la Cova Freda parece responder a un lugar de estabulación de rebaños durante el Neolítico Cardial, una función secundaria lógica dadas sus escasas condiciones de habitabilidad (alta humedad, baja temperatura y la propia morfología de la cueva). Otras opciones que deberemos explorar en el futuro son la función sepulcral multifase y la obtención de agua (de sus incontables coladas) entre otras. Las dos cuevas de Montserrat, durante el Neolítico Cardial, se integran en el conjunto de yacimientos definido como “Grupo del Llobregat” (Bernabéu 1999), recientemente reinterpretado como un complejo entramado de zonas con idiosincrasias propias y diferenciadas en cada uno de los territorios colindantes (Vallès, Penedès, Garraf-Ordal y Pla de Barcelona) (Oms 2017). Es una de las zonas donde más se ha intervenido en los últimos años y muestra un complejo mosaico de ocupación. Se conocen poblados principales al aire libre como Sant Pau del Camp (Molist et al. 2008; Gómez y Molist 2017) y Les Guixeres (Mestres y Esteve 2016; Oms et al. 2014); cuevas con fases de ocupación, inhumación y estabulación como la Cova de Can Sadurní y la Cova Bonica (Edo et al. 2011; Oms et al. 2017; Daura et al. 2019), de hábitat esporádico como la Cova del Frare y la Cova de la Guineu (Martín 2000; Oms et al. 2016), así como numerosos Trab. Prehist., 76, N.º 2, julio-diciembre 2019, pp. 335-344, ISSN: 0082-5638 https://doi.org/10.3989/tp.2019.12241 09-Prehistoria VOL76 N2-2019.indd 342 29/11/19 10:38 Nuevas intervenciones en la Cova Gran y la Cova Freda de Montserrat (Collbató, Barcelona)… campos de silos, como La Serreta, Mas d’en Boixos (Mestres y Esteve 2016), Pla de la Bruguera o Can Filuà entre otros. Por tanto, las “Coves de Montserrat” vienen a sumar información sobre las primeras fases de la neolitización en una de las regiones que dispone de más datos del Mediterráneo occidental. El proyecto iniciado durante el año 2018 en la Cova Gran y la Cova Freda se va a extender los próximos cuatro años. Ante su fuerte degradación durante los últimos 100 años, el objetivo principal era determinar la existencia y conservación de estratigrafía en ambas cuevas y la posibilidad de que las secuencias, previa extracción de potentes revueltos, pudiesen ser amplias. 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https://openalex.org/W4210622611	https://digitalcommons.wustl.edu/context/oa_4/article/2592/viewcontent/Microbiota_regulation_Trends_in_Microbiology.pdf	English	null	Microbiota regulation of viral infections through interferon signaling	Trends in microbiology	2,022	cc-by	12,500	Open Access Publications Open Access Publications Open Access Publications The triangular relationship between the host, the gut microbiota, and viral pathogens Coevolution of the intestinal microbiota (see Glossary) and the host innate immune system has resulted in a delicate balance of reciprocal interactions to maintain homeostasis in the gut. The microbiota plays a fundamental role in the induction, training, and maintenance of the host immune system [1]. At the same time, the immune system has evolved to constrain and maintain a symbi- otic relationship with these microbes [2]. A critical role for the gut microbiota in shaping host immu- nity becomes readily apparent in the context of eukaryotic viral infections. On the one hand, the gut microbiota can directly interact with virus particles and inﬂuence their infectivity. Numerous studies have described the mechanisms by which the gut microbiota is able to promote viral replication and pathogenesis (reviewed in [3,4]). On the other hand, the gut microbiota can prime and activate host antiviral immunity [5]. Together, the gut microbiota, host immunity, and viral patho- gens interact in a complex triangular relationship to determine infection and disease outcomes. The gut microbiota–IFN–virus axis holds therapeutic promise for antiviral therapies and viral vaccines in humans, but extrapolation to humans remains to be demonstrated. 1Department of Global Health – Amsterdam Institute for Global Health and Development (AIGHD), Amsterdam University Medical Center, Academic Medical Center, Amsterdam, The Netherlands 2Division of Infectious Diseases and Center for Experimental and Molecular Medicine (CEMM), Department of Medicine, Amsterdam University Medical Center, Academic Medical Center, Amsterdam, The Netherlands 3Division of Infectious Diseases, Department of Medicine, Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St Louis, MO, USA 4Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA 5All the authors wrote and edited the manuscript One recurring mechanism by which the gut microbiota has been found to inﬂuence antiviral control is via modulation of the IFN response. IFNs are a class of cytokines, secreted by host cells upon viral infection, that have a potent antiviral activity [6,7]. IFNs have dual actions: ﬁrst, they induce an immediate antiviral state in infected and neighboring cells, and second, they link the innate and adaptive immunity, mainly through priming of dendritic cells (DCs) [7,8]. Evidence to date indicates that the gut microbiota can either promote or suppress IFN signaling, depending on the speciﬁc virus and setting [9–19]. Highlights The interferon (IFN) response is the major early innate immune response against invading viral pathogens and is even capable of mediating sterilizing antiviral immunity without the support of the adaptive immune system. Cumulative evi- dence suggests that the gut microbiota can modulate IFN responses, indirectly determining virological outcomes. This review outlines our current knowledge of the interactions between the gut microbiota and IFN responses and dissects the different mechanisms by which the gut microbiota may alter IFN expression to diverse viral infections. This knowledge offers a basis for translating experimental evidence from animal studies into the human context and identiﬁes avenues for leveraging the gut microbiota–IFN–virus axis to improve control of viral infections and performance of viral vaccines. The gut microbiota modulates antiviral type I and III IFN responses, ultimately regulating viral infection outcomes. The gut microbiota is capable of modu- lating the IFN antiviral response both in the gastrointestinal tract and at extrain- testinal sites. The gut microbiota controls homeostatic IFN tone in the infection-naïve state, preparing the host for timely activation of antiviral responses upon infection. Both commensal bacteria-derived ligands and metabolites signal to different cell types to regulate IFN sig- naling pathways and thereby indirectly control viral infection. Microbiota regulation of viral infections through interferon Microbiota regulation of viral infections through interferon signaling signaling Nurul I. Wirusanti Megan T. Baldridge Vanessa C Harris Follow this and additional works at: https://digitalcommons.wustl.edu/oa_4 Part of the Medicine and Health Sciences Commons Please let us know how this document benefits you. Trends in Microbiology OPEN ACCESS 778 Trends in Microbiology, August 2022, Vol. 30, No. 8 https://doi.org/10.1016/j.tim.2022.01.007 © 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 778 Trends in Microbiology, August 2022, Vol. 30, No. 8 https://doi.org/10.1016/j.tim.2022.01.007 © 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY lic 1Department of Global Health – Amsterdam Institute for Global Health and Development (AIGHD), Amsterdam University Medical Center, Academic Medical Center, Amsterdam, The Netherlands 2Division of Infectious Diseases and Center for Experimental and Molecular Medicine (CEMM), Department of Medicine, Amsterdam University Medical Center, Academic Medical Center, Amsterdam, The Netherlands 3Division of Infectious Diseases, Department of Medicine, Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St Louis, MO, USA 4Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA 5All the authors wrote and edited the manuscript 1Department of Global Health – Amsterdam Institute for Global Health and Development (AIGHD), Amsterdam University Medical Center, Academic Medical Center, Amsterdam, The Netherlands Review Nurul I. Wirusanti,1,2,5 Megan T. Baldridge,3,4,5 and Vanessa C. Harris1,2,5,* Nurul I. Wirusanti,1,2,5 Megan T. Baldridge,3,4,5 and Vanessa C. Harris1,2,5,* Correspondence: v.c.harris@amsterdamumc.nl (V.C. Harris). Brief overview of IFN responses The IFN cytokine family contains three distinct types of IFN – types I, II, and III – with antiviral activity associated mainly with type I IFN and type III IFN, which we focus upon here [20]. The type I IFN family consists of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-δ, IFN-ω, IFN-ζ (mice), and IFN-τ (ruminants), though IFN-α and IFN-β are the best studied [21]. These cytokines are broadly associated with protection against systemic viral infections and contribute to restriction of infection at mucosal sites [7]. Type III IFNs, consisting of four different subtypes of IFN-λ, play a more prominent role in the protection of mucosal sites such as the intestinal and respiratory tracts [22]. Upon viral infection, the expression of IFNs is triggered by the sensing of viral nucleic acid by a variety of pattern-recognition receptors (PRRs). Both extracellular receptors, such as the membrane-bound Toll-like receptors (TLRs), and cytosolic receptors, such as RIG-I-like recep- tors (RLRs) and DNA sensors [which include cyclic GAMP synthase (cGAS)], can activate the expression of IFNs [8]. Subsequently, released IFNs bind to their respective receptors in an autocrine manner (the infected cell itself) or a paracrine manner (the neighboring cells) [23]. Type I IFNs bind to a heterodimer receptor complex composed of IFNAR1 and IFNAR2, while type III IFNs (or IFN-λ) bind to a heterodimer receptor complex comprised of IFNLR1 and IL-10RB. Despite distinct receptor complexes, type I and III IFNs share similar signaling cascades involving the phosphorylation of the JAK/STAT pathway and the translocation and binding of interferon regulatory factors (IRFs) to IFN-stimulated response elements (ISREs), inducing the expression of IFN-stimulated genes (ISGs) [24]. Expression of ISGs drives an The triangular relationship between the host, the gut microbiota, and viral pathogens Interestingly, the inﬂuence of the gut microbiota on IFN responses appears to be conserved across a wide range of viruses and, in parallel, numerous bacteria in the microbiota and their byproducts can activate IFN signaling. Understanding how the microbiota controls IFN responses will be critical to inform novel antiviral and viral vaccination strategies. 2Division of Infectious Diseases and Center for Experimental and Molecular Medicine (CEMM), Department of Medicine, Amsterdam University Medical Center, Academic Medical Center, A t d Th N th l d This review provides a brief overview of IFN biology followed by a detailed delineation of how the gut microbiota has been shown to modulate antiviral IFN responses at both local and remote OPEN ACCESS OPEN ACCESS Trends in Microbiology Correspondence: v.c.harris@amsterdamumc.nl (V.C. Harris). sites, discussing the speciﬁc mechanisms underlying microbiota and IFN interactions during viral infections. We focus on the gut microbiota, although these microbiota-driven mechanisms are likely at play across diverse anatomic sites, such as the lung and skin. Finally, the implications of these host–microbiota–viral pathogen interactions for antiviral therapies and viral vaccination strategies are explored. sites, discussing the speciﬁc mechanisms underlying microbiota and IFN interactions during viral infections. We focus on the gut microbiota, although these microbiota-driven mechanisms are likely at play across diverse anatomic sites, such as the lung and skin. Finally, the implications of these host–microbiota–viral pathogen interactions for antiviral therapies and viral vaccination strategies are explored. Trends in Microbiology, August 2022, Vol. 30, No. 8 779 Glossary LPS is considered a bacteria-associated molecular pattern and is often used as a proxy for bacterial infection. M cells: a type of epithelial cell, found in mucosal tissue, that has a specialized immune surveillance function. Mesenteric lymph node: a secondary lymphoid organ, located in the connective tissue that attaches the intestine to the abdominal wall (mesentery), which functions to drain (ﬁlter) lymphoid ﬂuid from the intestinal tract. In addition to directly inducing an antiviral state, IFNs are also important regulators of the adap- tive arm of the immune system. Nearly all immune cells express IFNAR1 and IFNAR2 and are therefore responsive to type I IFN. Conventional DCs (cDC) in particular rely on cues from type I IFNs for functional maturation and migration [29]. Functional cDCs are important in the priming of both T cells and B cells. Type I IFNs can also directly signal T cells to become acti- vated and proliferate [30]. The formation of germinal-center B cells and subclass distribution of IgG also depends on IFN-α/β signaling [31]. The role of IFN-λ in regulating adaptive immunity is only beginning to be understood. The extent to which human immune cells express IFNLR1, and thereby respond to IFN-λ stimulation, remains controversial. Naïve B cells are responsive to IFN-λ and require IFN-λ signaling to differentiate into plasmablasts and become functionally active, permitting cytokine release and antibody production [32]. CD8+ T cells do not directly respond to IFN-λ but still require IFN-λ to modulate the activation, antigen uptake, and migra- tion of lung DCs [33]. In addition, IFN-λ can indirectly regulate T cell and B cell responses through the thymic stromal lymphopoietin (TSLP) axis, a cytokine produced by M cells that is important for adaptive immune regulation [34]. In summary, IFNs are powerful antiviral cytokines, playing a central role in orchestrating innate and adaptive immune responses to viral infection. Box 1. Examples of local and distal IFN antiviral activity modulated by the gut microbiota 8 779 OPEN ACCESS OPEN ACCESS Trends in Microbiology Trends in Microbiology Glossary Epigenetic markers: alterations in gene activity and expression that do not involve alteration in the DNA sequence but rather the ‘packaging’ of the DNA. Epigenetic markers include, but are not limited to, DNA methylation and histone modiﬁcation. Histone acetyltransferase (HAT): an enzyme involved in the acetylation of the histone tail, a mechanism for gene regulation. In general, acetylated histone is a marker of increased gene transcription. Histone deacetylase (HDAC): the opposite of HAT, this enzyme is involved in the deacetylation of the histone tail, a mechanism for gene regulation. In general, deacetylated histone is a marker of reduced gene expression. Human organoid model: a three-dimensional tissue culture system, derived from stem cells of donated human tissue or differentiated pluripotent stem cells, thereby closely recapitulating the in vivo physiology and function of the represented organ. IFN-stimulated genes (ISGs): an array of genes whose expression is induced upon activation of the IFN signaling pathway. Expression of ISGs leads to the activation of an antiviral state in infected and neighboring cells. Intestinal/gut microbiota: the trillions of microbes, composed of bacteria, viruses, archaea, fungi, protozoa, and helminths, that reside in the gastrointestinal tract of the host and serve important functions in regulating host physiology and metabolism. Lipopolysaccharide (LPS): the main component of the outer membrane of Gram-negative bacteria. LPS is considered a bacteria-associated molecular pattern and is often used as a proxy for bacterial infection. M cells: a type of epithelial cell, found in mucosal tissue, that has a specialized immune surveillance function. Mesenteric lymph node: a secondary lymphoid organ, located in the connective tissue that attaches the intestine to the abdominal wall (mesentery), which functions to drain (ﬁlter) lymphoid ﬂuid from the intestinal tract. Pattern-recognition receptors (PRRs): receptors commonly found in sentinel cells such as macrophages and dendritic cells The function of PRRs is to antiviral state in infected and uninfected neighboring cells, resulting in direct interference with viral replication and dissemination [25]. antiviral state in infected and uninfected neighboring cells, resulting in direct interference with viral replication and dissemination [25]. Glossary Epigenetic markers: alterations in gene activity and expression that do not involve alteration in the DNA sequence but rather the ‘packaging’ of the DNA. Epigenetic markers include, but are not limited to, DNA methylation and histone modiﬁcation. Emerging paradigms suggest that type I and III IFNs each have unique and distinct roles in controlling viral infection. IFN-λ plays a more prominent role in the protection of mu- cosal sites, whereas IFN-α/β is more involved in the control of systemic infections. The localized function of IFN-λ results from the limited expression of IFNLR1 subunit of the type III IFN receptor to predominantly epithelial cells and only a subset of immune cells. By contrast, the receptor subunits for type I IFN, IFNAR1 and IFNAR2, are broadly expressed in nearly all nucleated cells [26]. Moreover, the IFN-α/β response is charac- terized by a rapid increase but also rapid decline of high-magnitude ISG expression, while ISG expression induced by IFN-λ is of lower magnitude, more delayed, but more sustained [27]. The differential localization and kinetics of IFN-λ responses may conﬁne antiviral responses to mucosal sites without inducing excessive inﬂammatory responses systemically, unless local responses fail to curb the infection [21]. Comprehensive com- parisons between IFN-α/β and IFN-λ for speciﬁc viral infections are available elsewhere [8,24,28]. Histone acetyltransferase (HAT): an enzyme involved in the acetylation of the histone tail, a mechanism for gene regulation. In general, acetylated histone is a marker of increased gene transcription. Histone deacetylase (HDAC): the opposite of HAT, this enzyme is involved in the deacetylation of the histone tail, a mechanism for gene regulation. In general, deacetylated histone is a marker of reduced gene expression. Human organoid model: a three-dimensional tissue culture system, derived from stem cells of donated human tissue or differentiated pluripotent stem cells, thereby closely recapitulating the in vivo physiology and function of the represented organ. IFN-stimulated genes (ISGs): an array of genes whose expression is induced upon activation of the IFN signaling pathway. Expression of ISGs leads to the activation of an antiviral state in infected and neighboring cells. Intestinal/gut microbiota: the trillions of microbes, composed of bacteria, viruses, archaea, fungi, protozoa, and helminths, that reside in the gastrointestinal tract of the host and serve important functions in regulating host physiology and metabolism. Lipopolysaccharide (LPS): the main component of the outer membrane of Gram-negative bacteria. 780 Trends in Microbiology, August 2022, Vol. 30, No. 8 The gut microbiota modulates IFN responses locally and remotely The gastrointestinal tract houses the body’s most densely populated microbiota, and thereby signals from the gut microbiota inﬂuence mucosal immune responses to enteric virus infec- tions [35]. However, it is also widely accepted that the gut microbiota can inﬂuence sites remote from the gastrointestinal tract. Correspondingly, the gut microbiota modulation of IFN responses occurs not only locally but also at extraintestinal compartments (Box 1). The means by which the resident intestinal microbiota inﬂuences distal sites remains an active area of study. There are at least two possible mechanisms: (i) commensal bacterial products or metabolites enter the systemic circulation and reach distal sites where they prime residing immune and epithelial cells; and (ii) circulating immune cells sample components of the gut microbiota in the intestine then migrate to other parts of the body to inﬂuence the local immune response. Further deﬁning the mechanisms of crosstalk between the gut microbiota and immune response will be essential to harnessing the potential of the gut microbiota in antiviral immunity. Box 1. Examples of local and distal IFN antiviral activity modulated by the gut microbiota The gut microbiota can modulate IFN-λ within the gastrointestinal tract during enteric viral infections [12,13,37]. One of the mechanisms is that the bacterial microbiota maintain the expression of a homeostatic tonic IFN-λ-mediated signature in intestinal epithelial cells (IECs). This is seen in murine rotavirus infections where depletion of the gut microbiota results in the loss of homeostatic tonic IFN-λ expression, limiting the host’s capacity to control murine rotavirus (MRV) infection [37]. In the proximal gut, commensal bacteria limit murine norovirus (MNV) infection, an inhibition associated with the pro- duction of microbiota-derived bile acids that prime IFN-λ [13]. Disruption of Ifnlr1 (the receptor for IFN-λ) also limits MNV’s dependence upon bacteria, again implicating IFN-λ in antiviral regulation in the intestine [12]. Interestingly, the gut micro- biota can also promote MNV infection in the distal intestine through an as-yet unclear mechanism [13]. A hypothesis is that the gut microbiota may express histo-blood group antigens (HBGAs) that are required for MNV infection [79]. These stud- ies suggest that patterns of microbiota-induced IFN-λ expression may be location-dependent, but ultimately support a model in which the gut microbiota maintains or supports IFN-λ induction to limit enteric viral infection. Gut microbiota modulation of type I IFN signaling at distal sites has been demonstrated in numerous murine studies in both germ-free (GF) and antibiotic-treated mice. A variety of extraintestinal virus infections have shown enhanced viral patho- genesis in the absence of microbiota-induced type I IFN . Reduced survival in antibiotic-treated mice upon infection with either LCMV [19] or VSV [9] is associated with reduced ISG expression in the spleen, a representation of the systemic im- mune compartment. During chikungunya virus (CHIKV) infection, viral loads in the spleen and serum of GF and antibiotic- treated mice are higher due to defective production of type I IFN by pDCs [14]. Similarly, absence of the gut microbiota leads to higher viral loads and lower type I IFN expression in the lung following infection of antibiotic-treated mice with in- ﬂuenza A virus [9,11] or respiratory syncytial virus (RSV) [17]. The inﬂuence of microbiota-induced type I IFN reaches as far as the brain, where reduced expression of IFN-β and ISGs in peripheral blood cells and spleen of antibiotic-treated mice leads to increased EMCV viral titers in the brain [15]. Trends in Microbiology, August 2022, Vol. 30, No. The gut microbiota controls homeostatic IFN and ISG expression Current evidence points to a role of the microbiota in inﬂuencing and maintaining IFN responses before infections occur [10,18,19,37]. The gut microbiota modulates the expres- sion of 'homeostatic IFN expression', a constitutive basal IFN expressed at a very low level that is crucial for timely activation of IFN antiviral activity upon infection [10,18,19,37]. Often, homeostatic ISG expression is used as a surrogate for IFN expression due to the extremely low and difﬁcult-to-detect levels of IFNs in this basal state [37]. Germ-free (GF) or antibiotic- treated mice devoid of residing gut microbiota have altered basal type I and type III IFN expression and signaling, predisposing these mice to defective or delayed viral clearance following infection [10,18,19,37]. transcriptomics, and metagenomics) to investigate the interconnected immune components and their interactions in a certain disease or environment. Systems vaccinology: a subset of system immunology where the integration of multiple omics data is used to unravel the complex molecular signatures of vaccine immunity. Plasmacytoid DCs (pDCs) are a subclass of DCs that are particularly important for the production of type I IFN. The bacterial microbiota can play a role in controlling the expression of homeostatic type I IFNs by pDCs, which is required for transcriptional, epigenetic, and metabolic programming of cDCs [10]. cDCs isolated from GF mice, when compared to cDCs isolated from control speciﬁc-pathogen-free (SPF) mice, lack numerous H3K4me3 epigenetic markers (indicative of transcriptionally active regions) for type I IFNs and ISGs. As a consequence, when cDCs from GF mice are stimulated with poly I:C (viral antigen) or LPS (bacterial antigen), transcription factors downstream of PRR activation can translocate to the nucleus but fail to bind to the pro- moter regions of these genes [18]. Impaired cDC programming and maturation therefore likely leads to an inability of cDCs to optimally prime CD8+ T cell and natural killer (NK) cell responses. Similarly, macrophages also depend on instructive signals from the bacterial microbiota to maintain homeostatic IFN responses. In murine models of lymphocytic choriomeningitis virus (LCMV) or inﬂuenza infection, depletion of the gut microbiota results in macrophages that are unresponsive to virus or IFN-β stimulation [19]. Further, genome-wide transcriptional analysis of these macrophages reveals that IFN-related genes, including PRRs and ISGs, are downreg- ulated. Potential mechanisms underlying gut microbiota–IFN–viral interactions signaling cascades as a response. Nonimmune cells are also equipped with PRRs to a lesser extent. g g p Nonimmune cells are also equipped with PRRs to a lesser extent. Prior studies have uncovered a variety of mechanisms by which the gut microbiota modulates IFN responses, delineating different components of the bacterial microbiota that contribute to these interactions. Here, we outline three key mechanistic themes underpinning microbiota–IFN interactions in relation to viral infections: microbiota-mediated control of homeostatic IFN; microbiota-derived PRR ligands that induce IFN activation; and microbiota-derived metabolites that regulate IFN expression (Figure 1, Key ﬁgure). It is important to note that these mechanistic themes do not work in isolation but rather they are interconnected and overlapping with each other. Homeostatic IFN production, for instance, may be maintained by bacterial ligands and/or metabolites. In turn, these ligands and metabolites can inﬂuence and alter microbiota composi- tion as well [36]. Peyer’s patches: one of the lymphoid tissues in the gastrointestinal tract which consists of aggregated lymphoid follicles. Peyer’s patches are important for immune surveillance and induction of immune tolerance. Poly I:C (polyinosinic:polycytidylic acid): an analog of double-stranded RNA, often used as a synthetic ligand for numerous PRRs and as a proxy for viral infection. Systems immunology: an approach using mathematical and computational modeling to integrate multiple types of - omics data (such as metabolomics, transcriptomics, and metagenomics) to investigate the interconnected immune components and their interactions in a certain disease or environment. Systems vaccinology: a subset of system immunology where the integration of multiple omics data is used to unravel the complex molecular signatures of vaccine immunity. Pattern-recognition receptors (PRRs): receptors commonly found in (PRRs): receptors commonly found in sentinel cells such as macrophages and dendritic cells. The function of PRRs is to recognize molecular patterns of pathogens and initiate inﬂammatory OPEN ACCESS OPEN ACCESS Trends in Microbiology Trends in Microbiology, August 2022, Vol. 30, No. 8 781 The gut microbiota provides PRR ligands that can lead to IFN induction Just like viruses, components of bacterial cells contain molecular patterns that can activate PRR signaling. Five different types of PRR can sense bacterial patterns, including TLRs, RLRs, NOD- like receptors (NLRs), DNA sensors, and AIM2-like receptors (ALRs). These bacterial PRRs are involved mainly in the induction of antibacterial signaling pathways, but a subset of these PRRs also induce IFN production during bacterial infection [40,41]. Figure 1. (A) The gut microbiota regulates basal homeostatic IFN expression. The gut microbiota can induce homeostatic type I IFN expression (shown in blue) from macrophages and plasmacytoid dendritic cells (pDCs) and homeostatic type III IFN (shown in orange) from intestinal epithelial cells (left panel). Type I IFN from macrophages is required for the priming of natural killer (NK) cell and CD8+ T cell function [18] (left panel). Type I IFN from pDCs is required for epigenetic programming of conventional dendritic cells (cDCs) so that they can prime NK cells and CD8+ T cells [19] (middle panel). When the gut microbiota is depleted, signals from the gut microbiota are diminished, leading to the reduction of basal homeostatic type I [18,19] and type III IFN, and impaired priming and functionality of cDCs, NK cells, and CD8+ T cells (right panel) [37]. (B) Gut microbiota-derived pathogen recognition receptor (PRR) ligands activate IFN expression. Components of the commensal gut microbiota generate molecular patterns that can bind to PRRs. For instance, Bacillus spp. poly-γ-glutamic acid [44] and Bacteroides fragilis polysaccharide A (PSA) [9] bind to TLR4, while the nucleic acids of lactic acid bacteria (LAB) can bind to either TLR3 [45], RIG-I-like receptors (RLRs) or cGAS [47]. This pattern recognition results in downstream signaling and type I IFN production. Depending on the type of PRR ligands and the PRR sensors, type I IFN production has been shown to block viral replication. (C) Gut microbiota-derived metabolites activate IFN expression. Gut commensals, such as members of the family Lachnospiraceae, can produce short-chain fatty acids (SCFAs) that can activate type I IFN expression in a GPR43-dependent manner to block the replication of respiratory syncytial virus (RSV) [17]. Clostridium scindens can transform primary bile acids (BAs) into secondary BAs. These secondary BAs can activate both the expression of type I IFN from pDCs to inhibit chikungunya virus (CHIKV) replication [14] and type III IFN from intestinal epithelial cells (IECs) to inhibit murine norovirus (MNV) replication [13]. The gut microbiota controls homeostatic IFN and ISG expression This impaired homeostatic IFN signaling translates to defects in induction of adaptive immune responses when mice are infected with either LCMV or inﬂuenza virus, such that CD8+ T cell function and antigen-speciﬁc antibody production are both impaired [19]. Similar observations have been made for encephalomyocarditis virus (EMCV) infection, wherein antibiotic-treated mice display unresponsive macrophages, along with impaired NK cell toxicity and decreased type I IFN and ISG expression, with reduced survival and exacerbated disease phenotypes [15]. Interestingly, signals from the gut microbiota of conventional mice are able to limit EMCV replication in distal target cells in the brain to protect them from neurological pathogenicity [15]. Altogether, these studies underscore the likely importance of gut microbiota-derived signals in inﬂuencing homeostatic type I IFN responses and viral control at extraintestinal sites. Trends in Microbiology, August 2022, Vol. 30, No. 8 781 OPEN ACCESS OPEN ACCESS Trends in Microbiology Potential mechanisms underlying microbiota regulation of interferon (IFN) antiviral immunity Potential mechanisms underlying microbiota regulation of interferon (IFN antiviral immunity Trends Trends in in Microbiology Microbiology (See ﬁgure legend at the bottom of the next page.) Trends Trends in in Microbiology Microbiology (See ﬁgure legend at the bottom of the next page.) Trends Trends in in Microbiology Microbiology (See ﬁgure legend at the bottom of the next page gy gy (See ﬁgure legend at the bottom of the next page.) 782 Trends in Microbiology, August 2022, Vol. 30, No. 8 OPEN ACCESS OPEN ACCESS Trends in Microbiology At mucosal sites, nonhematopoietic epithelial and stromal cells contribute signiﬁcantly to the maintenance of mucosal immunity alongside immune cells. Nonhematopoietic cells are also equipped with PRRs to sense pathogens and produce cytokines [38,39]. Lung stromal cells, for example, respond to tonic type I IFN during homeostasis and produce basal ISGs in a microbiota-dependent manner. The source of type I IFN in the lung remains an open question. Type I IFN signaling in the lung induces basal ISG expression in both stromal and hematopoietic cells; when the gut microbiota is absent, ISG expression is disrupted in lung stromal cells but not in immune cells for unclear reasons [11]. It is possible that the regulation of baseline IFN and ISGs in mucosal compartments and systemic compartments is distinct. However, given the heteroge- neity in microbiota models (antibiotic-treated versus GF mice), it also possible that differences in experimental models have led to observed discrepancies. The gut microbiota controls homeostatic IFN and ISG expression There is currently limited knowledge of the role of the gut microbiota in homeostatic IFN-λ signal- ing. Only one study so far has evaluated IFN regulation of basal ISG expression in the intestinal tract of mice [37]; it found that, in an antibiotic-treatment model, microbiota-associated IFN-λ sig- naling drives expression of homeostatic ISGs in a nonuniform manner along the intestinal epithelium. Speciﬁcally, IFN-λ-driven tonic ISG expression is localized to the tips of the intestinal villi, suggesting preferential expression of homeostatic ISGs by mature enterocytes. The physio- logical relevance of this preferential localized expression of microbiota-induced homeostatic ISGs remains to be further investigated but does appear to be required to mount a timely and robust antiviral response against enteric viruses that target mature enterocytes, such as MRV [37]. To date, no study has assessed homeostatic IFN-λ expression in airway epithelia. Considering the signiﬁcance of IFN-λ in antiviral defense against respiratory pathogens, and the capacity of the gut microbiota to induce type I IFN expression at extraintestinal sites, homeostatic IFN-λ expression in the airway in response to microbiota-derived signals is an important area for future investigation. Trends in Microbiology, August 2022, Vol. 30, No. 8 783 The gut microbiota provides PRR ligands that can lead to IFN induction cGAS, cyclic GMP-AMP synthase; IRFs, interferon regulatory factors; MAVS, mitochondrial antiviral-signaling protein; MYD88, myeloid differentiation primary response 88; TRIF, Toll–IL-1 receptor domain-containing adaptor inducing IFN-β; STING, stimulator of interferon genes. See references [9,11,15,18,19,30,38,41,43]. The ﬁgure was created with BioRender.com. Trends in Microbiology, August 2022, Vol. 30, No. 8 783 OPEN ACCESS OPEN ACCESS Trends in Microbiology A recent study showed that TLR4 sensing of the outer membrane of Bacteroides fragilis, specif- ically the polysaccharide A (PSA) domain, leads to the induction of IFN-β by DCs in the colon lam- ina propria [9]. In vitro incubation of bone-marrow-derived DCs with PSA prior to vesicular stomatitis virus (VSV) infection results in a reduced percentage of infected DCs and increased cell viability. Oral administration of PSA to antibiotic-treated mice protects mice against infection with VSV or inﬂuenza A virus, increasing their survival in comparison to untreated controls [9]. This ability of the PSA of B. fragilis to induce protective TLR4-dependent IFN-β activity is distinct from TLR4 activation by the canonical Escherichia coli LPS ligand, which often leads to the induction of (excessive) proinﬂammatory responses [42,43]. Another nontoxic TLR4 ligand is the biopolymer poly-γ-glutamic acid (ϒ-PGA), which is produced by Bacillus sp. Similar to PSA, ϒ-PGA induces IFN-β which can inhibit MNV entry and replication in vitro, while oral administration of ϒ-PGA to MNV-infected mice results in increased serum IFN-β and reduced MNV levels in Peyer’s patches and mesenteric lymph nodes [44]. Altogether, these studies highlight the potential of microbiota- derived PRR ligands to induce protective IFN antiviral responses. Intracellular sensing machinery was previously considered irrelevant for extracellular commensal bacteria and thought to be reserved for detection of invasive pathogens. However, several in vitro studies suggest that intracellular PRRs are also able to sense the nucleic acid of commensal bac- teria and thereby lead to the induction of IFN-β expression. TLR3, which detects double-stranded RNA, is discriminately activated by commensal lactic acid bacteria (LAB), but not by pathogenic bacteria. dsRNA is uncommon in bacteria and is synthesized only by speciﬁc species under certain conditions, a potential explanation behind the speciﬁc capacity of LAB to induce TLR3 activation [45]. In a separate study, LAB have been shown to induce IFN-β via cGAS-stimulator of interferon genes (STING) and RLR mitochondrial antiviral signaling protein (MAVS) activation in human macrophages. Both sensors recognize cytosolic DNA and RNA, respectively, and are classically associated with viral infections [46]. The gut microbiota provides PRR ligands that can lead to IFN induction Intriguingly, the ability of individual strains of LAB to induce type I IFN is inversely correlated with their ability to induce NF-κB, suggesting strain speciﬁcity in inducing pro- or anti-inﬂammatory responses [47]. Further work is required to determine whether these ﬁndings hold in in vivo experimental systems and if induction of IFN by intracellular sensors can provide protection against viral infections. The involvement of bacterial microbiota ligands in the induction of IFN-λ is currently understudied. Early evidence, derived from in vitro stimulation of mouse bone-marrow-derived DCs and human epithelial cell lines with a variety of bacterial TLR ligands, has shown that ligands of commensal bacteria can induce type III IFN expression, especially through TLR5 [48]. Thus, the possibility that the gut microbiota signals through TLRs to induce type III IFN merits further investigation. 784 Trends in Microbiology, August 2022, Vol. 30, No. 8 SCFAs SCFAs are the product of fermentation of nondigestible dietary ﬁber by the gut microbiota. The composition of both host diet and anaerobic bacteria in the gut determine the SCFA proﬁle of an individual. Acetate, propionate, and butyrate are the best-studied examples of SCFAs, and their role in shaping host immunity and physiology has been extensively described [53,54]. The immunomodulatory properties of SCFAs are ascribed to their function as histone deacetylase (HDAC) inhibitors, G-protein-coupled receptor (GPCR) agonists, and autophagy regulators [55]. SCFAs can modulate IFN responses to viral infections. Microbiota-derived acetate shows antiviral activity against RSV by increasing IFN-β in a GPR43-dependent manner. Mice administered either an SCFA-producer (species of the family Lachnospiraceae) or exogenous acetate exhibit reduced RSV pulmonary viral load, reduced migration of inﬂammatory cells into the lung, and overall improved survival [17]. Butyrate and propionate have been shown to have similar protec- tive effects [17]. While the link between GPR43 engagement and IFN-β production needs further clariﬁcation, NF-κB activation has been implicated as evidenced by increased NF-κB p65 trans- location to the nucleus following acetate supplementation [17]. Contrary to the capacity of acetate to restrict RSV replication in the mouse model, an in vitro study showed that butyrate supports the increased replication of several viruses [inﬂuenza A virus (IAV), reovirus, HIV, human metapneumovirus (hMPV), VSV] by reprogramming the expression of spe- ciﬁc ISGs. The ability of butyrate to suppress ISG induction is suspected to be secondary to the HDAC-inhibitor properties of butyrate since treatment with a synthetic HDAC inhibitor also sup- presses ISG expression, and a histone acetyltransferase (HAT) inhibitor reverses this sup- pression [16]. These contrasting proviral and antiviral effects may arise from different types of SCFA and virus tested. The use of immortalized cell lines may also drive divergence in research ﬁndings as transformed cells may have altered signaling pathways. The gut microbiota produces metabolites that can induce IFN production Alongside being the source of PRR ligands, the gut microbiota produces a broad repertoire of metabolites that can act as key mediators of microbiota–host interactions. These metabolites are either a product of cometabolism of a dietary compound between the host and the gut microbiota or synthesized de novo in bacterial cells [49]. The potential of microbial metabolites to regulate host immunity is well recognized and has been reviewed comprehensively elsewhere [50,51]. Here, we discuss two classes of metabolites, short-chain fatty acids (SCFAs) and bile acids (BAs), that have been shown to modulate viral replication through IFN responses. Another microbial metabolite, desaminotyrosine (DAT), is also capable of inducing IFN responses resulting in reduced disease pathology despite negligible alteration in viral titer [52]. We anticipate that future research will uncover more relevant classes of microbiota-derived metabolites that can act as IFN modulators. OPEN ACCESS OPEN ACCESS Trends in Microbiology Trends in Microbiology, August 2022, Vol. 30, No. 8 785 DAT DAT is a product of commensal bacteria degradation of plant-derived polyphenol compounds (ﬂavonoids) [63]. Administration of DAT into mice infected with inﬂuenza A virus protected mice from infection-associated mortality and morbidity but did not reduce viral titers [52]. DAT treatment reduced lung tissue immunopathology by augmenting the type I IFN response of phagocytic cells. Oral gavage with Clostridium, the commensal bacterium producing DAT, offered a similar protective effect [52]. Importantly, DAT treatment appears only to be protective when administered before infection occurs. DAT administration post-infection exacerbated disease outcomes instead [52]. 786 Trends in Microbiology, August 2022, Vol. 30, No. 8 Bile acids scindens colonization, highlighting the involvement of secondary BAs in induc- ing IFN responses [14]. Similarly, antibiotic-treated mice colonized with C. scindens or supplemented with DCA exhibit induction of IFN-λ signaling that protects against MNV infection in the proximal gut [13]. How the signals from BAs integrate into IFN signaling pathways is currently unclear. BA receptors FXR and TGR5 are the most obvious suspects given the involved signaling pathways. Infections by some viruses, such as LCMV, herpes simplex virus type 1 (HSV-1) and Sendai virus (Sev), can induce the expression of BA transporters, leading to BA accumulation in both hepatic and nonhepatic cell types [60–62]. This accumulation of BAs can subsequently induce type I IFNs through FXR and TGR5 activation. During LCMV infection in a murine model, CD8+ T cell- mediated destruction of LCMV-infected hepatocytes results in the release of BAs which then engage with FXR receptors in neighboring hepatocytes to induce the production of type I IFNs. In the absence of FXR receptors, type I IFN gene expression decreases, BAs accumulate, and immune cell migration is disturbed, all leading to a failure of LCMV control [62]. Similarly, intracel- lular BA accumulation following infection with HSV-1 and SeV in THP-1 cells activates the TGR5–β-arrestin–sarcoma (SRC) kinase pathway. Subsequently, SRC kinase phosphorylates important components of the IFN signaling pathways, including RIG-I, MAVS, STING, TBK1, and IRF3, and thereby induces the expression of IFN-β [61]. It has been suggested that TGR5 may itself be an ISG because its expression is increased upon viral infection or IFN-β stimulation in a STAT-1-dependent manner [60]. In contrast, a study using microbiota-derived BA showed that DCAs directly prime IFN-λ activation during stimulation with poly(I:C) or MNV infection, and that coincubation of DCAs and synthetic FXR agonists results in abrogation of DCA-dependent enhancement of IFN-λ [13]. TGR5 involvement was not tested in this system. More studies are needed to further clarify the roles of different BA receptors in the IFN response. Bile acids Primary BAs, such as cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized by the host in the liver. Primary BAs undergo conjugation with either glycine or taurine to become water-soluble before being excreted to the small intestine. In the small intestine, the gut microbi- ota remove these amino acid groups and transform primary BAs to secondary BAs such as lithocholic acid (LCA) and deoxycholic acid (DCA) [36]. Therefore, commensal bacteria help to shape the composition of BAs in the gut through their biotransformative activity. In addition to having important functional roles in digestion and lipid absorption, BAs can signal through numer- ous metabolic pathways. The two best-described BA receptors are the Farnesoid-X-receptor (FXR), a nuclear receptor preferentially activated by primary BA, and Takeda-G-protein receptor 5 (TGR5), a membrane-bound receptor preferentially activated by secondary BAs [56,57]. Evidence describing BA modulation of IFN signaling pathways and subsequent viral infection is conﬂicting. Some studies have reported that BAs can negatively regulate induction of IFN signal- ing pathways, facilitating viral replication. For example, BAs are required components in calicivirus cell culture propagation systems owning to their ability to suppress STAT1 activation and thereby facilitate calicivirus replication [58]. BA treatment of hepatitis C virus (HCV) cell culture systems, using autonomously replicating HCV replicons in hepatoma cells, similarly reduces IFN-α/γ anti- viral activity and thereby increases HCV RNA and protein expression [59]. In contrast, BAs have been reported to block chikungunya virus (CHIKV) and MNV replication in vivo using mouse models through the induction of type I and III IFN, respectively [13,14]. Colonization of antibiotic-treated or GF mice with the commensal bacterium Clostridium scindens, which pro- duces DCA, results in suppression of CHIKV viremia in serum and blood leukocytes in a type I Trends in Microbiology, August 2022, Vol. 30, No. 8 785 Trends in Microbiology, August 2022, Vol. 30, No. 8 785 OPEN ACCESS OPEN ACCESS Trends in Microbiology IFN-dependent manner. Oral administration of puriﬁed DCA is sufﬁcient to recapitulate this pro- tective effect of C. scindens colonization, highlighting the involvement of secondary BAs in induc- ing IFN responses [14]. Similarly, antibiotic-treated mice colonized with C. scindens or supplemented with DCA exhibit induction of IFN-λ signaling that protects against MNV infection in the proximal gut [13]. IFN-dependent manner. Oral administration of puriﬁed DCA is sufﬁcient to recapitulate this pro- tective effect of C. Perspective: translating gut microbiota–IFN–viral interactions to a human context The past several decades of research have transformed the understanding and appreciation of the numerous roles that the gut microbiota can have in the host defense against viral pathogens. Divergent microbiota proﬁles associate with both resistance and susceptibility to viral infections [64]. Similarly, the composition of the gut microbiota is an important factor in modulating the immune response to viral vaccination [65,66]. Full understanding of how the human microbiota can protect hosts from viral disease is needed to harness the microbiota’s therapeutic potential – either through antiviral therapies or through improvements in viral vaccine responses. Currently, research linking the gut microbiota to viral infections in humans is sorely lacking, with a preponder- ance of correlative studies in which microbiota-dependent mediators of immune responses to protection from viral infection remain largely unknown. Mechanistic insights from in vitro and animal studies can help to guide the exploration of microbiota-dependent antiviral immunity in the human context. It is also important to note that the human gut microbiota is a complex environment with numerous kingdoms of microbes beyond bacteria which can also strongly regulate IFN responses OPEN ACCESS OPEN ACCESS Trends in Microbiology (Box 2). In addition, other nonintestinal compartments also have their own microbiota communities that may modulate local and distal immune responses. The microbiota composition of the lower re- spiratory tract, for instance, is a better predictor of clinical outcomes in severe acute respiratory virus coronavirus 2 (SARS-CoV-2) infection than the composition of the gut microbiota [67,68]. Thus, in clinical settings, it is important to consider the contribution of both the intestinal and extra- intestinal microbiota in shaping host immunity. IFN responses have a long history as therapeutic targets for human viral disease. IFN-α/β has been used as an antiviral treatment for HCV and hepatitis B virus (HBV) infection, though drug resistance and drug toxicity are of concern [69,70]. IFN-λ has been compared to IFN-α/β in clinical trials as a potentially less toxic but therapeutically equivalent treatment for chronic HCV (NCT01001754i) and HBV infections (NCT01204762ii) [71,72]. In addition, IFN has been explored as a potential vaccine adjuvant. In preclinical testing, HSV-2 and HIV vaccines adjuvanted with IFN-λ confer improved protection against challenge with relevant viruses [73,74]. In light of the recent SARS-CoV-2 pandemic, there has been substantial interest in exploring both type I and III IFN as antiviral treatments (Box 3). Perspective: translating gut microbiota–IFN–viral interactions to a human context We propose that there may be several advantages to using microbiota-dependent induction of endogenous IFN responses in antiviral therapies and as vaccines adjuvants: (i) microbiota- induced IFN is localized; (ii) predeﬁned microbiota stimulants may be able to augment IFN responses to speciﬁc virus infections; and (iii) microbiota-induced IFN expression may result in fewer side effects due to endogenous feedback loops controlling IFN production. However, before translation into human applications is possible, several basic science questions remain to be addressed. Trends in Microbiology, August 2022, Vol. 30, No. 8 787 Box 2. Contributions of other members of the gut microbiota to IFN signaling The gut microbiota includes more than just bacteria, and the roles of nonbacterial kingdoms in shaping host immunity are currently understudied. Evidence is emerging that nonbacterial microbes can strongly modulate antiviral immune responses. The gut virome – the enteric viral community containing viruses that infect prokaryotes (bacteria) and viruses that infect eukaryotic cells – has been the most researched. Recent work suggests that Enterovirus B or Cosavirus coinfection correlates with reduced immunogenicity to live attenuated rotavirus vaccines in African infants [80]. Virome modulation of IFN responses may be mechanistically important as the virome regulates IFN responses in a manner that parallels the bacterial microbiota [81]. Depletion of the gut virome through antiviral treatment can reduce TLR3 and TLR7 activation, ultimately impairing production of IFN-β by pDC [82]. Chronic MNV infection, a resident commensal virus in immunocompetent mice, can replace the function of the commensal bacteria, reversing abnormalities in intestinal morphology and gut mucosal immunity in GF and antibiotic-treated mice through type I IFN activation [83]. Murine astrovirus can protect immunocompromised mice (deﬁcient in B and T cells, innate lymphoid cells, and gut-associated lymphoid tissue) against MNV and MRV infection by inducing IFN-λ signaling [84]. These ﬁndings highlight that the eukaryotic virome, just like bacterial microbiota, has signiﬁcant immunomodulatory properties that ultimately determine host susceptibility to disease and infection. Viral motifs derived from bacteriophages also trigger IFN production. Pseudomonas aeruginosa bacteriophages Pf induces IFN-β secretion that favors persistent infection of their bacterial host [85]. Whether bacteriophage-induced IFN production impacts eukaryotic virus infection warrants future investigation. Commensal fungi, namely Candida albicans and Saccharomyces cerevisiae, can also recapitulate functional beneﬁts of the bacterial microbiota through tonic signals calibrating immune responses to inﬂuenza A infection [86]. Debaryomyces hansenii, a fungus commonly found in the wounds of Crohn’s disease patients, is known to impair wound healing by inducing chemokine CCL5 through type I IFN axis [87]. Thus, commensal fungi likely also alter IFN-mediated immunity, shaping antiviral responses. Enteric helminths are also key members of the gut microbiome, modulating microbiome composition and host immunity [88]. Murine helminth Heligmosomoides polygyrus provides protection against RSV infection by inducing type I IFN expression in both the intestine and the lung [89]. Together, these studies emphasize the importance of characterizing interkingdom interactions within the gut microbiota and host as they may ultimately be strong determinants of IFN responses to viral infections. Trends in Microbiology, August 2022, Vol. 30, No. Outstanding questions SARS-CoV-2 and the COVID-19 pandemic have broadened our understanding of IFN’s potential as an antiviral therapeutic. Studies have had divergent ﬁndings. Some studies suggest that severe COVID-19 is associated with reduced and delayed IFN responses [90,91], while other studies implicate type I IFN, together with TNF and IL-1β, in hyperinﬂammation and pro- gression to severe COVID-19 [92]. What is the role of the gut microbiota in regulating IFN-mediated immunity in humans? How does human microbi- ota variation contribute to differential responses to viral infection? Clinical trials evaluating type I and III IFN therapies for patients hospitalized with COVID-19 have generally shown no or minimal clinical beneﬁt [93–96]. The DISCOVERY trial (NCT04315948iii), the largest randomized controlled trial testing subcutaneous IFN-β1a in 2063 hospitalized COVID-19 patients, showed no mortality beneﬁt compared to placebo [97]. However, in trials with smaller sample sizes, both type I (NCT04276688iv) [94] and type III IFNs (NCT04354259v) [95] appear to shorten the duration of viral shedding. To date, IFN therapy has not been broadly used in clinical COVID-19 practice. How do IFN responses develop along the trajectory of human life, and how does the gut microbiota contribute to this development? Just as for other COVID-19 therapeutics, IFN’s clinical beneﬁt may depend upon appropriate timing, route of administration, and use in appropriate patient populations [98]. Administering IFN early in disease was associated with a lower mortality in a retrospective cohort study [99]. A small randomized controlled trial of inhaled IFN treatment (NCT04385095vi) showed higher odds of improved clinical improvement than placebo [96]. Older adults infected with SARS-CoV-2 may beneﬁt more from IFN therapy as they have a disturbed IFN expression pattern, consisting primarily of IFN-α, IFN-β, and IFN-λ2, in contrast to infected children, who predominantly express IFN-λ1 [100]. Type III IFN may also offer a more protective response com- pared to type I IFN. The IFN proﬁle of patients with milder COVID-19 is dominated by expression of IFN-λ1 and IFN-λ3, while patients with more severe disease have higher type I IFN and IFN-λ2 [102]. Moreover, the antiviral state induced by type III IFN is conﬁned to mucosal sites, potentially preventing excessive proinﬂammatory responses that may be induced by type I IFN [103]. What are the roles of other microbiota kingdoms, such as the virome, in the regulation of IFN antiviral immunity? Outstanding questions How can we delicately regulate endogenous expression of IFN for sufﬁcient antiviral protection without overstimulation that can lead to auto- immune pathogenesis? What are the beneﬁts of endogenous stimulation by the gut microbiota as opposed to the use of exogenous IFNs for antiviral therapy and viral vaccine performance? Finally, as an alternative to exogenous IFN administration, ﬁne-tuning endogenous IFN production by modulating the gut microbiota composition may hold potential. The human gut microbiome has the capacity to modulate the production of inﬂammatory cytokines [104], and enhanced inﬂammatory cytokines in severe COVID-19 patients has been suggested to be induced by systemic bacterial products [105]. Thus, modulating the gut microbiota composition to favor the production of protective IFN responses may be an alternative for the control of SARS-CoV-2 infection. The ﬁrst key needed research area is the age-dependency of IFN responses. An example is with rotavirus infections. IFN responses to MRV infection are quite distinct between neonatal and adult mice: whereas neonatal mice require both IFN-λ and IFN-α/β to control infection, adult mice require IFN-λ only [75]. Considering that human infants and the elderly are the most susceptible to life-threatening respiratory and gastrointestinal virus infections, and that the microbiota changes dramatically with age [76–78], in-depth understanding of the maturation of IFN responses and the microbiota over the course of the human lifespan is crucial. The second key needed research area is understanding the risk for autoimmunity. The gut micro- biota may excessively prime IFN responses, resulting in unwanted T cell responses to harmless peripheral or self-antigens [10], manifesting as allergy or autoimmunity. The delicate balance between sufﬁcient priming for robust antiviral response versus overstimulation resulting in autoimmune pathogenesis needs further study and ﬁne-tuning. The ﬁnal key needed research area is a better understanding of microbiota interactions with IFN-λ. IFN-λ was discovered only in 2004, and studies relating to its fundamental biology and interactions with the microbiota have been limited. IFN-λ may be preferred as a human therapeutic over IFN-α/β owing to its ability to induce highly localized responses with less risk for proinﬂammatory excess. Major research gaps include whether induction of IFN-λ by PRR ligands from commensal bacteria is possible and sufﬁcient to protect from viral infection. Further, the mechanism(s) by which microbiota-dependent IFN-λ is linked to adaptive immunity, especially in its potential role as a vaccine adjuvant, requires further investigation. 788 Trends in Microbiology, August 2022, Vol. 30, No. 8 Box 2. Contributions of other members of the gut microbiota to IFN signaling 8 787 OPEN ACCESS Trends in Microbiology Trends in Microbiology Box 4. Tools for studying microbiota–host IFN–viral interactions in the human context The emerging ﬁelds of systems immunology and systems vaccinology offer promising approaches to identify relevant micro- biota targets that interact with and inﬂuence human antiviral and vaccine immunity. Via a systems vaccinology approach, IFN re- sponse was identiﬁed as an important early innate immune signature correlating with strong vaccine-induced antibody production for both the seasonal inﬂuenza vaccine [106] and the BNT162b2 mRNA SARS-CoV-2 vaccine [107]. However, mi- crobiota modulation through antibiotic treatment appears to have only minimal effects on systemic IFN regulation following sea- sonal inﬂuenza virus vaccination [108]. Future studies are warranted to investigate whether more subtle microbiota modulation impacts IFN responses for other vaccines. It is possible that the gut microbiota may be more tightly interlinked with enteric and mucosal vaccine immunity, and thus IFN responses at mucosal sites following viral vaccination warrant further investigation. The emergence of human organoids as a model system for studying host–virus interactions has dramatically enhanced scientists’ capacity to study IFN responses in mucosal compartments in a human context. Both human intestinal and lung organoid systems have been deployed to study the antagonism between IFN responses and numerous viruses, including human norovirus [109], adenovirus [110], astrovirus [111], enterovirus 71 [112], human rotavirus [113], and SARS-CoV-2 [114]. Efforts are currently underway to increase the complexity of intestinal organoid models by integrating gut microbiota components, immune cells, and the enteric nervous system in order to better mimic the human intestinal environment [115,116]. These models provide a platform by which the triangular interactions of gut microbiota–host IFN response–viral infection can be investigated, permitting therapeutic translation to human contexts. remain to be addressed before this vision can be realized (see Outstanding questions). The speciﬁc microbiota-derived signals that interact with IFN signaling pathways have been partially identiﬁed. Nonetheless, the possibility that other microbial components and metabolites, and even other kingdoms of the gut microbiota, may also induce a protective IFN response is deserv- ing of further exploration. The regulation and function of microbiota-dependent IFN-λ in particular needs to be better understood. In addition, there is an urgent need for the ﬁeld to move towards translation into the human setting. Recent advances in both computational approaches and experimental models (Box 4) have enabled more direct investigation of gut microbiota–host IFN–viral interaction in the human context, which can be leveraged in the near future for additional Trends Trends in in Microbiology Microbiology Figure 2. Concluding remarks IFN modulation by the gut microbiota represents an exciting opportunity to harness microbiota- based therapeutic approaches for viral control. However, both basic and translational questions 788 Trends in Microbiology, August 2022, Vol. 30, No. 8 OPEN ACCESS OPEN ACCESS Trends in Microbiology Trends in Microbiology, August 2022, Vol. 30, No. 8 789 References (2019) Type III interferons: Balancing tissue tolerance and resistance to pathogen invasion. J. Exp. Med. 217, 1–12 8. Ye, L. et al. (2019) Interferon-λ orchestrates innate and adap- tive mucosal immune responses. 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Fajrial for valuable discussion and input for ﬁgures. M.T.B. was supported by NIH grants R01 OD024917, R01 AI141716, R01AI139314, and R01 AI141478, the Pew Biomedical Scholars Program of the Pew Charitable Trusts, and the Children’s Discovery Institute of St Louis Children’s Hospital and Washington University (MI-II-2019-790). V.C.H. was supported by The Netherlands Organization for Health Research and Development (ZonMw) VENI 09150161810022 and 10430022010019, Health Holland TKI GLORIA fund, and Wellcome Trust 219775/Z/19/Z. The funders had no role in the de- cision to publish or the preparation of the manuscript. Box 4. Tools for studying microbiota–host IFN–viral interactions in the human context Future translation of microbiota–interferon(IFN)–viral interaction into the human context. The triangular relationship between the gut microbiota–host IFN response–viral infections has been extensively described in animal models. However, the translation of these interactions into the human setting for therapeutic purposes remains a major challenge. Recent computational tools, such as the systems biology approach and experimental tools such as human organoid platforms, may aid further exploration of this interaction, paving the way to microbiota-based therapies in viral control and viral vaccination. The ﬁgure was created with BioRender.com. Trends Trends in in Microbiology Microbiology Figure 2. Future translation of microbiota–interferon(IFN)–viral interaction into the human context. The triangular g ( ) g relationship between the gut microbiota–host IFN response–viral infections has been extensively described in animal models. However, the translation of these interactions into the human setting for therapeutic purposes remains a major challenge. Recent computational tools, such as the systems biology approach and experimental tools such as human organoid platforms, may aid further exploration of this interaction, paving the way to microbiota-based therapies in viral control and viral vaccination. The ﬁgure was created with BioRender.com. Trends in Microbiology, August 2022, Vol. 30, No. 8 789 Trends in Microbiology, August 2022, Vol. 30, No. 8 789 OPEN ACCESS OPEN ACCESS Trends in Microbiology Trends in Microbiology insights (Figure 2). The potential of microbiota-based therapy for viral control is promising, and IFN responses may serve as an important microbiota-regulated antiviral immunity component. The ability to regulate IFN responses in a speciﬁc and precise manner will allow the actualization of this promise. 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https://openalex.org/W2767334261	https://lexikos.journals.ac.za/pub/article/download/1402/856	English	null	Intellectualization through Terminology Development	Lexikos	2,017	cc-by	5,598	* This article was presented as a paper at the Twenty-first Annual International Conference of the African Association for Lexicography (AFRILEX), which was hosted by the Xitsonga and Sesotho sa Leboa National Lexicography Units, Tzaneen, South Africa, 4–6 July 2016. http://lexikos.journals.ac.za http://lexikos.journals.ac.za […] accommodate all children regardless of their physical, intellectual, social, linguistic or other conditions. This should include disabled and gifted children, street and working children, children from remote or nomadic populations, chil- dren from linguistic, ethnic, or cultural minorities and children from other dis- advantaged or marginalized area and groups. (UNESCO, 1994, Framework for Action on Special Needs Education: 6) Sleutelwoorde: INTELLEKTUALISERING, TERMINOLOGIEONTWIKKELING, OES, SKARE- DEELNAME, KONSULTASIE, VERIFIKASIE, STAWING, DEURLOPENDE TOEGANG Sleutelwoorde: INTELLEKTUALISERING, TERMINOLOGIEONTWIKKELING, OES, SKARE- DEELNAME, KONSULTASIE, VERIFIKASIE, STAWING, DEURLOPENDE TOEGANG Intellectualization through Terminology Development* Langa Khumalo, Linguistics Program, School of Arts, University of KwaZulu-Natal, South Africa (khumalol@ukzn.ac.za) Abstract: The term intellectualization was famously used in the Prague School to describe a process that a language undergoes in its development and refinement. In our South African context intellectualization entails a carefully planned process of hastening the cultivation and growth of indigenous official African languages so that they effectively function in all higher domains as languages of teaching and learning, research, science and technology. This article critically exam- ines the terminology development process that is being driven at the University of KwaZulu-Natal (henceforth UKZN) as one of the key agents of language intellectualization. The article critically evaluates the UKZN terminology development model that is used to harvest, consult and authenti- cate isiZulu terminology for Administration, Architecture, Anatomy, Computer Science, Environ- mental Science, Law, Physics, Psychology, and Nursing disciplines. Outflow platforms for the ter- minology in this development model are loosely listed as the 'database' and the 'development platform' but there is no clear end-user platform for students and lecturers, who seem to be the main end-user-targets of the whole terminology development initiative. The article will propose an improved model to cater for AnyTime Access, which is convenient for student needs between lec- tures, and improve the harvesting mechanism in the existing model. Keywords: INTELLECTUALIZATION, TERMINOLOGY DEVELOPMENT, HARVESTING, CROWDSOURCING, CONSULTATION, VERIFICATION, AUTHENTICATION, ANYTIME ACCESS Opsomming: Intellektualisering deur terminologieontwikkeling. Die term intellektualisering is oorspronklik deur die Praagse skool gebruik om 'n proses wat 'n taal tydens sy ontwikkeling en verfyning ondergaan te beskryf. In ons Suid-Afrikaanse konteks behels intellek- tualisering 'n sorgvuldig beplande proses om die ontwikkeling en groei van plaaslike amptelike Afrikatale te bespoedig sodat hulle effektief op alle hoëvlakterreine funksioneer as tale van onder- rig en leer, navorsing, wetenskap en tegnologie. Hierdie artikel doen 'n kritiese ondersoek na die terminologieontwikkelingsproses wat by die Universiteit van KwaZulu-Natal (voortaan UKZN) bestuur word as een van die sleutelfigure van taalintellektualisering. UKZN se terminologieont- wikkelingsmodel word gebruik om isiZulu-terminologie binne die dissiplines Administrasie, Argi- tektuur, Anatomie, Rekenaarwetenskap, Omgewingswetenskap, die Regte, Fisika, Sielkunde en Verpleegkunde te oes, te raadpleeg en te staaf. Die artikel evalueer hierdie model krities. Afvoer- Lexikos 27 (AFRILEX-reeks/series 27: 2017): 252-264 http://lexikos.journals.ac.za 253 Intellectualization through Terminology Development Intellectualization through Terminology Development platforms vir die terminologie in hierdie ontwikkelingsmodel word losweg gelys as die 'databasis' en die 'ontwikkelingsplatform', maar daar is geen duidelike eindgebruikerplatform vir studente en dosente nie, wat waarskynlik die belangrikste eindgebruikerteikengroep van die hele terminologie- ontwikkelingsinisiatief vorm. Die artikel stel 'n verbeterde model voor wat deurlopende toegang sal bied, wat gerieflik vir studente se behoeftes tussen lesings sal wees, en wat die oesmeganisme in die bestaande model sal verbeter. 1. Introduction It can be argued that South African Higher Education (henceforth SAHE) is increasingly embracing the imperative of transmuting African languages to be the kernel of the academy. This has been recently articulated in the #FeesMustFall initiative (cf. Chetty and Knaus 2016). Hitherto the dominant languages in SAHE have been English and Afrikaans. It has been persuasively argued that the high attrition rate in Africa's education system is in part due to the fact that Africa is uniquely one of the few continents where children receive education in foreign tongues (cf. Finlayson and Madiba 2002). It is in this vein that the UKZN language policy and plan (2006 revised 2014) recognizes the prominent role that language plays in the teaching and learning. Through its language policy and plan, UKZN has taken an initiative to cultivate, modernize and elaborate isiZulu so that it becomes a vehicle in knowledge production and knowledge dissemination. One of the stated aims of the UKZN language policy is to "achieve for isiZulu the institutional and academic status of English" and to "provide facilities to enable the use of isiZulu as a language of learning, instruction, research and administration." (Language Policy of the UKZN 2014: 2). It is in this regard that, while UKZN upholds the principle of multilingualism, English and isiZulu are the two official languages of the institution. It has thus been persua- sively argued that in order for African languages to be used in education as languages of instruction, innovation, science, mathematics and logic, there has to be a clear, conscious and careful process of the intellectualization of these languages. Through its language policy and plan, UKZN articulates a clear objective to remove language as a barrier of access and success. The notion of access and success is articulated in the 1994 UNESCO World Conference framework of action where the following was noted: that our education systems should http://lexikos.journals.ac.za 254 Langa Khumalo SAHE is thus encouraged to: […] recognize and respond to the diverse needs of their students, accommodat- ing both different styles of learning and ensuring quality education to all through appropriate curricula, organizational arrangements, teaching strategies, resource use and partnerships with their communities. (UNESCO, 1994, Framework for Action on Special Needs Education: 11-12) […] recognize and respond to the diverse needs of their students, accommodat- ing both different styles of learning and ensuring quality education to all through appropriate curricula, organizational arrangements, teaching strategies, resource use and partnerships with their communities. (UNESCO, 1994, Framework for Action on Special Needs Education: 11-12) Hence UKZN's strategy to cultivate isiZulu so that it becomes a language of administration and all academic activity in order to eliminate problems of lan- guage being a barrier to access and success. In order to achieve this, UKZN has committed huge financial and human resources. This article thus critically examines the UKZN's language program. The article locates terminology development as one of the ways through which the University seeks to intel- lectualize isiZulu. The article further evaluates the limitations of the Univer- sity's current terminology development model, and proposes an improved model to cater for the novel notion of AnyTime Access that we motivate here in order to satisfy end-user-needs. Busch and Press (2014) as the doyens in the scholarship of intellectualization. Sibayan (1999: 229) characterizes an intellectualized language as one: Busch and Press (2014) as the doyens in the scholarship of intellectualization. Sibayan (1999: 229) characterizes an intellectualized language as one: […] which can be used for educating a person in any field of knowledge from kindergarten to the university and beyond. Thus, an intellectualized language has the capacity to discuss any issue regardless of its complexity. According to Finlayson and Madiba (2002: 53), in the South African context intellectualization is a meticulous procedure aimed at expediting the growth and development of hitherto underdeveloped African languages to augment their capacity to effectively interface with modern developments, theories and concepts. It is imperative to note that germane to this process is the development of discipline specific terminology. The paucity of such specialized terminology is often cited as the reason why African lan- guages cannot be used as languages of teaching and learning, hence their dis- cernment as shallow and inadequate (cf. Shizha 2012). q Intellectualization in our context thus means the radical transformation of the capacity and role of indigenous African languages in carrying and convey- ing all forms of knowledge in all spheres of life. While the government through the Constitution (1996, section 6) has expressed commitment "to elevate the status and advance the use of" these hitherto underdeveloped languages, very little has actually been done to improve their status and role in SAHE (cf. Olivier 2014). The argument on the status and role of these languages has been sharply brought back to the center of SAHE through the #FeesMustFall. Writ- ing on the #FeesMustFall, Chetty and Knaus (2016) posit that at the center of the 2015 student protests was the disruption of the status quo with regards to "[…] institutions' language policies, high fees, structural inequalities and colo- nial symbols." 2. Intellectualization is a term originally used by Havranek (1932), a linguist from the Prague School, to characterize a process that a language undergoes in its advancement. By the intellectualization of the standard language, which we could also call its rationalization, we understand its adaptation to the goal of making possible pre- cise and rigorous, if necessary abstract, statements, capable of expressing the con- tinuity and complexity of thought, that is, to reinforce the intellectual side of speech. This intellectualization culminates in scientific (theoretical) speech, deter- mined by the attempt to be as precise in expression as possible, to make statements which reflect the rigor of objective (scientific) thinking in which the terms approximate concepts and the sentences approximate logical judgements. (My emphasis). (Havránek 1932: 32-84) Intellectualization thus is a clear process of (functionally) cultivating, develop- ing, elaborating and modernizing a language so that the terminology of the language can carry the full weight of scientific rigor and precision, and that its sentences can accurately express logical judgements resulting in a language that has the capacity to function in all domains. As the direct consequence of intellectualization the speakers of the language derive the pride, self-assurance and resourcefulness in the (new) ability to discuss the most complex of issues ranging from the mundane to academic and beyond. Intellectualization thus is a clear process of (functionally) cultivating, develop- ing, elaborating and modernizing a language so that the terminology of the language can carry the full weight of scientific rigor and precision, and that its sentences can accurately express logical judgements resulting in a language that has the capacity to function in all domains. As the direct consequence of intellectualization the speakers of the language derive the pride, self-assurance and resourcefulness in the (new) ability to discuss the most complex of issues ranging from the mundane to academic and beyond. Intellectualization has been famously associated with the development of Tagalog in the Philippines. The cultivation process involved Tagalog's lexical enrichment through terminology to enable its use in academia. Philippine lin- guistics and sociolinguists are recognized by Neville Alexander in Busch, http://lexikos.journals.ac.za 255 Intellectualization through Terminology Development Intellectualization through Terminology Development Busch and Press (2014) as the doyens in the scholarship of intellectualization. Sibayan (1999: 229) characterizes an intellectualized language as one: 3. UKZN has a University Language Board (henceforth ULB), which was set up through a committee charter (2006 amended 2010). The ULB meets quarterly and its mandate is to develop, implement, monitor and review the University Language Policy and Plan. The ULB is required to report to the University Sen- ate once a year, and importantly its annual report to Senate must be in both English and isiZulu. It is chaired by a Deputy Vice-Chancellor (henceforth DVC), who is also a member of the University's Executive. As reiterated above, UKZN first adopted its Language Policy and Plan in 2006, which was subse- quently revised in 2014 following the University's college reorganization and the introduction of the Use of Official Languages Act of 2012. In its strategy to institutionalize language planning and development, UKZN established the University Language Planning and Development Office (henceforth ULPDO), which is headed by a Director who is a member of the ULB, and reports to the DVC. http://lexikos.journals.ac.za 256 Langa Khumalo Langa Khumalo Langa Khumalo The program to intellectualize isiZulu so that it (ultimately) functions at par with English in all high function domains across the University is initiated by the ULPDO, and is reported quarterly at the ULB. The major thrust of the language program (see Fig. 1.) as approved by the ULB is the creation of disci- pline specific terminology in isiZulu, the building of an isiZulu National Cor- pus, and the development of a contemporary body of literature in isiZulu. Other language activities include the provision of training workshops, transla- tion and (simultaneous) interpreting services, Sign Language advocacy, the Sesotho Bua Le Nna (Let's Talk) Program, language research, and the develop- ment of computational tools. Figure 1: Language Program The article critically evaluates the UKZN terminology development model (see Fig. 2.) used to harvest, consult and authenticate isiZulu terminology for Administration, Architecture, Anatomy, Computer Science, Environmental Science, Law, Physics, Psychology, and Nursing to date. In this model, outflow platforms for the authenticated terminology are loosely indicated as the 'data- base' and 'development platform'. There is no clear end-user platform for stu- dents in the model, yet the students seem to be the main end-user-target of the whole terminology development initiative. Other important end-users are lec- turers, teachers, lexicographers, educational interpreters and translators, isiZulu speakers, and authors. 3. The article will propose an improved model to cater for AnyTime Access, which is convenient particularly for student needs between lectures, and propose an improved harvesting mechanism for the existing model. We critically examine the terminology development model in section 4, discuss in section 5 and conclude in section 6. Figure 1: Language Program Figure 1: Language Program Figure 1: Language Program The article critically evaluates the UKZN terminology development model (see Fig. 2.) used to harvest, consult and authenticate isiZulu terminology for Administration, Architecture, Anatomy, Computer Science, Environmental Science, Law, Physics, Psychology, and Nursing to date. In this model, outflow platforms for the authenticated terminology are loosely indicated as the 'data- base' and 'development platform'. There is no clear end-user platform for stu- dents in the model, yet the students seem to be the main end-user-target of the whole terminology development initiative. Other important end-users are lec- turers, teachers, lexicographers, educational interpreters and translators, isiZulu speakers, and authors. The article will propose an improved model to cater for AnyTime Access, which is convenient particularly for student needs between lectures, and propose an improved harvesting mechanism for the existing model. We critically examine the terminology development model in section 4, discuss in section 5 and conclude in section 6. http://lexikos.journals.ac.za 257 4. Terminology Development Model The harvested terms are a wordlist of key terms created from a main course/module or a major reference work. Because of the expenses involved in convening a single terminology develop- ment workshop (each workshop ca. R65 000–R95 000), the ULPDO has imposed a minimum requirement of at least 500 terms to be submitted for them to be taken through the development processes. Each workshop is a minimum of three days and can go up to five days depending on the complexity of the subject or discipline. j p Once the terms are harvested and have been submitted to the ULPDO, the latter convenes a consultative workshop with a panel of at least two discipline experts, lexicographers, linguists, terminologists, and students of up to 25 indi- viduals to describe, create and codify the isiZulu terms. This approach can be rightly criticized as top-down, elitist and exclusionary because it seems to tar- get experts to the exclusion of general isiZulu speakers and other stakeholders who are outside the academe. A counter-argument is that terminology devel- opment is inherently a specialized task for people with certain levels of exper- tise for use by people working or training in a particular specialized discipline. A balance of the top-down and bottom-up approach is argued for in section 5 below. Once the terms have been described and codified the ULPDO convenes a second workshop for the PanSALB subcommittee of isiZulu national language body to consider the terms. This is called the verification workshop. After the verification committee has thoroughly engaged academics and students in these often grueling verification workshops, the agreed terms are then submit- ted to the full isiZulu national language body (UMZUKAZWE) for authentica- tion and standardization. The UMZUKAZWE is convened by the PanSALB regional office through the request of the ULPDO to consider and approve the consulted and verified terminology. Once the full UMZUKAZWE has approved the terms in the presence of the discipline experts, ULPDO notifies the ULB, and afterwards the terms are ready to be used in the lecture room and in formal academic discourses. In the current terminology development model the authenticated terms are stored in a database. It is crucial to observe that these workshops (consultative, verification and authentication) are arduous to both academics and students, and the members of each committee involved in the process. 4. Terminology Development Model UKZN has identified terminology development as one of the main cogs (together with corpus building and computer assisted language tools) to drive the goal of isiZulu intellectualization. One of the important principles in the development of specialized terminology at UKZN is to observe statutory man- datory processes. These processes are driven by the Pan South African Lan- guage Board (henceforth PanSALB), which is a Board constituted through an Act of Parliament. Through the provisions of the PanSALB Act 59, (1999), there exists an isiZulu National Language Body, UMZUKAZWE, which is short for UMkhandlu WesiZulu Kuzwelonke. In light of these provisions, UKZN has designed a unique terminology development process with five important stages that incorporate the statutory processes facilitated by PanSALB through its KwaZulu-Natal Provincial office. There are five stages in the UKZN terminology development model and these include 1) harvesting of existing usage terms; 2) description and translation of terminology that has been harvested or created; 3) consultation and verification with end-users about terminology proposed, and 4) authentication and standardi- zation through official national structures. The "finalization" of the process in the current model takes place in 5) through the listing of these terms on the termi- nology databases for wider institutional and national usage. The five stages are illustrated in Figure 2 below. Figure 2: UKZN Terminology Development Model (cf. Khumalo 2016) The current terminology development model as shown in Fig. 2 relies on the discipline lecturers, as experts in their fields, to initiate the harvesting of terms that are central to the module or course that they lecture in. While the UKZN Figure 2: UKZN Terminology Development Model (cf. Khumalo 2016) The current terminology development model as shown in Fig. 2 relies on the discipline lecturers, as experts in their fields, to initiate the harvesting of terms that are central to the module or course that they lecture in. While the UKZN http://lexikos.journals.ac.za 258 Langa Khumalo Langa Khumalo language policy exists as an instrument that enables the development of teaching materials in both English and isiZulu, the enforcement of the policy is tepid, cautious and not compulsory. Hence harvesting is thus done voluntarily by discipline lecturers who are committed to the principles of the UKZN lan- guage policy, and also realize the value in making their teaching materials available to students in both languages. Intellectualization through Terminology Development Intellectualization through Terminology Development Science, Environmental Science, Law, Nursing Science, Physics, Psychology, and Research. The terminology development model is cyclical. Once a discipline has gone through all the processes, ULPDO moves on to the term list submitted by the next discipline experts and the process is repeated. It is clear that the pro- cess requires a huge investment both in terms of time and financial resources. 4. Terminology Development Model It is through sheer individual commitment and dedication by aca- demics in each disciplines, and the members of the isiZulu language commit- tees, that UKZN has completed in a narrow sense (given that terminology development is a continuous process) the terminology development processes in the following disciplines: Administration, Architecture, Anatomy, Computer http://lexikos.journals.ac.za 259 Intellectualization through Terminology Development 5. Discussion In this section we critically evaluate the terminology development model. The current model relies on the discipline lecturers to initiate harvesting. This har- vesting work is done over and above the discipline lecturer's other teaching, research, and administrative commitments. Those who have done so have demonstrated outstanding commitment. However the harvesting has become slow and erratic. This is attributable to the University's Teaching Workloads Framework (used to assess teaching) and Performance Measurers (used to assess research, among other things). According to the Teaching Workloads Framework the norm is that a lecturer must have at least 810 hours of teaching. The approved performance measures are 60 Productivity Units (henceforth PU) for lecturers, 90 PUs for senior lecturers, 120 PUs for associate professors and 150 PUs for professors per year. A single publication in a Department of Higher Education and Training (DHET) approved journal carries 60 PUs. This means a lecturer has to publish at least a single journal article per annum, while a pro- fessor has to publish two and a half articles in the same period. There are indeed some lecturers who have abandoned terminology development for their disciplines citing huge workloads, while expressing the obvious need for isiZulu terminology as a useful intervention to improve conceptual and epis- temic access. To mitigate erratic terminology harvesting, and the effects of a clearly top- down and exclusionist approach to terminology development, ULPDO is introducing (computational) technology in term harvesting. Crowdsourcing has been advanced as a useful strategy to harness discipline specific terminol- ogy. This argument was advanced when ULPDO was developing isiZulu ter- minology in computer science. The two discipline experts, Dr Maria Keet and Dr Graham Barbour created a novel method (cf. http://www.commuterm.co.za/) of harnessing terms in computer science using computational resources (cf. Keet and Barbour 2014). This is a useful strategy to improve the collection of termi- nology, which can also be further extended to include online commentary in order to augment the role of the consultative workshop, with a view of replacing it in the long term in order to reduce costs. The current UKZN terminology development model can be criticized for being a top-down approach to language planning. Figure 3: Zulu Lexicon Screenshot In order to also improve access to the authenticated and finalized terminology we propose a clearer end-user platform(s) for students, lecturers, teachers, lexi- cographers, educational interpreters and translators, and isiZulu speakers. Hitherto a lot of terminology has been developed by many universities in the country, the National Language Services in the Department of Arts and Culture (DAC), and the South Africa Norway Tertiary Education Development Pro- gram (SANTED) multilingual glossary project, among others. Terminology 5. Discussion Language planning is often viewed as a top-down process, which is characterized by Cooper (1989: 45), and Grin (1996: 31) as a considered, planned, systematic effort to influence and http://lexikos.journals.ac.za 260 Langa Khumalo affect the language behavior of a particular society through crafting and implementing regulations or policies, thus regulating a speech community's language behavior. According to Webb (2002: 42) for language planning to be effective, top-down processes need to be complemented by a bottom-up approach, which means that the interest, views, attitudes and linguistic com- petences of the targeted speech community must be accommodated. y In explicating the bottom-up approach, Kaplan and Baldauf (1997: 50), distinguish between activities of governments and agencies and activities of pressure groups and individuals in the language planning process (cf. Sithole 2017). According to these distinctions, a bottom-up approach to language plan- ning refers to the language cultivation activities of individual and pressure group agencies. We argue in this article that a judicious mixture of the top- down and bottom-up approach may render a better outcome for the UKZN terminology development model. In order to also improve access to the authenticated and finalized terminology we propose a clearer end-user platform(s) for students, lecturers, teachers, lexi- cographers, educational interpreters and translators, and isiZulu speakers. Hitherto a lot of terminology has been developed by many universities in the country, the National Language Services in the Department of Arts and Culture (DAC), and the South Africa Norway Tertiary Education Development Pro- gram (SANTED) multilingual glossary project, among others. Terminology In order to also improve access to the authenticated and finalized terminology we propose a clearer end-user platform(s) for students, lecturers, teachers, lexi- cographers, educational interpreters and translators, and isiZulu speakers. Hitherto a lot of terminology has been developed by many universities in the country, the National Language Services in the Department of Arts and Culture (DAC), and the South Africa Norway Tertiary Education Development Pro- gram (SANTED) multilingual glossary project, among others. Terminology http://lexikos.journals.ac.za 261 Intellectualization through Terminology Development Intellectualization through Terminology Development developed is stored away in some inaccessible databases without any dissemina- tion strategy. ULPDO has developed two open-source platforms to cater for a broad-based end-user uptake of its authenticated terminology. ULPDO has also introduced the notion of AnyTime access so that students and other users can access the terms anytime they need them. ULPDO has exploited technol- ogy to develop improved end-user access through the development of a mobile compatible application. The application is called the Zulu Lexicon, which is short for isiZulu Lexicon, and is a free download from iTunes and Google Play in Smartphone and Android. The Zulu Lexicon affords students, lecturers, and other end-users anytime access at their fingertips to all the authenticated (mul- tidisciplinary) terminology, with search functions on the APP in both English and isiZulu as shown in Fig. 3. g Fig. 3 shows the landing page for the Zulu Lexicon App (on the left hand side) the word-of-the-day in the discipline of anatomy in the middle, and the a- z lexicon with random entries under the letter I of the Roman alphabet (on the far right). The APP has other filters to make searching very easy, and user- friendly for users to find the kind of information they want. y y The second open source terminology resource is the isiZulu Term Bank. ULPDO developed the isiZulu Term Bank (ITB) as an open source database that is freely accessible on the following URL: https://ukzntermbank.ukzn. ac.za. It is a flexible system that can be incrementally populated with more authenticated terms for existing disciplines that are already in the database or with new disciplines whose terms have been developed and authenticated. Figure 4 below shows the ITB screenshot. Figure 4: ITB Screenshot Figure 4: ITB Screenshot Figure 4: ITB Screenshot Figure 4: ITB Screenshot http://lexikos.journals.ac.za 262 Langa Khumalo Langa Khumalo The left screen shows the landing page for the isiZulu Term Bank. The landing page will flash the most popular word of the day, week and month on the screen. The screen window is closed upon clicking the close button at the bot- tom of flashing page. On the right side of Fig. 4 is the screen showing the home page with easy to use functionalities. Intellectualization through Terminology Development The end-user can search for a term by language (English or isiZulu), word structure (prefix, suffix or complete word) or by discipline, by following a drop down menu. The Zulu Lexicon APP and the isiZulu Term Bank are two open source technology platforms that the ULPDO has developed in order to effectively disseminate the authenticated isiZulu terminology that has been developed to date in the disciplines of Administration, Architecture, Anatomy, Computer Science, Environmental Science, Law, Physics, Psychology, and Nursing Science. As open source resources, the Zulu Lexicon application and the isiZulu Term Bank are freely available to students, lecturers, teachers, lexicographers, educational interpret- ers and translators, isiZulu speakers, and authors. Intellectualization through Terminology Development Intellectualization through Terminology Development It is clear that stages 1 and 2 in the current ULPDO terminology develop- ment model (term harvesting and consultation workshops) can be enhanced using computational resources. It must be argued that the discipline expert knowledge is required consistently throughout the terminology development process. In order for these experts to be able to also participate in the online platforms (and at a later stage in verification and authentication workshops), a process must be initiated by the ULPDO to submit to the ULB and ultimately to Senate an argument to formally recognize participation in the terminology development within the Teaching and Workloads Framework. This will ensure that expert standards are retained, and rebut the argument that terminology development for African languages is an exercise in dumbing down education. The strategy to improve access to multidisciplinary authenticated termi- nology using a novel mobile application the Zulu Lexicon and the isiZulu Term Bank introduces to the UKZN terminology development model new end-user platforms. The multidisciplinary terms are now available anytime to students, lecturers, teachers, lexicographers, educational interpreters and translators, isiZulu speakers, and authors. 6. Conclusion The article has argued that intellectualization is a gradual process, which cul- minates in a scientific language capable of reflecting the rigor of objective thinking. While the Constitution (1996) recognizes the importance of develop- ing the official indigenous African languages, the imperative to cultivate them for higher level functions in order to improve access and success at higher edu- cation institutions in order to arrest rampant attrition rates has been brought sharply to the fore in the recent #FEESMUSTFALL initiative. SAHE is thus embracing the importance of bringing these hitherto marginalized languages at the very center of teaching and learning. This is arguably a very length process. y g g g y y g p The article has argued that terminology development is one of the vital cogs in the intellectualization process. The paucity of terminology is often cited as the reason why African languages cannot contribute meaningfully to the knowledge economy, and this is articulated in Bamgbose (2002: 2), when citing lack of political will by those in authority as an impediment: "[…] They are quick to point out that African languages are not yet well developed to be used in certain domains or that the standard of education is likely to fall […]." We thus argue in this article that terminology development should not be exclu- sively a top-down and selective process involving workshops often with a few discipline experts, terminologists, lexicographers and linguists, which is very resource intensive, but must embrace a bottom-up approach through crowd- sourcing (cf. Keet and Barbour 2014) in cases of terminology harvesting, including usurping certain functions of the consultative workshops in order to improve efficiency and reach, enhance acceptance, and preclude exclusion while cutting back on expenditure. Thus, the UKZN terminology development process must embrace a mixed approach in which the top-down and bottom- up approaches complement each other. http://lexikos.journals.ac.za 263 Webb, V.N. 2002. Language in South Africa: The Role of Language in National Transformation, Recon- struction and Development. Amsterdam/Philadelphia, Penn: John Benjamins. http://lexikos.journals.ac.za Sithole, E. 2017. From Dialect to 'Official' Language: Towards the Intellectualization of Ndau in Zim- babwe. Unpublished D.Phil. Thesis. Grahamstown: Rhodes University. UNESCO. 1994. The Salamanca Statement and Framework for Action on Special Needs Education. Paris: UNESCO. p y UNESCO. 1994. The Salamanca Statement and Framework for Action on Special Needs Education. Paris: UNESCO. Webb, V.N. 2002. Language in South Africa: The Role of Language in National Transformation, Recon- struction and Development. Amsterdam/Philadelphia, Penn: John Benjamins. Sithole, E. 2017. From Dialect to 'Official' Language: Towards the Intellectualization of Ndau in Zim- babwe. Unpublished D.Phil. Thesis. Grahamstown: Rhodes University. UNESCO. 1994. The Salamanca Statement and Framework for Action on Special Needs Education. Paris: UNESCO. Webb, V.N. 2002. Language in South Africa: The Role of Language in National Transformation, Recon- struction and Development. Amsterdam/Philadelphia, Penn: John Benjamins. Sithole, E. 2017. From Dialect to 'Official' Language: Towards the Intellectualization of Ndau in Zim- babwe. Unpublished D.Phil. Thesis. Grahamstown: Rhodes University. UNESCO. 1994. The Salamanca Statement and Framework for Action on Special Needs Education. Paris: UNESCO. Webb V N 2002 Language in South Africa: The Role of Language in National Transformation Recon References Bamgbose, A. 2002. Launch of the Activities of the African Academy of Languages: Mission and Vision of the ACALAN. ACALAN Special Bulletin 2002: 24-25. Chetty, R. and C.B. Knaus. 2016. #FeesMustFall: SA's Universities are in the Grip of a Class Strug- gle. Mail&Guardian, 14 January 2016. Accessed on 31 July 2016. Cooper, R.L. 1989. Language Planning and Social Change. Cambridge: Cambridge University Press. Finlayson, R. and M. Madiba. 2002. The Intellectualization of the Indigenous Languages of South Af i Ch ll d P t C t I i L Pl i 3(1) 40 61 Finlayson, R. and M. Madiba. 2002. The Intellectualization of the Indigenous Languages of South Africa: Challenges and Prospects. Current Issues in Language Planning 3(1): 40-61. nges and Prospects. Current Issues in Language Planning 3(1): 4 Grin, F. 1996. The Economics of Language: Survey, Assessment, and Prospects. Grin, F. (Ed.). 1996. Economic Approaches to Language and Language Planning. International Journal of the Sociology of Language 121: 17-44. Havranek, B. 1932. The Functions of Literary Language and its Cultivation. Havranek, B and M. Weingart (Eds.). 1932. A Prague School Reader on Esthetics, Literary Structure and Style: 32- 84. Prague: Melantrich. Kaplan, R.B. and R.B. Baldauf. 1997. Language Planning from Practice to Theory. Clevedon: Multi- lingual Matters. Keet, C.M. and G. Barbour. 2014. Limitations of Regular Terminology Development Practices: The Case of isiZulu Computing Terminology. Alternation: Special Edition 12: 13-48. Khumalo, L. 2016. Disrupting Language Hegemony: Intellectualizing African Languages. Samuel, M., R. Dhunpath and N. Amin (Eds.). 2016. Disrupting Higher Education Curriculum. Undoing Cog- nitive Damage: 247-264. Rotterdam/Boston/Taipei: Sense Publishers. Olivier, J. 2014. Compulsory African Languages in Tertiary Education: Prejudices from News Web- site Commentary. Southern African Linguistics and Applied Language Studies 32(4): 483-498. Shizha, E. 2012. Reclaiming and Re-visioning Indigenous Voices: The Case of the Language of Instruction in Science Education in Zimbabwean Primary Schools. 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https://openalex.org/W3107868738	https://www.researchsquare.com/article/rs-15025/v2.pdf?c=1603314781000	English	null	Clinical outcomes and prognostic factors in bloodstream infections due to extended-spectrum β-lactamase-producing Enterobacteriaceae among patients with malignancy: a meta-analysis	Annals of clinical microbiology and antimicrobials	2,020	cc-by	7,814	Ai-Min Jiang Hao-Ran Zheng Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xue Chen Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Chao-Xin Fan Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Rui Zhang Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xiao-Qiang Zheng Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xiao Fu Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Yu Yao Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Tao Tian (  tiantao0607@163.com ) Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Clinical outcomes and prognostic factors in bloodstream infections due to extended-spectrum β-lactamase-producing Enterobacteriaceae among patients with malignancy: a meta-analysis Ai-Min Jiang Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Na Liu Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Rui Zhao Huazhong University of Science and Technology Hao-Ran Zheng Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xue Chen Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Chao-Xin Fan Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Rui Zhang Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xiao-Qiang Zheng Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Xiao Fu Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Yu Yao Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Tao Tian (  tiantao0607@163.com ) Xi'an Jiaotong University Medical College First Affiliated Hospital Department of Medical Oncology Research Version of Record: A version of this preprint was published on November 23rd, 2020. See the published version at https://doi.org/10.1186/s12941- 020-00395-7. Page 1/14 Page 1/14 Page 1/14 Page 1/14 Background In recent years, the incidence of bloodstream infections (BSIs) caused by Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) has been increasing over time all over the world. (1) There is a growing body of evidence to show that BSIs caused by ESBL-PE are more worrisome in clinical practice. Extended-spectrum β-lactamases (ESBLs) mediates resistance to a wide variety of antibiotics, including third-generation cephalosporins, aminoglycosides, and quinolones. Furthermore, most of empirical antimicrobial regimens can not cover these pathogens. (2, 3) Therefore, the antimicrobial therapeutic regimens are often limited in these infections. (1) Patients with malignancy are more vulnerable to developing severe infection, including those caused by ESBL-PE since they are more likely to be immunocompromised due to chemotherapy, radiotherapy, surgery, invasive procedures, malnutrition, and malignancy itself. (4, 5) As a result, these infections have become significant therapeutic challenges for clinicians due to delayed initiation of chemotherapy, reduced standard dosage, prolonged hospitalization, increased financial burden on healthcare, and raised severe morbidity and mortality. (3, 6) Therefore, rapid initiation of appropriate antibiotic therapy is pivotal for oncological patients with BSIs caused by ESBL-PE, (4) while inappropriate empirical antibiotic treatment is associated with worse outcomes and survival. (3) Previous meta-analyses have investigated the prevalence of BSIs caused by ESBL-PE in patients with malignancy. (7, 8) However, there was no further analysis of clinical outcomes and risk factors in these populations. Therefore, we conducted this study to assess the prognosis and risk factors of BSIs due to ESBL-PE in patients with malignancy and provide updated evidence via meta-analysis. Search strategy Our meta-analysis was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. (9) We conducted an overall literature retrieval for PubMed, EMBASE, and Cochrane Library published up to 10 December 2019. Both MeSH terms and free words were used to search for title/ abstract. Our search terms were: “(ESBL OR (extended-spectrum beta-lactamase) OR (extended-spectrum β-lactamase)) AND (tumor OR neoplasia OR malignancy OR cancer OR carcinoma OR sarcoma OR leukemia OR leukaemia OR lymphoma OR hematolog* OR haematolog* OR oncolog*)”. We manually screened other relevant studies and reference lists. The search was performed independently by two investigators (AM Jiang and N Liu). Abstract Background: The colonization of Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) in bloodstream infections (BSIs) has been increased dramatically worldwide, and it was associated with worse clinical outcomes in patients with malignancy. We performed the meta- analysis to investigate the prognosis and risk factors in BSIs caused by ESBL-PE in oncological patients. Methods: PubMed, EMBASE, and Cochrane Library were searched for related studies. All-cause mortality was considered as the primary outcome. Subgroup analyses, meta-regression analyses, and sensitivity analysis were used to investigate heterogeneity and reliability in results. Results: 6,729 patients from 25 studies were eligible. Six studies enrolled oncological patients with BSIs caused by ESBL-PE only, while 19 studies both enrolled ESBL-PE and non-ESBL-PE infections. The results showed that BSIs caused by ESBL-PE in patients with malignancy was associated with higher mortality than non-ESBL-PE infections (RR = 2.21, 95 % CI: 1.60–3.06, P < 0.001), with a significant between-study heterogeneity (I2 = 78.3%, P < 0.001). Subgroup analyses showed that children (RR = 2.80, 95 % CI: 2.29–3.43, P < 0.001) and hematological malignancy (RR =3.20, 95 % CI: 2.54–4.03, P < 0.001) were associated with a higher mortality. Severe sepsis/ septic shock, pneumonia, and ICU admission were the most common predictors of mortality. Conclusions: Our study identified that BSIs caused by ESBL-PE in patients with malignancy were associated with worse clinical outcomes compared with non-ESBL-PE infections. Furthermore, children and hematological malignancy were associated with higher mortality. Severe sepsis/ septic shock, pneumonia, and ICU admission were the most common predictors of mortality. Data extraction and quality assessment Definitions and study outcomes Neutropenia was defined as an absolute neutrophil count of <500 neutrophils/mm3. (11) Empirical antibiotics treatment was considered inappropriate once the antibiotics could not suppress the activity of the isolated pathogens according to the results of antimicrobial susceptibility tests during the first 24 h after the blood culture was obtained. (11) The all-cause mortality at the end of the study and the predictors in BSIs due to ESBL-PE in patients with malignancy were the primary outcomes in the study. (12) Study characteristics and quality assessment Our literature search identified 1,260 studies. After excluding repeated records and the initial screening based on titles and abstracts, 25 articles were eligible in this study. Of these, six studies enrolled oncological patients with BSIs caused by ESBL-PE only, while 19 studies both enrolled ESBL-PE and non-ESBL-PE infections. Of the 25 studies, there were eight prospective cohort studies, 14 retrospective cohort studies, and three case- control studies. All included studies were published between 2009 and 2019, and there were six studies published in 2019, accounting for 24%. There were 15 studies conducted in Asia, seven studies conducted in Europe, and three studies were conducted in North America. The detailed flow chart of the study selection process was described in Figure 1. Table 1 summarized the characteristics of 25 selected studies. The majority of the included studies had a NOS score of more than 5 points, and 21 studies included in this article were high-quality studies. Table S1 presented the results of the quality assessment. Statistical analysis The RRs and 95% CIs for mortality were calculated to assess the outcomes of BSIs caused by ESBL-PE in oncological patients. All results were depicted as forest plots. Heterogeneity was assessed using Cochran’s Q test and the I2 statistic test. When the heterogeneity was statistically significant (P < 0.05 and I2 > 50%), a random-effects model was applied to obtain the pooled RRs; otherwise, a fixed-effects model was performed. Subgroup analyses and meta-regression analyses were conducted to explore the sources of heterogeneity. We also performed a sensitivity analysis to evaluate the quality and stability of results by omitting one study in each turn. Begg’s test and Egger’s test were used to assess the publication bias. Statistical tests were two-tailed at the significance level of P < 0.05. All analyses were used with STATA V.14.0 (Stata Corporation, College Station, TX). Mortality In all the studies, the time of death records was not similar. The majority of studies used 30-day mortality to evaluate the clinical outcomes of BSIs caused by ESBL-PE in patients with malignancy, (13-29) only one study did not report a particular time of death. (30) In studies that only enrolled oncological patients with ESBL-PE infections, the mortality of BSIs varied from 10.7% to 31.0%. However, the mortality of BSIs varied from 4.8% to 51.0 % in studies that both enrolled ESBL-PE and non-ESBL-PE infections oncological patients, respectively. We finally included 19 studies that enrolled both ESBL-PE and non-ESBL-PE infected oncological patients into analyses to estimate the mortality of BSIs caused by ESBL-PE in patients with malignancy. The results showed that in patients with malignancy, ESBL-PE infections were associated with a higher mortality risk from BSIs than non-ESBL-PE infections (RR = 2.21, 95 % CI: 1.60–3.06, P < 0.001) (Figure 2), with a significant between-study heterogeneity (I2 = 78.3%, P < 0.001). Study selection Studies were considered as eligible based on the following criteria: (1) population: patients with solid or hematological malignancies; (2) intervention (exposure): BSIs caused by ESBL-PE; (3) comparison: BSIs caused by non-ESBL-PE; (4) outcome: the mortality of BSIs. Literature that satisfied the following criteria were excluded: (1) letters, case reports, editorials, expert opinion or reviews without original data; (2) overlapping or duplicate data; (3) incomplete data about outcomes; (4) not English literatures; (5) the sample size of BSIs caused by ESBL-PE in oncological patients less than 10; (6) studies only focusing on risk factors for ESBL-PE infections. Data extraction and quality assessment Page 2/14 Two investigators (AM Jiang and N Liu) independently extracted the data using a standardized approach. Any disagreement in the study selection and data extraction phases was resolved through discussion with the third investigator (R Zhao). The following data information was retrieved from each article: first author’s name, year of publication, country, study population, infection type of BSIs, the total number of screened subjects, the total number of ESBL-PE caused BSIs, the total number of BSIs caused death and ESBL-PE BSIs caused death, and ESBL detection method. The data was extracted from texts or tables in articles. Newcastle Ottawa Quality Assessment Scale (NOS) was used in our research to assess the quality of selected studies (10). The scale included three aspects: selection, comparability, and outcome. Studies that scored more than five were considered of high quality. Discussion Over the past ten years, the colonization and prevalence of ESBL-PE infections have continued to increase rapidly all over the world,(36) and these infections generally associated with worse clinical outcomes, prolonged hospitalization, extra healthcare burden, and delayed initiation of treatment for malignancy.(3) Patients with malignancy are more easily to develop BSIs caused by ESBL-PE since oncological patients are easily immunocompromised due to a series of mechanisms as mentioned before.(5) Therefore, timely and appropriate empirical antimicrobial therapeutic regimen is pivotal for patients with malignancy who developed BSIs caused by ESBL-PE. (5) In this meta-analysis, we included 19 studies that both enrolled oncological patients with ESBL-PE and non-ESBL-PE infections, and the results showed that the mortality in BSIs caused by ESBL-PE among patients with malignancy was higher compared with non-ESBL-PE infections (RR = 2.21, 95 % CI: 1.60–3.06, P < 0.001). Consistent with our findings, Trecarichi EM et al. reported that ESBL-PE caused BSIs in patients with malignancy was associated with high mortality compared with non-ESBL-PE infections. (13, 14, 16) This result suggests that we should think highly of BSIs caused by ESBL-PE in patients with malignancy during hospitalization, and the rapid initiation of antibiotics treatment should be considered as early as possible once it was recognized. The results of the subgroup analyses showed that the mortality of ESBL-PE BSIs varies from different regions. We found that the mortality in North America and Europe was higher than in Asia. It could be explained by the fact that the majority of studies were conducted in Asia, and the study region was confirmed as a source of heterogeneity after further meta-regression analyses. Alevizakos M et al. reported that ESBL-PE are the causative agents of approximately 10.0% BSIs among patients with malignancy in Southeast Asia, and it has been associated with increased mortality in these subjects compared with Europe and America. (37) Therefore, more relevant studies need to be included in the future to draw a more reliable conclusion. We also found that children and hematological malignancy were also associated with worse prognosis in BSIs caused by ESBL-PE. It may be attributed to the fact that the included children population were mainly diagnosed with hematological malignancies, which were more vulnerable to develop immunosuppression, prolonged neutropenia, and septic shock. (38, 39) Interestingly, we observed that FN was not associated with higher mortality in oncological patients with BSIs caused by ESBL-PE. Predictors of mortality in BSIs caused by ESBL-PE among patients with malignancy Predictors of mortality in BSIs caused by ESBL-PE among patients with malignancy We then summarized the risk factors for BSIs caused by ESBL-PE in patients with malignancy. It showed that the most commonly studied risk factors for BSIs caused by ESBL-PE in patients with malignancy were age, gender, ESBL production, neutropenia, inadequate initial antimicrobial treatment, ICU admission, intra-abdominal infection, pneumonia, Pitt bacteremia score, severe sepsis/ septic shock, solid tumor, and concurrent corticosteroid therapy. However, metastasis and mechanical ventilation were the least studied variables. We also found that the most common independent risk factors of mortality were severe sepsis/ septic shock, pneumonia, ICU admission, and neutropenia. At the same time, indwelling urinary catheter, (23) pneumonia, (31) Pitt bacteremia score, (32) and severe sepsis/ septic shock (31) were the most common independent risk factors of mortality in studies that only enrolled patients with BSIs caused by ESBL-PE. In studies that both included ESBL-PE and non-ESBL-PE infections, severe sepsis/ septic shock, (14, 16, 18, 19, 22, 24, 28, 33) ICU admission, (14, 19, 27, 33, 34) neutropenia, (13, 14, 24, 35) and pneumonia (14, 16, 21, 28) were the most commonly investigated independent risk factors, respectively. Interestingly, there were only three studies (13, 14, 16) identified that ESBL production was associated with unfavorable outcomes in these patients. The detailed information was in Table 3. Subgroup analyses and meta-regression analyses Page 3/14 Subgroup analyses in all selected studies were conducted by study design, region, study population, malignancy type, FN, ESBL detection methods, and NOS score. Most of the subgroups (study design, region, study population, malignancy type, and ESBL detection methods) were consistent with the overall trend and showed statistically significant increases, except for the subgroup without FN, and the subgroup with NOS score < 6. The subgroup analyses suggested that study region was identified as potential sources of the heterogeneity (test for subgroup difference: P =0.014), and the RR of mortality in studies from Asia (RR = 1.49, 95 % CI: 1.22–1.82) was lower compared with Europe and North America, with no evidence of heterogeneity (I2 = 27.3%, P = 0.177). The detailed information was in Table 2 and Figure S1. Page 3/14 Page 3/14 Sensitivity analysis and publication bias We then carried out the sensitivity analysis by omitting each study in turn. As summarized in Figure S2, the pooled RRs and 95% CIs of mortality ranged from 2.03 (1.53-2.68) to 2.36 (1.72-3.25). The results of the sensitivity analysis show that our results are stable and reliable since there were no individual studies influenced the overall results. Begg’s test and Egger’s test showed no evidence of publication bias (P = 0.944 for Begg’s test; P = 0.538 for Egger’s test, respectively) (Figure S3). Discussion However, only fewer studies concluded that the Pitt bacteremia score and Charlson Index Score (CCI) were more common in patients who died. An interesting finding of our study is that only three studies confirmed ESBL production was an independent risk factor in mortality. Rottier WC et al. reported that ESBL production was associated with higher mortality compared with bacteremia with non- ESBL-PE. (41) This could be due to the small sample size of some studies we included in this study. Therefore, more prospective multicenter studies and clinical trials were urgently needed in the future to provide sufficient evidence. To our knowledge, this is the first study that evaluated the clinical outcomes and risk factors in BSIs caused by ESBL-PE among patients with malignancy using Meta-analysis. However, our study has several limitations. First, all of the included articles were observational studies and published in English. Besides, high-estimated heterogeneity was observed, which is probably related to different design, study region, and study population. Moreover, some studies included oncological patients with FN, but the data was not available for subgroup analysis. Therefore, the clinical outcomes of BSIs caused by ESBL-PE in these patients should be further validated since these patients are more vulnerable to severe infections. To sum up, clinical conclusions need to be comprehensive assessment in combination with other indicators, and more sample sizes and studies need to be added to verify our results. Conclusions In summary, our study provided a systematic analysis for the prognosis and risk factors of BSIs due to ESBL-PE in oncological patients. Our findings suggested that BSIs caused by ESBL-PE in patients with malignancy were associated with worse clinical outcomes compared with non- ESBL-PE infections. Furthermore, children and hematological malignancy were associated with higher mortality. We also identified that severe sepsis/ septic shock, pneumonia, and ICU admission were the most common predictors of mortality. Large-scale and prospective studies are warranted to verify the results of our study. Discussion This can be explained by the fact that only three studies included patients with FN. Besides, some studies only enrolled a subset of FN patients, but the data were not accessible to analyze. (13, 14, 16, 21, 24, 25, 30, 34, 40) Among these studies, Kang CI et al. reported that FN/ neutropenia was not the risk factor for mortality in BSIs caused by ESBL-PE among patients with malignancy. (14, 16, 21, 24) Hence, the accuracy of the conclusion needs to be further confirmed. The combined RR of sensitivity analysis further confirmed the stability of the results. Besides, the meta-regression analyses also suggested that the study region might be the source of heterogeneity in this meta-analysis, despite other relevant factors such as age, comorbidities, and antimicrobial treatment regimens cannot be analyzed due to lack of relevant data. However, Begg’s test and Egger’s test showed there was no evidence of publication bias in our study. In our study, approximately 72.0% of studies analyzed the relationship between age and mortality of BSIs caused by ESBL-PE in patients with malignancy. However, there were no studies that identified age as independent predictor. Furthermore, more than 50.0% of studies concluded that severe sepsis/ septic shock, pneumonia, and ICU admission were the most common independent risk factors in mortality. Besides, fewer studies confirmed that neutropenia was more common in patients who died. According to a study conducted by Vardakas KZ et al., they reported that underlying diseases and severity scores were the most commonly identified prognostic factors of mortality in patients with infections due to multi- drug resistant Gram-negative bacteria (MDRGNB). (12) Similar to the previous study, (12) we also found that severe sepsis/ septic shock was the Page 4/14 Page 4/14 most common risk factor in mortality. However, only fewer studies concluded that the Pitt bacteremia score and Charlson Index Score (CCI) were more common in patients who died. An interesting finding of our study is that only three studies confirmed ESBL production was an independent risk factor in mortality. Rottier WC et al. reported that ESBL production was associated with higher mortality compared with bacteremia with non- ESBL-PE. (41) This could be due to the small sample size of some studies we included in this study. Therefore, more prospective multicenter studies and clinical trials were urgently needed in the future to provide sufficient evidence. most common risk factor in mortality. Abbreviations ESBL-PE: extended-spectrum β-lactamase-producing Enterobacteriaceae; BSI: bloodstream infection; SC: single-center; MC: multicenter; NR: not reported; FN: febrile neutropenia; RR: relative risk; CI: confidence interval; NOS, Newcastle-Ottawa scale; CCI: Charlson Index Score; ICU: intensive care unit. Acknowledgments Not applicable. Not applicable. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Author Contributions T.T. and Y.Y. conceived the study. A.M.J., N.L., and R.Z. performed the search, extracted the data and performed the analyses. H.R.Z., X.C., C.X.F, and R.Z. participated in the study design and helped with the data analyses. X.Q.Z. and X.F. participated in the study design and manuscript revision. All authors interpreted the data, and A.M.J. and N.L. wrote the paper. All authors read and approved the final manuscript. Acknowledgments Availability of data and material Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. d not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Page 7/14 Page 7/14 Page 7/14 Tables Tables Page 8/14 Table 1 Characteristics of the studies and their populations included in the review. First author Design Year Country Population Infection type - bacteria No. of screened No. of ESBL- PE BSIs (%) No. of BSIs caused death No. of ESBL- PE BSIs caused death (%) ESBL detection method ESBL-PE only Wang SS (31) MC retrospective cohort 2011 Taiwan, China Adults with solid or hematological malignancy E. coli and Klebsiella 351 113 (32.2%) 35 35 (31.0%) MicroScan Wu UI (15) SC prospective cohort 2012 Taiwan, China Adults with solid or hematological malignancy E. coli 97 39 (40.2%) 10 10 (25.6%) Disk diffusion Kang CI (17) MC retrospective cohort 2013 Korea Adults with solid malignancy E. coli 92 36 (39.1%) 6 6 (16.7%) Microdilution or disk diffusion Gudiol C (29) MC retrospective cohort 2017 Spain Adults with acute leukemia and neutropenia E. coli, Klebsiella, and Enterobacter cloacae NR 425 65 65 (15.3%) Disk diffusion Cattaneo C (23) MC prospective cohort 2018 Germany Adults with hematological malignancy E. References coli, Klebsiella, and other Enterobacter spp 137 61 (44.5%) 10 10 (16.4%) Microdilution Benanti GE (32) SC retrospective cohort 2019 USA Adults with hematological malignancy E. coli NR 103 11 11 (10.7%) NR ESBL-PE and non-ESBL-PE Trecarichi EM (13) SC retrospective cohort 2009 Italy Adults and children with hematological malignancy E. coli 107 26 (24.3%) 13 11 (42.3%) Disk diffusion Gudiol C (27) SC prospective cohort 2010 Spain Adults and children with solid or hematological malignancy E. coli 531 17 (3.2%) 29 6 (35.3%) MicroScan Cornejo- Juarez P (40) Case-control study 2012 Mexico Adults and children with hematological malignancy E. coli 670 100 (14.9%) 102 51 (51%) MicroScan Kang CI (14) MC retrospective cohort 2012 Korea Adults with hematological malignancy E. coli and Klebsiella 142 29 (20.4%) 29 13 (44.8%) Microdilution or disk diffusion Ha YE (16) SC retrospective cohort 2013 Korea Adults with solid or hematological malignancy E. coli 350 95 (27.1%) 52 21 (22.1%) Disk diffusion Kim SH (18) SC retrospective cohort 2013 Korea Adults with hematological malignancy and FN E. coli and Klebsiella 96 23 (24.0%) 8 4 (17.4%) MicroScan cohort hematological malignancy and Enterobacter cloacae Bodro M (19) SC prospective cohort 2014 Spain Adults with solid or hematological malignancy Klebsiella and other Enterobacter spp 392 19 (4.8%) 18 4 (21.1%) Disk diffusion Kim SJ (28) SC prospective cohort 2014 Korea Adults and children with solid or hematological malignancy Enterobacter spp 203 31 (15.3%) 34 6 (19.4%) Disk diffusion Han SB (20) SC retrospective cohort 2015 Korea Children with solid or hematological malignancy and FN E. coli and Klebsiella 59 21 (35.6%) 3 1 (4.8%) VITEK®2 automated system Cattaneo C (21) MC prospective cohort 2016 Italy Adults with acute leukaemia Enterobacter spp 433 36 (8.3%) 37 5 (13.9%) Microdilution or disk diffusion Ma J (22) SC retrospective cohort 2017 China Adults with hematological malignancy E. coli 168 97 (57.7%) 21 15 (15.5%) Disk diffusion Çeken S (24) SC retrospective cohort 2018 Turkey Adults with solid or hematological malignancy E. coli and Klebsiella 122 70 (57.4%) 31 23 (32.9%) VITEK®2 automated system Islas- Muñoz B (25) SC prospective cohort 2018 Mexico Adults with solid or hematological malignancy E. coli, Klebsiella, and Enterobacter spp 496 123 (24.8%) 89 37 (30.1%) Disk diffusion Ben- Chetrit E (42) SC retrospective cohort 2019 Israel Adults with solid or hematological malignancy E. a p for heterogeneity within each subgroup. b p for heterogeneity between subgroups with meta-regression analyses. b p for heterogeneity between subgroups with meta-regression analyses. eity within each subgroup. a p for heterogeneity within each subgroup. Abbreviations: RR, relative risk; CI, confidence interval; FN, febrile neutropenia; ESBL, extended-spectrum β scale. References coli and Klebsiella 88 26 (19.5%) 17 6 (7.5%) NR Isendahl J (26) Case-control study 2019 Sweden Adults and children with solid or hematological malignancy E. coli and Klebsiella 945 238 (25.2%) 107 45 (18.9%) NR Kim YJ (35) SC prospective cohort 2019 Korea Adults with solid or hematological malignancy and FN E. coli and Klebsiella 179 23 (12.8%) 51 8 (34.8%) MicroScan Namikawa H (30) Case-control study 2019 Japan Adults with solid or hematological malignancy E. coli, Klebsiella, and Enterobacter spp 65 42 (64.6%) 13 9 (21.4%) NR Zhang Q (33) SC retrospective cohort 2019 China Adults with solid or hematological malignancy E. coli 324 160 (49.3%) 71 39 (24.4%) VITEK®2 automated system Abbreviations: ESBL-PE, extended-spectrum β-lactamase-producing Enterobacteriaceae; BSI, bloodstream infection; SC, single-center; MC, multicenter; NR, not reported; FN, febrile neutropenia. Abbreviations: ESBL-PE, extended-spectrum β-lactamase-producing Enterobacteriaceae; BSI, bloodstream infection; SC, single-center; MC, multicenter; NR, not reported; FN, febrile neutropenia. Page 10/14 Table 2 Subgroup analysis for meta-analysis of mortality. Variables No RR (95% CI) I2 Pa Pb Design Retrospective cohort 10 1.72(1.38-2.14) 62.2% 0.005 0.395 Prospective cohort 6 2.02(1.57-2.60) 75.7% 0.001 Case-control study 3 3.11(2.47-3.92) 90.2% <0.001 Region Europe 5 2.79(2.10-3.69) 78.2% 0.001 0.014 Asia 12 1.49(1.22-1.82) 27.3% 0.177 North America 2 3.47(2.73-4.41) 93.5% <0.001 Population Adults and children 7 2.80(2.29-3.43) 88.3% <0.001 0.303 Adults 12 1.80(1.50-2.15) 28.4% 0.167 Malignancy type Hematological 7 3.20(2.54-4.03) 83.0% <0.001 0.355 Solid or hematological 12 1.81(1.54-2.14) 60.9% 0.003 FN Yes 3 1.48(0.87-2.53) 0.0% 0.418 0.530 No 16 2.21(1.92-2.54) 80.8% <0.001 ESBL detection methods Disk diffusion 6 2.21(1.72-2.83) 64.6% 0.015 0.554 MicroScan 4 3.99(3.10-5.15) 85.9% <0.001 NOS <6 2 0.76(0.39-1.49) 16.5% 0.274 0.065 ≥6 17 2.30(2.00-2.64) 77.3% <0.001 Abbreviations: RR, relative risk; CI, confidence interval; FN, febrile neutropenia; ESBL, extended-spectrum β-lactamase; NOS, Newcastle-Ottawa scale. a p for heterogeneity within each subgroup. b p for heterogeneity between subgroups with meta regression analyses Table 2 Page 11/14 Page 11/14 Table 3 Table 3 The most commonly studied characteristics as predictors of mortality in the reviewed studiesa. -spectrum β-lactamase-producing Enterobacteriaceae; CCI, Charlson Index Score; ICU, intensive care unit; a Refers to the variables for which data were reported in the individual studies. s: ESBL-PE, extended-spectrum β-lactamase-producing Enterobacteriaceae; CCI, Charlson Index Score; ICU References Risk factor ESBL-PE only studies n/N (%) ESBL-PE vs non- ESBL-PE studies n/N (%) Total n/N (%) Identified as independent predictor for mortality Age 5/6 (83.3) 13/19 (68.4) 18/25 (72.0) 0+0 Gender 4/6 (66.7) 11/19 (57.9) 15/25 (60.0) 0+1(22) CCI 1/6 (16.7) 4/19 (21.1) 5/25 (20.0) 0+2(16, 42) ESBL production 1/6 (16.7) 12/19 (63.2) 13/25 (52.0) 0+3(13, 14, 16) Neutropenia 2/6 (33.3) 11/19 (57.9) 13/25 (52.0) 0+4(13, 14, 24, 35) Inadequate initial antimicrobial treatment 3/6 (50.0) 13/19 (68.4) 16/25 (64.0) 0+3(13, 19, 25) ICU admission 2/6 (33.3) 6/19 (31.6) 8/25 (32.0) 0+5(14, 19, 27, 33, 34) Immunosuppressant use 0 3/19 (15.8) 3/25 (12.0) 0+1(14) Indwelling urinary catheter 2/6 (33.3) 1/19 (5.3) 3/25 (12.0) 1(23)+1(14) Infecting organism, Klebsiella pneumoniae 0 5/19 (26.3) 5/25 (20.0) 0+1(18) Intra-abdominal infection 4/6 (66.7) 3/19 (15.8) 7/25 (28.0) 0+1(28) Mechanical ventilation 0 2/19 (10.5) 2/25 (8.0) 0+1(16) Metastasis 0 1/19 (5.3) 1/25 (4.0) 0+1(33) Organ failure 1/6 (16.7) 2/19 (10.5) 3/25 (12.0) 0+1(33) Pneumonia 1/6 (16.7) 5/19 (26.3) 6/25 (24.0) 1(31)+4(14, 16, 21, 28) Pitt bacteremia score 4/6 (66.7) 4/19 (21.1) 8/25 (32.0) 1(32)+2(14, 42) Severe sepsis/ septic shock 3/6 (50.0) 11/19 (57.9) 14/25 (56.0) 1(31)+8(14, 16, 18, 19, 22, 24, 28, 33) Solid tumor 3/6 (50.0) 8/19 (42.1) 11/25 (44.0) 0+1(27) Simultaneous corticosteroid therapy 0 8/19 (42.1) 8/25 (32.0) 0+2(19, 27) Abbreviations: ESBL-PE, extended-spectrum β-lactamase-producing Enterobacteriaceae; CCI, Charlson Index Score; ICU, intensive care unit; Page 12/14 Page 12/14 Page 12/14 Figure 1 Flow chart of the eligible studies for meta-analysis. Page 13/14 Figure 1 Flow chart of the eligible studies for meta-analysis. Flow chart of the eligible studies for meta-analysis. Flow chart of the eligible studies for meta-analysis. Page 13/14 Figure 2 Forest plot of mortality in BSIs due to ESBL-PE among patients with malignancy. RR, relative risk; CI, confidence interval; BSIs, bloodstream infections; Weights are from random-effects analysis. The size of the squares is analogous to the study's weight. Diamonds represent the pooled RRs and their confidence interval. Additionalfile1.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile1.pdf Page 14/14 Page 14/14 Page 14/14 Page 14/14
https://openalex.org/W4214835543	https://www.zora.uzh.ch/id/eprint/222428/1/2022_Tsagkaris_The_sitting_vs_standing_spine_NASSJ.pdf	English	null	The sitting vs standing spine	North American Spine Society journal	2,022	cc-by	7,769	Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Christos Tsagkaris a , b , Jonas Widmer a , b , Florian Wanivenhaus a , Andrea Redaelli c , Claudio Lamartina c , Mazda Farshad a , ∗ a Department of Orthopedics, Balgrist University Hospital, Zurich, Switzerland b Spine Biomechanics, Department of Orthopaedics, Balgrist University Hospital, Zurich, Switzerland c GSpine4 - I.R.C.C.S. Istituto Ortopedico Galeazzi, Milan, Italy a b s t r a c t Editor: Dr J Grauer. Keywords: Sitting radiographs Seated imaging Spine, spinal fusion Spine surgery EOS imaging sedentary lifestyle Background: Planning of surgical procedures for spinal fusion is performed on standing radiographs, neglecting the fact that patients are mostly in the sitting position during daily life. The awareness about the diﬀerences in the standing and sitting conﬁguration of the spine has increased during the last years. The purpose was to provide an overview of studies related to seated imaging for spinal fusion surgery, identify knowledge gaps and evaluate future research questions. Methods: A literature search according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) extension for Scoping Reviews (PRISMASc) was performed to identify reports related to seated imaging for spinal deformity surgery. A summary of the ﬁnding is presented for healthy individuals as well as patients with a spinal disorder and/or surgery. Results: The systematic search identiﬁed 30 original studies reporting on 1) the pre- and postoperative use of seated imaging of the spine (n = 12), 2) seated imaging of the spine for non – surgical evaluation (n = 7) and 3) seated imaging of the spine among healthy individuals (12). The summarized evidence illuminates that sitting leads to a straightening of the spine decreasing thoracic kyphosis (TK), lumbar lordosis (LL), the sacral slope (SS). Further, the postural change between standing and sitting is more signiﬁcant on the lower segments of the spine. Also, the adjacent segment compensates the needed postural change of the lumbar spine while sitting with hyperkyphosis. Conclusions: The spine has a diﬀerent conﬁguration in standing and sitting. This systematic review summarizes the current knowledge about such diﬀerences and reveals that there is minimal evidence about their consideration for surgical planning of spinal fusion surgery. Further, it identiﬁes gaps in knowledge and areas of further research. The sitting vs standing spine ☆ Christos Tsagkaris a , b , Jonas Widmer a , b , Florian Wanivenhaus a , Andrea Redaelli c , Claudio Lamartina c , Mazda Farshad a , ∗ Christos Tsagkaris a , b , Jonas Widmer a , b , Florian Wanivenhaus a , Andrea Redaelli c Claudio Lamartina c , Mazda Farshad a , ∗ https://doi.org/10.1016/j.xnsj.2022.100108 Received 18 January 2022; Received in revised form 21 February 2022; Accepted 24 February 2022 Available online 2 March 2022 2666-5484/© 2022 The Author(s). Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) The sitting vs standing spine Tsagkaris, Christos ; Widmer, Jonas ; Wanivenhaus, Florian ; Redaelli, Andrea ; Lamartina, Claudio ; Farshad, Mazda DOI: https://doi.org/10.1016/j.xnsj.2022.100108 DOI: https://doi.org/10.1016/j.xnsj.2022.100108 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-222428 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Tsagkaris, Christos; Widmer, Jonas; Wanivenhaus, Florian; Redaelli, Andrea; Lamartina, Claudio; Farshad, Mazda (2022). The sitting vs standing spine. North American Spine Society Journal, 9:100108. DOI: https://doi.org/10.1016/j.xnsj.2022.100108 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-222428 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-222428 Journal Article Published Version Originally published at: Tsagkaris, Christos; Widmer, Jonas; Wanivenhaus, Florian; Redaelli, Andrea; Lamartina, Claudio; Farshad, Mazda (2022). The sitting vs standing spine. North American Spine Society Journal, 9:100108. DOI: https://doi.org/10.1016/j.xnsj.2022.100108 g y p Tsagkaris, Christos; Widmer, Jonas; Wanivenhaus, Florian; Redaelli, Andrea; Lamartina, Claudio; Farshad, Mazda (2022). The sitting vs standing spine. North American Spine Society Journal, 9:100108. DOI: https://doi org/10 1016/j xnsj 2022 100108 North American Spine Society Journal (NASSJ) 9 (2022) 100108 Methods Studies in healthy individuals comparing a standard standing and sitting position reported that sitting aﬀects predominantly the thora- columbar spine, from T10-T11 to L5-S1 [23] . Seated position leads to an increase in cervical lordosis (CL) and decrease in thoracic kypho- sis (TK) [24] and approximately 50% decrease in lumbar lordosis (LL) [20 , 24–28] . The extent of the LL decrease varied signiﬁcantly with age [24] . More speciﬁcally, the decrease in LL was signiﬁcantly (by approx- imately 15%) reduced among the middle aged and elderly in compar- ison to young adults [25 , 26] . LL was positively correlated with tho- racic kyphosis [24 , 27] . The sacral slope (SS) is also decreased by up to 50% when sitting [20 , 24–28] and again the SS decrease among the elderly is reduced by approximately 15% in comparison to young adults [25 , 26] . On the contrary, the PT is increased up to 25% in seated po- sition [20 , 24–28] . The PI remains constant [27 , 28] . Sitting leads to a retroversion of the pelvis [20] , but lumbopelvic mobility appears poor among the elderly [25] . The realignment of the spine in sitting position leads to greater loading towards the intervertebral discs (IVDs) [28] , translocating the nucleus pulposus posteriorly [21] . Details regarding the changes in spinal alignment are presented in Table 2 . Although all studies are consistent in terms of the decrease or increase of certain spinal alignment parameters, there is a numerical variability which can be associated with the mixed age groups included and the lack of a strict deﬁnition for the standard sitting position among others. To identify relevant peer reviewed publications and grey literature the authors searched PubMed-Medline, Web of Science, Cochrane Li- brary ‒Cochrane Central Register of Controlled Trials (CENTRAL) and Clinicaltrials.gov until September 10, 2021. The reference lists of the selected sources were also hand – searched to identify potentially rele- vant resources. The authors used the search terms: “sitting radiographs ”, “seated imaging ”, “spine surgery ”, “spine fusion [MeSH] ” in combina- tion with Boolean operators (AND, OR), when appropriate. Introduction tilt – PT, pelvic incidence and lumbar lordosis mismatch - PI-LL mis- match, T1 pelvic angle - TPA) assistive planning software, 3D spinal anatomy reconstruction of biplanar radiographic images and 3D print- ing of patient – speciﬁc instrumentation [6–8] . To date, preoperative planning has been greatly based on standing radiographies – with supine computed tomography (CT) and magnetic resonance imaging (MRI) as an adjunct. Spinal fusion surgery has increased in frequency over the last two decades. Complications rates remains high aﬀecting between 29 and 62% of individuals undergoing this type of surgery [1–4] . The same upward trend applies to the relevant healthcare expenses and disability [5] . Thorough preoperative planning has a major potential to decrease the likelihood of complications and tailor the treatment to the condition and the needs of the patient. A wealth of techniques has been employed to optimize and personalize preoperative planning. These include sagit- tal alignment parameters’ mapping (sagittal vertical axis – SVA, pelvic This stated, it appears that conventional imaging and subsequent planning have ignored the fact that people spend a signiﬁcant part of the daytime sitting. Sedentary behavior is a complex phenomenon involv- ing physiological and kinematic adaptation of the body and oftentimes associated with musculoskeletal, cardiovascular and metabolic implica- ☆Summary Sentence: Planning of surgical procedures for spinal fusion is performed on standing radiographs, neglecting the fact that patients are mostly in the sitting position during daily life. Increasing awareness regarding the diﬀerent conﬁguration of the spine in standing and sitting position, can help improve surgical planning and outcomes nding author at: Mazda Farshad, Balgrist University Hospital, Department of Orthopedics, University of Zurich, Balgrist Cam Switzerland. E-mail address: mazda.farshad@balgrist.ch (M. Farshad). C. Tsagkaris, J. Widmer, F. Wanivenhaus et al. North American Spine Society Journal (NASSJ) 9 (2022) 100108 were based on plain radiographs, while few studies used EOS radio- graphs (3) or positional MRI (5). The majority of studies focused on the lower segments of the spine (lumbar, sacral), with a limited num- ber assessing the thoracic spine or global spinal alignment. Only one study focused exclusively on the cervical spine. The majority of rele- vant studies have been published after 2018, with only two studies be- ing published before 2010. All the studies, apart from Moon et al. 2018 [16] evaluated subjects on a common natural sitting position comparing it with conventional standing lateral and/or sagittal radiographies. Methods Studies were included if they fulﬁlled all the following eligibility criteria: (1) ongo- ing or published clinical studies and systematic reviews reporting the use of sitting imaging in spine deformity surgery, (2) prospective and retrospective, human and animal studies reporting on the same, and (3) cohort or cross-sectional studies. A study was excluded if it met at least one of the following criteria: (1) non-English or German publication lan- guage, (2) study types: opinion articles and perspectives. No sample size restriction was applied when screening for eligible studies. Disputes in the selection of relevant studies were discussed between the primary au- thors and a senior author until a consensus was reached. The literature was searched and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) extension for Scop- ing Reviews (PRISMASc). IBM SPSS Statistics 26 was used for statistical analysis of the included studies’ characteristics. Studies that compared more variations of sitting provide further in- sights. It seems that kneel sitting [16] and sitting on a chair with back support [29] do not diﬀer signiﬁcantly from standing in terms of spinal alignment. However, LL, SS and PT were signiﬁcantly diﬀerent in cross legs sitting. LL was decreased by up to 75% in comparison to standing and 40% in comparison to chair sitting, SS was decreased by up to 63% and 33%, and PT was increased by 64% and 44% respectively [16] . Floor sitting also leads to a signiﬁcant decrease in LL, approximately 74% and 57% in comparison to standing and sitting accordingly. Segmental lor- dosis is greatly altered in the L4-S1, where it decreases by 60% in chair sitting and by approximately 70% in ﬂoor sitting [18] . Similar decrease of the L4-5 segmental angulation, ranging between 60-70%, was mea- sured in hard-back-chair and stool sitting. Posterior disc heights were increased by approximately 10% in L1-L2. The diﬀerence in disc height in other lumbar segments did not change signiﬁcantly between natural sitting, sitting on a kneeling chair, sitting on a vertical angled chair, sitting on a hard- back chair, sitting on a chair with anterior support, sitting on stool, and cross leged sitting [19] . An overview of the alter- ations in spinal alignment between standing and sitting is provided in Fig. 2. Introduction Few studies included more seated position variants in their analysis; namely erect sitting [17] , ﬂoor sitting [16 , 18] , sitting on a kneeling chair, sit- ting on a chair with back support, sitting on 90° angled chair, sitting on chair with anterior support, sitting on stool, sitting cross-legged [19] , kneel sitting [16] , anteﬂexed sitting [20] , upright sitting [20 , 21] , seated ﬂexion [21 , 22] , seated right and left axial rotation [21] , reclined and forward inclined sitting [23] . An overview of the included studies’ and subjects’ characteristics is presented in Table 1 . tions. Numerous sitting patterns have been documented across diﬀerent population groups and cultures [9–11] . According to a recent study, nearly 20% of the population of Europe spends more than 7.5 hours per day on a chair [12] . People in North Europe sit more, with an av- erage of 6,5 hours daily, while people in southern and eastern Europe sit for at least 3 hours daily. A number of factors including urbanization and the shift of the labor markets towards white collar professions have promoted sedentary lifestyle among almost all age groups [13 , 14] . Par- ticularly since the beginning of the COVID-19 pandemic in early 2020, lockdowns and home – oﬃce mandates have signiﬁcantly increased sit- ting time and its musculoskeletal implications in all population groups including children and young adults [15] . Therefore, sitting radiographs and adjunct imaging acquired at a seated position should potentially be integrated in spinal fusion preoperative planning. To achieve so, a ro- bust body of evidence investigating the beneﬁts and challenges of sitting radiographies in spinal fusion needs to be created. The ﬁrst step in this regard is to map the available evidence and identify knowledge gaps. Aim To provide a systematic overview of the available evidence on diﬀerences on the spinal conﬁguration in sitting and standing, identify research gaps and discuss their implications in future research and clin- ical practice. Results The initial search including hand – searching of reference lists yielded 404 studies. After removing duplicates and screening titles and abstracts the authors evaluated the full texts of 44 studies. As per in- clusion and exclusion criteria 31 studies were eligible for inclusion. A literature search ﬂow is presented in Fig. 1 . The authors divided the studies into 3 categories based on the study design and purpose; namely based on the use of seated imaging in pe- rioperative (preoperative and/or postoperative settings), in non – oper- ative settings and in the assessment of healthy individuals or individ- uals without a history of spine condition. Most studies were observa- tional (retrospective, cross – sectional or prospective). Approximately one third were related to the perioperative assessment of patients, ap- proximately one third of studies assessed spinal conditions by means of sitting imaging irrespective of surgery and the remaining evaluated the radiological characteristics of the sitting spine in healthy individu- als (not diagnosed with a spine condition). The majority of the studies 2 North American Spine Society Journal (NASSJ) 9 (2022) 100108 C. Tsagkaris, J. Widmer, F. Wanivenhaus et al. Table 1 Overview of the included studies’ and subjects’ characteristics. Study Date Study type Context Sample Spine conditions Spine segment Imaging type Seated position Yoshida et al. 2020 Observational clinical study Perioperative imaging 113 Adult spinal deformity Thoracic, Lumbar, sacral Sitting XR Natural sitting Hey et al. 2020 Observational clinical study Perioperative imaging 120 Low back pain Lumbar, sacral Sitting XR Natural sitting Zhao et al. 2019 Observational clinical study Perioperative imaging 36 Thoracolumbar kyphosis Lumbar, sacral EOS Natural sitting Janjua et al. 2018 Observational clinical study Perioperative imaging 20 Thoracolumbar deformity Thoracic, Lumbar, sacral Sitting XR Natural sitting Zhu et al. 2018 Observational clinical study Perioperative imaging 44 Idiopathic thoracic scoliosis Thoracic, Lumbar, sacral Sitting XR Natural sitting Chiu et al. 2018 Observational clinical study Perioperative imaging 28 Osteoporotic thoracolumbar fractures Lumbar, sacral Sitting XR Natural sitting Hey et al. 2017 Observational clinical study Perioperative imaging 70 Low back pain Thoracic, Lumbar, sacral Sitting XR Natural sitting Hey et al. 2017 Observational clinical study Perioperative imaging 58 Low back pain Lumbar, sacral EOS Natural sitting Vaughn et al. 2014 Observational clinical study Perioperative imaging 26 Idiopathic scoliosis Thoracic, Lumbar, sacral Sitting XR Natural sitting Siddiqui et al. Results 2005 Observational clinical study Perioperative imaging 12 Symptomatic lumbar spinal stenosis Lumbar, sacral MRI Natural sitting Zhou et al. Ongoing Clinical trial Perioperative imaging 200 Adult degenerative scoliosis Lumbar, sacral Sitting XR, MRI Natural sitting Sun et al. 2020 Observational clinical study Postoperative imaging 63 Lumbar degeneration Lumbar, sacral Sitting XR Natural sitting, Erect sitting Nishida et al. 2020 Observational clinical study Physiological imaging 113 Healthy Global spinal alignment Sitting XR Natural sitting Maekawa et al. 2019 Observational clinical study Physiological imaging 253 Healthy Lumbar, sacral Sitting XR Natural sitting Berry et al. 2019 Observational clinical study Physiological imaging 13 Healthy Lumbar, sacral MRI Natural sitting, Seated right axial rotation, Seated left axial rotation Chevilotte et al. 2018 Observational clinical study Physiological imaging 15 Healthy Lumbar, sacral Sitting XR Natural sitting, Upright sitting, Seated ﬂexion Suzuki et al. 2018 Observational clinical study Physiological imaging 25 Healthy Lumbar, sacral Sitting XR Natural sitting, Anteﬂexed sitting, Upright sitting Moon et al. 2018 Observational clinical study Physiological imaging 16 Healthy Lumbar, sacral Sitting XR Floor sitting, Kneel sitting Alamin et al. 2018 Observational clinical study Physiological imaging 20 Healthy Lumbar, sacral Sitting XR Natural sitting, Sitting on a kneeling chair, Sitting on a vertical angled chair, Sitting on a chair with back support, Sitting on a chair with anterior support, Sitting on stool, Cross leged sitting Suzuki et al. 2016 Observational clinical study Physiological imaging 73 Healthy Lumbar, sacral Sitting XR Natural sitting Cho et al. 2015 Observational clinical study Physiological imaging 30 Healthy Lumbar, sacral Sitting XR Natural sitting Bae et al. 2012 Observational clinical study Physiological imaging 30 Healthy Lumbar, sacral Sitting XR Natural sitting, Floor sitting Endo et al. 2012 Observational clinical study Physiological imaging 16 Healthy Lumbar, sacral Sitting XR Natural sitting Baumgartner et al. 2012 Observational clinical study Physiological imaging 6 Healthy Global spinal alignment MRI Upright sitting, Reclined Sitting, Forward inclined sitting Zhou et al. 2021 Observational clinical study Non-surgical evaluation 62 Symptomatic spondylolisthe- sis (lumbar degeneration) Lumbar, sacral Sitting XR Natural sitting Inoue et al. 2021 Observational clinical study Non-surgical evaluation 23 Lumbar spondylosis Lumbar, sacral Sitting XR Natural sitting Sielatycki et al. 2021 Observational clinical study Non-surgical evaluation 70 Low back pain Lumbar, sacral Sitting XR Natural sitting, Seated ﬂexion Kusakabe et al. 2019 Observational clinical study Non-surgical evaluation 108 Spinal degeneration Cervical Sitting XR Natural sitting Rouissi et al. Results 2016 Imaging protocol Non-surgical evaluation 36 Neuromuscular scoliosis Lumbar, sacral EOS Natural sitting Bouloussa et al. 2015 Imaging protocol Non-surgical evaluation 41 Neuromuscular scoliosis Lumbar, sacral EOS Natural sitting Karadimas et al. 2006 Observational clinical study Non-surgical evaluation 30 Low back pain Lumbar, sacral MRI Natural sitting 3 Table 1 Overview of the included studies’ and subjects’ characteristics. 3 North American Spine Society Journal (NASSJ) 9 (2022) 100108 North American Spine Society Journal (NASSJ) 9 (2022) 100108 C. Tsagkaris, J. Widmer, F. Wanivenhaus et al. Fig. 1. Literature search ﬂow diagram. Fig. 1. Literature search ﬂow diagram. Fig. 1. Literature search ﬂow diagram. Fig. 1. Literature search ﬂow diagram. Fusion surgery A number of studies included patients who underwent (or were eli- gible to undergo) fusion surgery. Hey and colleagues (2017) compared spinal alignment between standing and sitting in patients with low back pain of various etiologies and noticed forward SVA displacement, su- perior movement of the apex vertebra towards the lumbar curve and inferior towards the thoracic curve by one vertebral level. There were signiﬁcant diﬀerences with a TK decrease by 30%, LL decrease by 50%, SS by 40%, PT increase by 53% and the thoracolumbar junctional angle tended to become less kyphotic and more lordotic. Although these ob- servations are consistent with the ones on healthy individuals, a slight reduction in SS decrease (9%) and in PT increase (8%) was observed [30] , implying that patients undergone lumbar fusion are more likely to have residual lordosis, particularly at the lower lumber spine, in natural sitting position [17] . The shape of the spine can aﬀect this type of predictions according to a study of Hey and colleagues (2020) assessing the implications of a predilection towards S- or C-shaped spine to spine realignment surgery. Although, the LL decreases by an average of 75% in both types of spinal deformity when sitting, LL can be up to 20% larger in S-shaped spines in sitting position. SS diﬀers signiﬁcantly between the two types of spine alignment and while in S-shaped spines SS tends to decrease by 75% in sitting, in C-shaped spines the angle can be retroverted (from 32 degrees to - 0.9 degrees). PT increased by 68% in C-shaped spines and by 58% in S-shaped spines. The second study of the same group provided more clariﬁcations by investigating the spinal ROM in diﬀerent postures including slump sit- ting. It appears that the greatest ROM of the lumbar spine is achieved in slump sitting, particularly in the L4-L5 segment, whose mobility reaches approximately 50%. In this position, L1-L4 may even become kyphotic [31] . Pre- to postoperative changes in kyphosis can be predicted by the diﬀerence between sitting to standing radiographs. To date, the most re- liable predictor is a plumb line distance between the upper instrumented vertebra (UIV) and the C2 with a cutoﬀvalue of 11,5 cm [7 , 32] . Scoliosis Five studies have focused on scoliosis (adult degenerative scolio- sis, idiopathic thoracic scoliosis and neuromuscular scoliosis). Rouissi et al. (2017) and Bouloussa et al. (2016) described a protocol for EOS imaging in neuromuscular scoliosis; their results were oriented towards feasibility and satisfaction measures and did not have direct reference to surgery [33 , 34] . The studies of Vaughn, Chiu, Hey and their col- leagues assessed preoperative and, in some cases, postoperative spine seated imaging. Preoperative sitting imaging in patients with idiopathic scoliosis reveals a decrease in TK, LL, SS [35–37] . The decrease in TK appears reduced in comparison to healthy or non - scoliotic individuals (approximately 10% instead of 30%). Postoperative changes should also be considered. Following posterior thoracic fusion in patients with id- iopathic thoracic scoliosis a signiﬁcant reduction in LL and SS decrease (9.7% and 5.7% in contrast to 42.1% and 31.1% preoperatively respec- tively) and a signiﬁcant reduction in PT increase (39.0% in contrast to 193.6%) from standing to sitting occur [36] . Fusion surgery Con- sidering these together with the fact that extended L1-L5 fusion would decrease lumbar ﬂexion by 47.6° and lumbar extension by up to 5.9°, it becomes evident that lower lumbar fusion can lead to a malalignment of their adjacent segments. Failure to address so with suitable spine instru- mentation can precipitate adjacent segment degeneration (ASD) [31] . Clinical implications in patients An overview of the impact of fusion surgery on spinal alignment is pro- vided in Fig. 3 . Studies involving patients pre- and/or postoperatively or regard- less of surgical intervention provide useful information regarding spinal alignment in standing position in a number of spinal deformities and its potential implications on spine surgery. Fig. 3. Graphical overview of spinal alignment in sitting and standing posture in patients undergone spinal fusion. Fig. 3. Graphical overview of spinal alignment in sitting and standing posture in patients undergone spinal fusion. Clinical implications in patients Table 2 Table 2 Lumbar Lordosis (LL), Sacral Slope (SS) and Pelvic Tilt (PT) in standing and sitting position, all values are expressed in (°). LL Stand. LL Sit. SS Stand. SS Sit. PT Stand. PT Sit. Reference 36.2 ± 12.1 0.7 ± 26.3 ( ↓) 35.8 ± 21.8 16.6 ± 9.38 ( ↓) 14.4 ± 7.27 65.17 ± 8.24 ( ↑ ) Nishida et al. 2020 Young adults 49.3 ± 14.2 23.3 ± 13.4 ( ↓) 34.6 ± 7.7 18.1 ± 10.1 ( ↓) 19.7 ± 16.4 32.5 ± 12.7 ( ↑ ) Maekawa et al. 2019 Middle aged 40.8 ± 11.5 24.9 ± 16.2 ( ↓) 31.3 ± 8.5 18.8 ± 10.1 ( ↓) 22.2 ± 15.1 33.3 ± 14.0 ( ↑ ) Elderly 42.1 ± 14.1 27.1 ± 14.8 ( ↓) 31.6 ± 8.9 20.1 ± 9.6 ( ↓) 24.3 ± 15.8 33.2 ± 14.7 ( ↑ ) 54.8 ± 9.8 15.9° ± 14.6 ( ↓) 37.1 ± 6.3 11.3 ± 10.8 ( ↓) 12.1 ± 6.3 37.7 ± 10.4 ( ↑ ) Chevillote et al. 2018 31.9 ± 10.4 7.9 ± 10.8 ( ↓) 35.9 ± 8.7 14.9 ± 11.7 ( ↓) 7.7 ± 9.5 31.5 ± 8 ( ↑ ) Suzuki et al. 2018 Young adults 31.3 ± 10.4 15.5 ± 10.1 ( ↓) 36.4 ± 7.2 19.0 ± 9.7 ( ↓) 10.3 ± 7.3 27.6 ± 10.5 ( ↑ ) Suzuki et al. 2016 Elderly 26.6 ± 12.8 16.0 ± 13.9 ( ↓) 32.6 ± 8.6 21.2 ± 10.9 ( ↓) 15.0 ± 7.3 27.5 ± 10.9 ( ↑ ) 33.3 ± 11.2 16.7 ± 11.2 ( ↓) 37.2 ± 7.1 18.5 ± 10.9 ( ↓) 9.9 ± 7.4 28.2 ± 10.8 ( ↑ ) Endo et al. 2012 X 24.7 ± 8.3 ( ↓) X X X X Baumgartner et al. 2012 Mean LL decrease 56% ↓ Mean SS decrease 49% ↓ Mean PT increase 58% ↑ Table 2 Lumbar Lordosis (LL), Sacral Slope (SS) and Pelvic Tilt (PT) in standing and sitting position, all values are expressed in (°). Fig. 2. Graphical overview of spinal alignment in sitting and standing position. Fig. 2. Graphical overview of spinal alignment in sitting and standing position. North American Spine Society Journal (NASSJ) 9 (2022) 100108 C. Tsagkaris, J. Widmer, F. Wanivenhaus et al. Limitations and future research Even if we were not able to perform a lack of risk of bias analysis and metanalysis due to the heterogeneity of the included studies and we did not include studies published in languages diﬀerent than English and German, we could identify following areas of future research. At preclinical level, more attention should be given to the loading alterations in sitting position, the eﬀect of sitting on muscles, tendons, proprioceptive networks and reﬂexes involved in spine biomechanics and the ﬁnite elements’ behavior and pathogenetic adaptation – coping mechanisms. At clinical level, it is important to delve into instrumen- tation fatigue and screw loosening associated with sitting posture and relevant radiological markers, diﬀerent instrumentation techniques and their biomechanical eﬀect on the fused and adjacent segments in sitting vs standing, the contribution of sitting to the development of particular spinal fusion complications, the standard sitting position which emu- lates the most frequent sitting patterns and can be used by radiologists in the future and the sensitivity and speciﬁcity of speciﬁc seated imag- ing modalities (radiographs, EOS, MRI) in the form of indications for pre- and postoperative imaging in speciﬁc conditions and operations. In patients with scoliosis, TK, LL, SS are also signiﬁcantly decreased in sitting position, but the extent of decrease is reduced by approxi- mately 10% (JJ Vaughn & RM Schadjacwend, 2014). These parame- ters can be decreased up to an additional 10% after surgery [36] . Their spine is less straightened in sitting position. This means that preopera- tive planning with sitting radiographs should be potentially diﬀerent in scoliosis surgery compared to other types of spinal fusion. Further re- search is needed to ﬁnd the relevance of these ﬁnding for management of scoliotic deformities. Subsequently, epidemiological studies should seek sitting – associ- ated risk factors for spinal surgery failure among patients and investigate whether preoperative planning based on sitting imaging is more eﬀec- tive in particular population or patients’ groups depending on their age, gender, weight, occupation or disease history. Research in population health and epidemiology should also provide input regarding the sitting patterns in modern societies through cross sectional and participant ob- servation studies and educate the public on healthy sitting behaviors. Certainly, the integration of seated imaging in clinical practice will require meta-research to generate speciﬁc guidelines and feasibility studies to assess the implementation, ﬁnancial and educational aspects of the matter. Discussion The spine has a diﬀerent conﬁguration in standing and sitting. Sitting tends to straighten the spine decreasing TK, LL and SS up to approx. 50% and increasing PT by 50% as well. After spinal fusion, the upper adja- cent segment needs to compensate for the decrease in LL and therefore it becomes more kyphotic while sitting. The hyperkyphosis of the adjacent segment might result in the formation of a zone of increased compres- sion in the anterior spine and increased tension on the posterior spine. This phenomenon, which is known as proximal junctional kyphosis and is an ASD feature per se, sheds light to potential biomechanical ASD risk factors [32 , 42 , 43] . These include increased intradiscal pressure, poste- rior translocation of the nucleus pulposus, altered angular mobility at the proximal kyphotic level, adaptation of the paraspinal muscles and ligaments to the new loading conditions and subsequent injury of these structures. Structural damage as a result of this pathomechanical cas- cade can alter loading mechanics, severe the local vasculature and disc nutrition and trigger reactive inﬂammation [44] . Cumulatively, these can accelerate the degeneration of the involved level. The amount of this phenomenon might be dependent on the fusion angle, the quality of instrumentation and the diﬀerent patterns of tissue damage caused by anterior and posterior fusion techniques (laminectomy, iatrogenic mus- cle damage, ligament rupture, soft tissue defect). Sitting radiographs could therefore be used to predict the postoperative reciprocal change and might inﬂuence surgical planning (alteration of fusion angle, soft landing techniques etc). Further, these ﬁndings might lead the surgeon to advise patients against ﬂoor sitting after fusion surgery, given that it leads to adjacent segments hyperﬂexion [18] . Non-surgical insights Non-surgical insights Additional insights can be traced in studies that did not report on surgical associated sitting imaging. The combination of sitting radio- graphs with supine sagittal MRIs is more accurate in revealing high vertebral slip percentages in comparison to standing radiographs [39] . Seated imaging is also more accurate in revealing kyphosis, particularly in the lower lumbar segments (L4-S1) [22] . Sitting does not signiﬁcantly aﬀect the cervical spine, unless there is signiﬁcant vertebral imbalance leading to decreased cervical lordosis (CL) and large LL-PI mismatch that leads to increased CL [39] . Nevertheless, sitting radiographs were not as accurate as the lateral decubitus position for the assessment of spinal instability Inoue et al. (2021) [40] . Nonetheless, seated MRI was more accurate in detecting degeneration associated decrease in end – plate angles than supine or standing imaging. Seated MRI also revealed an increase in the anterior and middle disc heights by approximately 16% when sitting, regardless of the degree of lumbar degeneration [41] . The exponential increase in sitting and its musculoskeletal sequelae during the COVID-19 pandemic enhances the relevance of this review further. Since early 2020, numerous studies have stressed that low back pain and sagittal imbalance has become quite more frequent in individ- uals of all ages and particularly students and young workers [15 , 47–51] . Oftentimes, these individuals enter a vicious circle, where they seek re- lief from pain in sitting, but bad sitting postures only deteriorate their pain and the underlying spinal condition [52] . This is expected to in- crease the demand for spine surgery among younger or middle – aged individuals in the foreseeable future. Given the demanding lifestyle and the increased life – expectancy of this patients’ group, decreasing the rate of complications and revision surgery is a dire need. Sitting imaging has a major potential to address this need, because sitting will consti- tute both a pathogenetic mechanism and a signiﬁcant lifestyle factor in this population. Furthermore, sitting imaging research will help attract funding and investments to counter the damage that the pandemic has inﬂicted to elective spine surgery and related research and entrepreneur- ship [53–55] . Henceforth, this review also prompts the need for more preclinical and clinical research in the ﬁeld. Osteoporotic fractures Sitting radiographs can also be useful in vertebroplasty for osteo- porotic fractures. Zhu et al. (2018) evaluated dynamic stress mobility radiographs (including sitting radiographs) in an attempt to predict the vertebral height restoration, kyphosis correction, and cement volume re- quired in vertebroplasty for osteoporotic thoracolumbar vertebral frac- 5 C. Tsagkaris, J. Widmer, F. Wanivenhaus et al. North American Spine Society Journal (NASSJ) 9 (2022) 100108 tures with intravertebral cleft. Calculating the supine stress versus sit- ting diﬀerence (SSD) enabled the researchers to predict that following vertebroplasty the kyphotic wedge endplate angle (WEPA) and the re- gional kyphotic angle (RKA) would decrease by 50% [38] . advantages over radiographs acquired in sitting position. In particular, they reﬂect the weight – bearing condition of the spinal structures, they can be obtained more easily from children, overweight and obese in- dividuals [45] . 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https://openalex.org/W2894278838	http://openaccess.sgul.ac.uk/111247/1/document.pdf	English	null	Relaxation and Exercise in Lymphoma Survivors (REIL Study): A Randomised Clinical Trial Protocol	Journal of global oncology	2,018	cc-by	6,817	Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 https://doi.org/10.1186/s13102-019-0127-7 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 https://doi.org/10.1186/s13102-019-0127-7 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation https://doi.org/10.1186/s13102-019-0127-7 Open Access © 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: Lymphoma survivors commonly report ongoing complaints including fatigue, pain, depression and decreased quality of life (QoL) following treatment. Although evidence suggests that both relaxation and exercise can significantly improve such symptoms, there is no consensus on which intervention is more effective. This paper presents the REIL (Relaxation and Exercise In Lymphoma) Study protocol. The REIL study aims to compare the effect of two home-based interventions – relaxation and exercise – on QoL in lymphoma survivors. Methods: Eligible participants (n = 36) will be randomised to a relaxation or exercise programme to perform at least three times per week. The primary outcome measure is QoL, assessed by the European Organisation for Research and Treatment of Cancer QoL Questionnaire Core 30 (EORTC QLQ-C30). Secondary outcome measures include body composition, cardiovascular status, pulmonary function, grip strength, functional exercise capacity (six minute walk test), well-being assessed by the FACT-Lym questionnaire, and psychological status assessed by the Hospital Anxiety and Depression Scale. Total duration of the study will be twelve weeks and outcome measures will be assessed at baseline, six weeks and at the end of the study. Discussion: It is anticipated that results from this preliminary study will begin to highlight effective pathways to improve QoL following chemotherapy for this population. This will better inform healthcare professionals to optimise QoL of lymphoma patients, and enable a smooth transition from being a cancer patient to survivor. Trial registration: The REIL study has been registered on a publicly accessible database, ClinicalTrials.gov, Registration Number: NCT02272751, October 2014. Trial registration: The REIL study has been registered on a publicly accessible database, ClinicalTrials.gov, Registration Number: NCT02272751, October 2014. Keywords: Lymphoma survivors, Exercise, Relaxation, Mindfulness, Rehabilitation, Cancer survivors, Self-management, Quality of life Keywords: Lymphoma survivors, Exercise, Relaxation, Mindfulness, Rehabilitation, Cancer survivors, Self-management, Quality of life Keywords: Lymphoma survivors, Exercise, Relaxation, Mindfulness, Rehabilitation, Cancer survivors, Self-management, Quality of life Relaxation and exercise in lymphoma survivors (REIL study): a randomised clinical trial protocol Suchita Hathiramani1,4* , Ruth Pettengell2, Hannah Moir3 and Ahmed Younis1,4 Background treatment exposures. Commonly reported long-term and late effects of treatment in lymphoma survivors include both physical and psychosocial symptoms such as fa- tigue, pain, muscle weakness, neuropathies, depression, anxiety and decreased self-esteem; as well as decreased function and quality of life (QoL) [1–3]. Hence there has been a call for further research to address the ongoing needs of lymphoma survivors [1]. Significant improvements have been made in the survival rates in cancer patients due to earlier detection and ad- vances in treatment. Although initial research in the field of cancer survivorship focused on tumours such as breast and prostate, dramatic advances in haematological malignancies have also led to increased survival [1], but at a cost of increasing complications. Like survivors of other cancers, lymphoma survivors are at an increased risk of morbidity and adverse effects due to disease and When medical treatment is completed successfully and the patient is in clinical remission, the close support from medical staff is usually reduced and the patient is expected to return to ‘normal life’. This transition how- ever does not always occur smoothly and studies have highlighted that post-treatment survivors at this * Correspondence: s.hathiramani@sgul.kingston.ac.uk 1Kingston University and St. George’s University of London, London, UK 4Faculty of Health, Social Care & Education, Cranmer Terrace, London SW17 0RE, UK Full list of author information is available at the end of the article * Correspondence: s.hathiramani@sgul.kingston.ac.uk 1Kingston University and St. George’s University of London, London, UK 4Faculty of Health, Social Care & Education, Cranmer Terrace, London SW17 0RE, UK Full list of author information is available at the end of the article Page 2 of 9 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 2 of 9 has been a call for further studies to move beyond wait- list control groups and to compare with active control or other empirically supported interventions [15, 16], for instance relaxation to exercise. As a result, there have been studies and study protocols looking at comparison of two interventions in cancer survivors, but the majority of these have focused on breast cancer [17, 18] and au- thors have called for future research to focus on other survivor groups, including haematologic cancer sites [1, 19]. transition phase often feel unprepared and uninformed [1, 3]. Survivors at this phase are sometimes in need of further support and advice on how to progress activity, manage ongoing symptoms and return to pre-morbid status and often do not receive this [2, 3]. There are cur- rently no recommended care pathways for lymphoma patients in remission following treatment, despite the fact that this transition period shortly after treatment has been highlighted to be particularly difficult [4]. This lack of care pathways has been highlighted by the government in the United Kingdom (UK). The National Cancer Survivorship Initiative (NCSI) was launched in 2008 and calls for research into the development of pathways for cancer survivors [5], recommending holis- tic assessment and personalised care-planning, emphasis on the use of patient-reported outcome measures and a shift towards self-management [6]. The REIL study aims to address some of these issues highlighted by studying a sample of lymphoma survivors post-chemotherapy, supporting patients during the tran- sition phase, comparison of two interventions, use of pa- tient-reported outcome measures and emphasis on self- management. Results from this preliminary study would provide an indication of efficacy of interventions, and with the findings from the REIL study we aim to build towards the development of evidence-based practice guidelines for lymphoma survivors. Self-management in cancer survivorship has been de- fined as ‘awareness and active participation by the per- son in their recovery, recuperation and rehabilitation, to minimise the consequences of treatment, and promote survival, health and well-being” [7]. Increasing numbers of survivors together with limited healthcare resources has led to a focus on self-management as an important approach for cancer survivors. Furthermore, self-man- agement can empower cancer patients, increase their confidence to manage problems associated with disease and treatment, and enhance QoL [8]. This paper presents the REIL study protocol using SPIRIT 2013 guidelines [20]. Study design The REIL study is a prospective, randomised, clinical intervention trial. Participants will be randomised to ex- ercise or relaxation intervention. Participants will be assessed at baseline prior to commencing the interven- tion programme, at six weeks, and at the end of the twelve week intervention. Aims The primary aim of the REIL Study is to compare the ef- fect of two interventions – relaxation and exercise – on QoL in a sample of lymphoma patients in remission post-chemotherapy. The null hypothesis is that there is no difference in QoL between the relaxation and exer- cise groups post-intervention. Literature indicates that interventions such as relax- ation and exercise both have a positive impact on QoL of cancer survivors [9–12]. It has been demonstrated that exercise interventions have a positive impact on physical and psychological factors related to QoL such as peak oxygen consumption, physical functioning, fa- tigue, self-esteem and social functioning; as well as over- all QoL of cancer patients and survivors [9, 10]. Studies on relaxation interventions including mindfulness-based stress reduction and progressive muscular relaxation have also demonstrated significant positive impact on physical and psychological symptoms such as fatigue, pain, anxiety and depression, as well as on QoL in can- cer survivors [10–12]. Hence both relaxation and exer- cise interventions are recommended by organisations providing expert advice and support to cancer patients and survivors such as Lymphoma Action [13] and Mac- millan Cancer Support [14]. Secondary aims are to investigate the effects of the two interventions on body composition, cardiovascular status, pulmonary function, muscle strength, functional exercise capacity, well-being and psychological status; and explore perceptions about participation in the post- treatment intervention programme. Ethical approval However in the majority of trials studying the effects of relaxation or exercise on cancer survivors, the benefits have only been relative to a control group; and to rule out potential placebo effects future studies need to spe- cify more rigorous comparison conditions, for instance a control intervention with similar elements that may in- fluence outcome such as attention from study personnel or time spent on the procedure [15, 16]. Hence there The REIL study has received ethical approval from Cam- den and Islington National Research Ethics Service (13/ LO/1327), who are the responsible for approval of final protocol as well as any modifications or amendments. Local site approval from St. George’s Hospital Joint Re- search and Enterprise Office (JREO) has also been ob- tained (13.0108). The JREO is an independent office Page 3 of 9 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 3 of 9 Page 3 of 9 (2019) 11:17 providing external review and monitoring of any re- search undertaken at St. George’s NHS Trust in order to maintain clinical research governance guidelines and standards. Randomisation A random allocation list will be prepared by the depart- ment statistician, generated using GraphPad randomisa- tion software (GraphPad Prism version 6.04 for Windows, GraphPad software, CA). On entry into the study each participant will be assigned an anonymous ID number and each number will be allocated an interven- tion (exercise or relaxation) on the list. As all assessment sessions will be carried out by the single principal inves- tigator (SH), it will not be possible to blind the investiga- tor to intervention. Also due to the nature of the intervention participants cannot be blinded to group allocation. The study has been registered on a publicly accessible database, ClinicalTrials.gov, NCT02272751. Interventions Interventions for this study are reported using the tem- plate for intervention description and replication (TIDieR) checklist and guide [24]. Interventions for this study are reported using the tem- plate for intervention description and replication (TIDieR) checklist and guide [24]. For both groups, advice, instruction, demonstration and practice will be carried out at baseline. This will be delivered by the principal investigator SH, an experi- enced physiotherapist with a special interest and experi- ence in oncology. Participants will be able to contact SH by telephone in between sessions if additional support is required. All participants will be advised on potential ad- verse events and what to report including pain, fatigue, muscle soreness, lymphoedema, nausea, dyspnoea, dizzi- ness, tachycardia and cramp. A logbook will be provided to all participants to encourage adherence and document self-reported participation levels, intervention type car- ried out, frequency, intensity, duration and any adverse events. Exercise intervention While there are no guidelines for the delivery of exercise in cancer survivors, it is recommended to follow the guidelines for general UK population [25]. Hence partici- pants randomised to the exercise intervention in this study will be advised to carry out the programme for at least 50 min three times per week. Eligibility criteria l Inclusion criteria include patients with histologically confirmed lymphoma in remission post–chemotherapy, chemotherapy treatment completed within the last six weeks, age 18 years or older, able to give informed con- sent, good performance status (assessed by the Eastern Cooperative Oncology Group {ECOG} status 0–2) [21] and medically able to carry out an exercise training programme. Patients with active disease, unstable angina or unexplained electrocardiogram, poor performance status (ECOG status 3 or more), pregnancy, difficulty breathing at rest, persistent cough, fever or illness, or any cognitive impairment limiting ability to give in- formed consent or complete QoL questionnaires will be excluded. Written informed consent will be obtained from all individual participants included in the study. Recruitment Participants will be recruited from a single specialist clinical setting – the Haematology-Oncology Out-Pa- tient (HOOP) Clinic at St George’s Hospital, London. Assessment for eligibility, recruitment, medical screening and obtaining of informed consent will be carried out by the patients’ medical consultant (RP). In order to en- courage participant enrolment, potential participants will be introduced to the principal investigator (SH) who will explain about the study, answer questions and provide with the written participant information sheet to take away and read before making a decision. Flow of participants in the study is shown in Fig. 1. Sample size Sample size was calculated to determine clinically rele- vant effects on the primary outcome measure, the Euro- pean Organisation for Research and Treatment of Cancer – QoL core questionnaire (EORTC QLQ-C30, version 3.0). A minimally important difference of 5 to 10 points is generally accepted as clinically meaningful [22], and calculations were based on comparison of means be- tween two groups. Assuming a two-sided significance level level (α) of 0.05, power of 80% (ß = 0.20) and stand- ard deviation from EORTC website reference values [23], it was determined that a sample of minimum 46 participants will be required to detect a significant change in the EORTC summary score, 23 in each inter- vention group. The exercise programme includes elements of aerobic, upper and lower limb resistance training, core stability and stretches and is designed to be performed independ- ently at home. For the aerobic component, participants will be advised to walk indoors or outdoors for a mini- mum of 30 min and to aim for moderate intensity as rec- ommended by the Borg Rate of Perceived Exertion (RPE) scale [26]. Where unable to maintain moderate in- tensity due to fatigue, weakness or other complaints, participants will be advised to aim for moderate intensity aerobic exercise for any duration of time able, and to gradually increase over the next few weeks aiming to build up to 150 min over one week. They will be taught Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 4 of 9 (2019) 11:17 Page 4 of 9 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation Fig. 1 Flow of participants in the REIL Study. CONSORT flow diagram demonstrating participant flow through each phase of the randomised controlled trial (enrolment, intervention allocation, follow-up and data analysis) Relaxation intervention resistance exercises for the major upper and lower limb muscles using body weight or TheraBand™isometric re- sistance bands to carry out following aerobic activity. The resistance training follows American College of Sports Medicine (ACSM) guidelines [27], and partici- pants will be advised to carry out three sets of each exer- cise for 8–12 repetitions. They will also be taught stretches for upper and lower limb muscles, and core abdominal exercises in supine. Participants will be also be provided with written instruction, leaflet with photographs and advice sheet. Similar programmes in- corporating aerobic, resistance and flexibility training three times a week over a twelve week period have been used with good results in improving QoL in cancer survivors [9]. The relaxation intervention consists of a bed- or chair- based program, developed based on the literature on mindfulness-based interventions in cancer survivors [10–12]. An audio CD was produced to guide partici- pants through relaxation techniques incorporating mind- fulness meditation, breathing exercises, guided visualisation and progressive muscle relaxation. Here also participants will be advised to carry out the programme for 50 min three times a week. Participants will also be provided with written information on relax- ation and tips on how to incorporate into daily life. Par- ticipants in this group will not be advised to perform any exercise outside of their normal habits, nor asked to avoid activity. Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 5 of 9 (2019) 11:17 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation answered on a four-point scale and scores are derived according to the EORTC scoring manual [30]. Participants will be followed-up at six weeks, midway through the programme. At this stage all assessments will be repeated, any change noted and they will be ad- vised on how to modify the programme (relaxation or exercise) as appropriate. Once participants have com- pleted twelve weeks of their intervention, they will be provided with resources of the other intervention group, for their information or to carry out as desired, in ac- cordance with the Declaration of Helsinki. Body composition Due to the illness itself and treatment and side-effects (including steroid use, loss of appetite, nausea and vomiting), lymphoma patients may experience a fluctu- ation in weight and body fat percentage during the dur- ation of their treatment. Therefore body composition will be assessed using a bioelectrical impedance analyser (Tanita BC-418) to monitor whether it will stabilise to within the desired range. Participants’ standing stature will be measured using a stadiometer (SECA, Germany). Height measured will be inserted into the body compos- ition analyser to obtain weight, body mass index (BMI), and body fat percentage. Participants will be measured without footwear and in light clothing using the same equipment each time. Outcome measures All outcome measures will be assessed at baseline prior to commencing intervention, midway (six weeks) and on completion of intervention programme (twelve weeks). Outcome measures, instruments and guidelines used and time assessed are summarised in Table 1. At base- line, patient demographics including gender, age, social history and medical history will be recorded. Psychological status The Hospital Anxiety and Depression Scale (HADS) is a valid tool for assessing the severity of anxiety disorders and depression in various populations including psychi- atric, primary care patients and the general population [37]. The HADS is also recommended in cancer settings [38]. Questions are answered using a four-point scale and higher scores represent higher levels of anxiety and depression. Muscle strength Grip strength is a good indication of global muscle strength and assessment of grip is common method that is used to assess general strength characteristics [32]. Here, isometric grip strength will be measured in kilo- grams using an adjustable hand-held dynamometer (Jamar® Hydraulic Hand Dynamometer, USA) with the elbow flexed at 90 degrees and the forearm and wrist in neutral position following standardised American Col- lege of Sports Medicine (ACSM) guidelines [32]. End of study questionnaire During their final assessment at twelve weeks, partici- pants will be invited to complete the end of study ques- tionnaire. The open questions aim to explore their thoughts and feelings about the intervention programme, and to analyse their views on their particu- lar intervention including preferences, perceived advan- tages and disadvantages and reasons for participation and adherence. Quality of life p y Resting Blood Pressure (BP) and Heart Rate (HR) will be assessed using an electronic BP monitor (Omron M10- IT, Japan) and finger pulse oximeter (Solaris S10A, China) in sitting. In addition, measures of pulmonary capacity and function will be assessed using a hand-held microspirometer (Micro 1 Medical Microspirometer, England) following British Thoracic Society guidelines [31]. Forced expiratory volume in one second (FEV1), The primary outcome measure is QoL, assessed by the EORTC QLQ-C30. This self-reported questionnaire has been demonstrated to be a valid and reliable tool, takes approximately eleven minutes to complete and most subjects require no assistance [28]. This has been used extensively in cancer survivorship research, and also spe- cifically with lymphoma patients [29]. The EORTC QLQ-C30 consists of thirty items. Each question is Table 1 Primary and Secondary Outcome Measures Outcome measured Instrument Table 1 Primary and Secondary Outcome Measures Outcome measured Instrument Guidelines When assessed Baseline Midway 6 weeks End of intervention 12 weeks Primary outcome Quality of Life EORTC QLQ-C30 Self-reported Questionnaire ☑ ☑ ☑ Secondary outcomes Body Composition Height, Weight, Bioimpedance analysis – BMI, Body Fat % - TANITA BC-418 Body Composition Analyzer Same investigator and equipment, time of day and clothing kept similar where possible ☑ ☑ ☑ Cardiovascular status – Resting BP, HR, SpO2 Omron M10-IT electronic BP monitor, Solaris Finger Pulse Oximeter S10A ☑ ☑ ☑ Pulmonary Function – FEV1, FVC, FER, PEF Hand-held Micro 1 Medical Microspirometer British Thoracic Society, 2013 ☑ ☑ ☑ Muscle Strength – Grip Strength Jamar® Hydraulic Hand Dynamometer American College of Sports Medicine, 2014 ☑ ☑ ☑ Functional Exercise Capacity Six Minute Walk Test American Thoracic Society, 2002 ☑ ☑ ☑ Physical Well-being, Social/Family Well-being, Emotional Well-being, Functional Well-being FACT-Lym Self-reported Questionnaire Anxiety and Depression HADS ☑ ☑ ☑ Feelings and Perceptions about participating in programme End of Study Questionnaire Open-ended Questionnaire mailed to participants’ home ☑ Guidelines Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 6 of 9 (2019) 11:17 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation forced vital capacity (FVC), forced expiratory ratio (FER) and expiratory peak flow (PEF) will be recorded. lymphoma subscale. The FACT-Lym has good internal consistency and validity [35]. All questions are answered on a five-point scale and scoring guidelines are recom- mended together with the questionnaire [36]. Functional exercise capacity Due to the variation in the population being studied in terms of age, co-morbidity and physical ability, a sub- maximal test was considered appropriate for this study. The Six-Minute Walk Test (6MWT) is considered a good indicator of functional ability and exercise capacity and is a common outcome measure to evaluate progress in rehabilitation programmes [33]. This test was chosen as it can be undertaken in a clinical setting with ease, takes a short duration of time to complete and appeared appropriate for this sample of patients following chemo- therapy who are generally deconditioned and fatigue quickly. The 6MWT has been validated and used exten- sively in a variety of patient populations, and has been recommended for use in cancer patients also [34]. Data analysis ll b y Data will be entered into the Microsoft Excel (2013) database by SH and analysed by all authors using the IBM SPSS version 22 (SPSS, Inc., Chicago, IL) statis- tical software package. The intent-to-treat principle will be applied and the significance level will be set at 0.05. Descriptive statistics will be used to present baseline demographic characteristics. To compare dif- ference in the primary outcome measure, QoL score between the two groups post-intervention, ANCOVA (analysis of covariance) will be used, with pre-test values as covariate to adjust for any baseline differ- ences. Data will be analysed to check fit for ANCOVA assumptions. Post-hoc paired-samples t- tests for pre-post intervention will be tested within groups with Bonferroni corrections. Missing data will be treated as recommended by scoring guidelines of questionnaires [24, 30]. These analyses will be re- peated for secondary outcome measures, providing data fit normal distribution and other assumptions for ANCOVA. The influence of potential confounding factors such as age, gender, number of co-morbidities and ECOG status will be explored using correlation analyses. Adherence and drop-out rates will also be analysed. Qualitative data from the end of study ques- tionnaire will be analysed for codes and themes using qualitative content analysis [39]. The 6MWT will be carried out following guidelines recommended by the American Thoracic Society (ATS) [33]. The test will be carried out in a 30 m marked dis- tance, and the total distance covered in 6 min will be re- corded, as well as post-test Heart Rate and oxygen saturation, RPE and Rate of Dyspnoea [26]. The test will cease prior to 6 min if the patient chooses to stop or the tester terminates testing (indications for terminating ex- ercise as per the ATS guidelines include the following: severe exhaustion or shortness of breath, wheezing, diz- ziness, chest pain or muscle cramps) [33]. At no point will the patient be encouraged to continue beyond the level at which they wish to stop. Discussion A large proportion of lymphoma survivors continue to experience unmet needs following treatment [1–3]. Long-term and late effects are often overlooked, and survivors do not routinely receive advice or interventions to target these and maximise well-being [40]. Previous studies have supported the use of both relaxation and exercise interventions to treat physical and psychosocial complaints of cancer survivors [9–12]. No trial to date has compared efficacy of these two interventions in lymphoma survivors. It will not be possible to stratify participants by factors such as age, gender, etc. during randomization due to the small sample size. However, sample size was calcu- lated using reference values (including standard devi- ation) and power calculations recommended by the EORTC reference manual [23]; similar trials on the ef- fects of exercise in cancer patients have been carried out with comparable sample sizes, and these were able to de- tect significant results [9]. The REIL study aims to study a sample of lymph- oma survivors shortly post-chemotherapy, and com- pare the effectiveness of relaxation and exercise on improving QoL. This study was developed to address some issues highlighted in survivorship literature in- cluding focus on survivors of cancers other than breast or prostate, emphasis on the transition phase immediately following treatment, addressing physical, psychological and social needs of lymphoma survivors, use of patient-reported outcome measures, self-man- agement, and moving from control groups to com- parison between two interventions. In addition to quantitative data from outcome mea- sures, the end of study questionnaire will explore partici- pants’ perceptions and feelings towards an intervention programme post-chemotherapy. Such information will highlight behaviours and patterns in this sample of lymphoma survivors including reasons for participation, adherence and non-adherence, and any preferences; it is anticipated that this data will ultimately help in the pro- motion of a healthy lifestyle in lymphoma and cancer survivors. The authors will aim to publish results from this study to add to the evidence to inform healthcare professionals on effective interventions to improve QoL of lymphoma survivors. In this study lymphoma patients in remission will be recruited within six weeks of their last chemother- apy session. Patients during this early phase of sur- vivorship who have completed initial cancer treatment have been relatively neglected [19], and this period immediately following treatment has been identified as a key time to address side effects of treatment and facilitate return to pre-morbid health [41]. Well-being h The Functional Assessment of Chronic Illness Therapy (FACIT) questionnaires have been validated in studies of cancer management and are designed to encompass a range of psychosocial factors [35]. Like the EORTC QLQ-C30, these questionnaires are also a patient re- ported measure of QoL. However these questionnaires focus on aspects of well-being including physical, social, emotional and functional well-being subscales. Here, the lymphoma-specific scale (FACT-Lym) will be used (comprising the general or FACT-G questionnaire plus a Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 Page 7 of 9 (2019) 11:17 (2019) 11:17 Page 7 of 9 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation flexibility, familiar surroundings, family support and reduced travel requirements [45]. Discussion Studies on cancer survivors have indicated that they would prefer to begin an exercise programme immediately or soon after treatment [42], and at this point the focus can move from the disease and treatment to ‘moving on’ to the next phase of life [43]. Conclusion It is well documented that lymphoma survivors commonly suffer from consequences of treatment such as pain, fa- tigue, decreased function, anxiety and depression which have a negative impact on their quality of life. However, there is no standardize care pathway recommended for lymphoma survivors following chemotherapy. The current proposed REIL study aims to determine the more effective intervention of two in improving QoL in lymphoma survivors. It is anticipated that re- sults from this preliminary study will help build to- wards the development of feasible and effective practice guidelines to improve QoL of lymphoma sur- vivors post-chemotherapy. While the additional psychosocial benefits of super- vised group classes for cancer survivors have been recog- nised [44], the need for a shift towards self-management in cancer survivorship has been highlighted [6, 7]. Here participants are supported to self-manage in a variety of ways, but the onus will be on them to initiate contact with the healthcare professionals for additional support when needed [7, 8]. Both the relaxation and exercise programmes in this study are designed to be performed independently and participants will be supplied with re- sources to allow convenient home-based performance, including written instructions and advice. Participants will be able to contact the investigator whenever re- quired. This study will provide results including ad- herence and drop-out rates that are more generalizable to ‘real-world’ lymphoma survivors than controlled clinic-based research. Home-exercise pro- grammes also afford patients better scheduling 6MWT: Six Minute Walk Test; ACSM: American College of Sports Medicine; ANOVA: Analysis of Variance; ATS: American Thoracic Society; BMI: Body Mass Index; BP: Blood Pressure; CD: Compact Disc; COPD: Chronic Obstructive Pulmonary Disease; ECOG: Eastern Cooperative Oncology Group; EORTC QLQ-30: European Organisation for Research and Treatment of Cancer – QoL core questionnaire; FACIT: Functional Assessment of Chronic Illness Therapy; FACT-Lym: Functional Assessment of Cancer Therapy - Lymphoma; FER: Forced Expiratory Rate; FEV1: Forced Expiratory Volume; FVC: Forced Vital Capacity; HADS: Hospital Anxiety and Depression Scale; HOOP: Haematology and Oncology Out-Patients; HR: Heart Rate; NCSI: National Cancer Survivorship Initiative; NHS: National Health Service; PEF: Peak Expiratory Flow; QoL: Quality of Life; REIL: Relaxation and Exercise In Lymphoma; RPE: Rate of Perceived Exercise; TIDieR: Template for Intervention Description and Replication; UK: United Kingdom Received: 29 November 2018 Accepted: 18 July 2019 25. Campbell A, Stevinson C, Crank H. The BASES expert statement on exercise and cancer survivorship. J Sports Sci. 2012;i:1–4. Availability of data and materials 13. Lymphoma Action. 2019. https://lymphoma-action.org.uk/ (Accessed 22 July 2019). The written material and datasets used and/or analysed during the current study will be available from the corresponding author on reasonable request. 14. Macmillan Cancer Support. 2019. https://www.macmillan.org.uk/information- and-support (Accessed 22 July 2019). 14. Macmillan Cancer Support. 2019. https://www.macmillan.org.uk/information- and-support (Accessed 22 July 2019). 15. Bower JE. Mindfulness interventions for cancer survivors: moving beyond wait-list control groups. J Clin Oncol. 2016;34:3366–8. 15. Bower JE. Mindfulness interventions for cancer survivors: moving beyond wait-list control groups. J Clin Oncol. 2016;34:3366–8. Acknowledgments Not applicable. 8. Foster C, Fenlon D. Recovery and self-management support following primary cancer treatment. Br J Cancer. 2011;105:S21–8. 8. Foster C, Fenlon D. Recovery and self-management support following primary cancer treatment. Br J Cancer. 2011;105:S21–8. Foster C, Fenlon D. Recovery and self-management support following primary cancer treatment. Br J Cancer. 2011;105:S21–8. 9. Gerritson JKW, Vincent AJPE. Exercise improves quality of life in patients with cancer: a systematic review and meta-analysis of randomised controlled trials. Br J Sports Med. 2016;50:796–803. 9. Gerritson JKW, Vincent AJPE. Exercise improves quality of life in patients with cancer: a systematic review and meta-analysis of randomised controlled trials. Br J Sports Med. 2016;50:796–803. Funding 12. Reich RR, Lengacher CA, Alinat CB, et al. Mindulness-based stress reduction in post-treatment breast cancer patients: immediate and sustained effects across multiple symptom clusters. J Pain Symptom Manag. 2017;53:85–95. There are no sources of funding for this study. Abbreviations 6MWT Si Mi 6MWT: Six Minute Walk Test; ACSM: American College of Sports Medicine; ANOVA: Analysis of Variance; ATS: American Thoracic Society; BMI: Body Mass Index; BP: Blood Pressure; CD: Compact Disc; COPD: Chronic Obstructive Pulmonary Disease; ECOG: Eastern Cooperative Oncology Group; EORTC QLQ-30: European Organisation for Research and Treatment of Cancer – QoL core questionnaire; FACIT: Functional Assessment of Chronic Illness Therapy; FACT-Lym: Functional Assessment of Cancer Therapy - Lymphoma; FER: Forced Expiratory Rate; FEV1: Forced Expiratory Volume; FVC: Forced Vital Capacity; HADS: Hospital Anxiety and Depression Scale; HOOP: Haematology and Oncology Out-Patients; HR: Heart Rate; NCSI: National Cancer Survivorship Initiative; NHS: National Health Service; PEF: Peak Expiratory Flow; QoL: Quality of Life; REIL: Relaxation and Exercise In Lymphoma; RPE: Rate of Perceived Exercise; TIDieR: Template for Intervention Description and Replication; UK: United Kingdom Page 8 of 9 Page 8 of 9 Page 8 of 9 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:17 (2019) 11:17 Hathiramani et al. BMC Sports Science, Medicine and Rehabilitation Consent for publication Not applicable. Not applicable. 20. Chan A, Tetzlaff JM, Gøtzche PC, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ. 2013;346:e7586. Author details 1 22. Osaba D, Rodrigues G, Myles J, et al. Interpreting the significance of changes in health-related quality-of-life scores. J Clin Oncol. 1998;16:139–44. 1Kingston University and St. George’s University of London, London, UK. 2St. George’s Healthcare NHS Trust, London, UK. 3Kingston University, Kingston Upon Thames, London, UK. 4Faculty of Health, Social Care & Education, Cranmer Terrace, London SW17 0RE, UK. 23. Scott NW, Fayers PM, Aaronson NK, et al. EORTC QLQ-C30 Reference Values. 2008. https://www.eortc.org/app/uploads/sites/2/2018/02/reference_values_ manual2008.pdf (Accessed 22 July 2019). 24. Hoffman TC, Glasziou PP, Boutron I, et al. Better reporting of interventions: template for intervention description and replication (TIDier) checklist and guide. BMJ. 2014;348:g1687. Received: 29 November 2018 Accepted: 18 July 2019 Authors’ contributions All authors contributed to the development of study protocol. SH was a major contributor in writing the manuscript. RP contributed to aims, eligibility and recruitment sections; AY to sample size, randomisation and statistical analysis; HM contributed to outcome measures, and feedback to all sections of paper. All authors read and approved the final manuscript. 10. Duncan M, Moschopoulou E, Herrington E, et al. Review of systematic reviews of non-pharmacological interventions to improve quality of life in cancer survivors. BMJ Open. 2017;7:e015860. 11. Parás-Bravo P, Salvadores-Fuentes P, Alonso-Blanco C, et al. 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Improving provision of care for long-term survivors of lymphoma. Clin Lymphoma Myeloma Leuk. 2017;17:e1–9. 28. Aaronson NK, Ahmedzai S, Begman B, et al. The European Organisation for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst. 1993;85: 365–76. 3. Raphael D, Frey R, Gott M. Maintaining psychosocial wellbeing for post- treatment haematological cancer survivors: strategies and potential barriers. Eur J Oncol Nurs. 2019;38:36–41. 29. Heutte N, Flechtner HH, Mourier N, et al. Quality of life after successful treatment of early-stage Hodgkin's lymphoma: 10-year follow-up of the EORTC–GELA H8 randomised controlled trial. Lancet Oncol. 2009;10:1160–70. 4. Bhuva N, Li SP, Maher J. Living with and beyond Cancer: new challenges. In: Mohan R, editor. Topics in Cancer survivorship. Croatia: InTech Europe; 2012. p. 1–12. 30. Fayers PM, Aaronson NK, Bjordal K, et al. 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National Cancer Survivorship Initiative Vision. 2010. https://webarchive.nationalarchives.gov. uk/20100809113601/http://www.improvement.nhs.uk/cancer/LinkClick. aspx?fileticket=4apVUSvGcow%3d&tabid=214. (Accessed 22 July 2019). 33. American Thoracic Society. ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med. 2002;166:111–7. 33. American Thoracic Society. ATS statement: guidelines for the six-minute walk test. Ethics approval and consent to participate The REIL study has received ethical approval from Camden and Islington National Research Ethics Service (13.LO.1327), as well as local site approval from St. George’s Hospital Joint Research and Enterprise Office (13.0108). The study has been registered on a publicly accessible database, ClinicalTrials.gov (NCT02272751). 16. Buchanan DR, White JD, O’Mara AM, et al. 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https://openalex.org/W2897730463	https://europepmc.org/articles/pmc6195872?pdf=render	English	null	Appraisal of systemic inflammation and diagnostic markers in a porcine model of VAP: secondary analysis from a study on novel preventive strategies	Intensive care medicine experimental	2,018	cc-by	11,945	© 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. Appraisal of systemic inflammation and diagnostic markers in a porcine model of VAP: secondary analysis from a study on novel preventive strategies Gianluigi Li Bassi1,2,3,4†, Raquel Guillamat Prats3,5†, Antonio Artigas3,5, Eli Aguilera Xiol1,2,3, Joan-Daniel Marti1, Otavio T. Ranzani1, Montserrat Rigol1,2,6, Laia Fernandez1,2,3,4, Andrea Meli1,7, Denise Battaglini1,8, Nestor Luque1, Miguel Ferrer1,2,3,4, Ignacio Martin-Loeches9, Pedro Póvoa10,11, Davide Chiumello7, Paolo Pelosi8 and Antoni Torres1,2,3,4* * Correspondence: atorres@clinic.cat †Gianluigi Li Bassi and Raquel Guillamat Prats contributed equally to this work. (2018) 6:42 (2018) 6:42 Intensive Care Medicine Experimental Li Bassi et al. Intensive Care Medicine Experimental https://doi.org/10.1186/s40635-018-0206-1 Intensive Care Medicine Experimental RESEARCH Open Access Open Access Appraisal of systemic inflammation and diagnostic markers in a porcine model of VAP: secondary analysis from a study on novel preventive strategies * Correspondence: atorres@clinic.cat †Gianluigi Li Bassi and Raquel Guillamat Prats contributed equally to this work. 1Division of Animal Experimentation, Department of Pulmonary and Critical Care Medicine, Thorax Institute, Hospital Clinic, Calle Villarroel 170, Esc 6/8 Pl 2, Barcelona, Spain 2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Full list of author information is available at the end of the article © 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. Background Ventilator-associated pneumonia (VAP) is a common iatrogenic pulmonary complica- tion in critically ill patients on mechanical ventilation (MV) [1–3]. Clinical presentation of VAP is highly heterogenous ranging from mild to highly severe [4], potentially caus- ing a systemic cytokine storm and septic shock [5]. Many efforts have been made to fully characterize pathophysiology of the disease and the host inflammatory response, improve diagnostic accuracy, and develop efficacious preventive strategies [2, 3]. Among the available preventive interventions [6], body position plays a critical role. Currently, intensive care unit (ICU) patients are kept with the head of the bed oriented above 30° to avoid gastro-pulmonary aspiration, namely the semirecumbent position [7]. A recent clinical trial has also tested preventive efficacy of the Trendelenburg pos- ition, which limits gravity-driven aspiration of oropharyngeal secretions [8]. Neverthe- less, to date, the effects of body position on the host inflammatory response and potential association with the development of VAP are still unknown. Inflammatory biomarkers in blood or bronchoalveolar lavage fluids of patients with VAP [9–11] not only have been tested to characterize inflammation but also to accurately and promptly diagnose VAP. Indeed, VAP is currently diagnosed using clinical criteria and microbiology cultures, which yield low specificity/sensitivity and are often too slow for clin- ical needs [12]. Unfortunately, aforementioned clinical studies were biased by the well-recognized challenges in VAP diagnosis, the extreme heterogeneity among ICU popula- tions and degrees of severity. As a result, VAP still lacks a clinical diagnostic gold-standard. We previously developed a reliable animal model of VAP [13] to circumvent some of aforementioned limitations encountered in clinical settings and to specifically evaluate novel diagnostic and preventive strategies. This model was recently used to study efficacy of (1) inverse-ratio ventilation with positive end-expiratory pressure (PEEP) or (2) the Trendelen- burg position in the prevention of VAP. These ventilatory settings were applied because mucus clearance is enhanced through inverse-ratio ventilation [14, 15] and the Trendelen- burg position [16], while gravity-driven pulmonary aspiration is reduced through PEEP [17]. Abstract In this reliable model of VAP, ETA culture showed good diagnostic accuracy, whereas systemic IL-10 and TNF-α marginally improved accuracy. Further clinical studies will be necessary to confirm clinical value of the Trendelenburg position as a measure to hinder inflammation during mechanical ventilation and significance of systemic IL-10 and TNF-α in the diagnosis of VAP. Keywords: Trendelenburg, Semirecumbent, Inflammation, Interleukin, Mechanical ventilation, Ventilator-associated pneumonia Conclusions: Our findings demonstrate anti-inflammatory effects associated with the Trendelenburg position. In this reliable model of VAP, ETA culture showed good diagnostic accuracy, whereas systemic IL-10 and TNF-α marginally improved accuracy. Further clinical studies will be necessary to confirm clinical value of the Trendelenburg position as a measure to hinder inflammation during mechanical ventilation and significance of systemic IL-10 and TNF-α in the diagnosis of VAP. Keywords: Trendelenburg, Semirecumbent, Inflammation, Interleukin, Mechanical ventilation, Ventilator-associated pneumonia Abstract Background: We previously evaluated the efficacy of a ventilatory strategy to achieve expiratory flow bias and positive end-expiratory pressure (EFB + PEEP) or the Trendelenburg position (TP) for the prevention of ventilator-associated pneumonia (VAP). These preventive measures were aimed at improving mucus clearance and reducing pulmonary aspiration of bacteria-laden oropharyngeal secretions. This secondary analysis is aimed at evaluating the effects of aforementioned interventions on systemic inflammation and to substantiate the value of clinical parameters and cytokines in the diagnosis of VAP. Methods: Twenty female pigs were randomized to be positioned in the semirecumbent/ prone position, and ventilated with duty cycle 0.33 and without PEEP (control); positioned as in the control group, PEEP 5 cmH2O, and duty cycle to achieve expiratory flow bias (EFB+PEEP); ventilated as in the control group, but in the Trendelenburg position (Trendelenburg). Following randomization, P. aeruginosa was instilled into the oropharynx. Systemic cytokines and tracheal secretions P. aeruginosa concentration were quantified every 24h. Lung biopsies were collected for microbiological confirmation of VAP. 1Division of Animal Experimentation, Department of Pulmonary and Critical Care Medicine, Thorax Institute, Hospital Clinic, Calle Villarroel 170, Esc 6/8 Pl 2, Barcelona, Spain 2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Full list of author information is available at the end of the article Results: In the control, EFB + PEEP, and Trendelenburg groups, lung tissue Pseudomonas aeruginosa concentration was 2.4 ± 1.5, 1.9 ± 2.1, and 0.3 ± 0.6 log cfu/mL, respectively (p = 0.020). Whereas, it was 2.4 ± 1.9 and 0.6 ± 0.9 log cfu/mL in animals with or without VAP (p < 0.001). Lower levels of interleukin (IL)-1β (p = 0.021), IL-1RA (p < 0.001), IL-4 (p = 0.005), IL-8 (p = 0.008), and IL-18 (p = 0.050) were found in Trendelenburg animals. VAP increased IL-10 (p = 0.035), tumor necrosis factor-α (p = 0.041), and endotracheal aspirate (ETA) P. aeruginosa concentration (p = 0.024). A model comprising ETA bacterial burden, IL-10, and TNF-α yielded moderate discrimination for the diagnosis of VAP (area of the receiver operating curve 0.82, 95% CI 0.61–1.00). (Continued on next page) Page 2 of 15 Page 2 of 15 (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (Continued from previous page) Conclusions: Our findings demonstrate anti-inflammatory effects associated with the Trendelenburg position. Animal preparation and handling We studied 21 Large White-Landrace female pigs, orotracheally intubated and mechan- ically ventilated. Animals were anesthetized and paralyzed. Endogenous pneumonia was prevented with ceftriaxone. The femoral artery and jugular vein were cannulated for hemodynamic monitoring and blood sampling. Clinical parameters Body temperature, white blood cell count, and arterial partial pressure of oxygen were assessed before bacterial challenge and at 24, 48, and 72 h thereafter. The arterial par- tial pressure of oxygen per inspiratory fraction of oxygen ratio (PaO2/FIO2) was com- puted. At the same time points, serum creatinine and alanine transaminase were measured. Of note, reference values of aforementioned parameters in pigs are similar to those in humans. Finally, at 72 h, we collected tracheal secretions for quantitative microbiology culture, we qualitatively evaluated purulence, and we computed the clin- ical pulmonary infection score (CPIS), as described in Table 1. Aim, design, and settings We performed a secondary analysis of a previous study [18], conducted at the Division of Animal Experimentation, University of Barcelona, Barcelona, Spain. The primary goals of this secondary analysis were to evaluate dynamics of inflammatory biomarkers during ap- plication of novel VAP preventive strategies and to ascertain significance of various clin- ical parameters and cytokines in the diagnosis of VAP. Animals were managed according (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Page 3 of 15 Li Bassi et al. Intensive Care Medicine Experimental to the local guidelines and regulations for the use and care of animals. The animal experi- mentation ethical committee reviewed and approved the study protocol. Additional details on animal handling and methods are reported in previous publications [13, 18]. Randomization Randomization Following surgical preparation, pigs were randomized as follows: Following surgical preparation, pigs were randomized as follows: 1. Control: Pigs were placed in prone position and ventilated as reported above, but without PEEP. As previously reported [17, 19], the surgical bed was oriented approximately 30° in the anti-Trendelenburg position to achieve an orientation of the respiratory system as in the semirecumbent position in humans. 2. Expiratory flow bias and PEEP (EFB + PEEP): Pigs were positioned as in the control group. The duty cycle (TI/TTOT) was adjusted daily to achieve a mean expiratory-inspiratory flow bias of 10 L/min and PEEP was set at 5 cm H2O. As previously described [17], this ventilatory strategy was aimed at improving mucus clearance through the resulting expiratory flow bias [14], and hindering pulmonary aspiration of colonized subglottic secretions through PEEP. 2. Expiratory flow bias and PEEP (EFB + PEEP): Pigs were positioned as in the control group. The duty cycle (TI/TTOT) was adjusted daily to achieve a mean expiratory-inspiratory flow bias of 10 L/min and PEEP was set at 5 cm H2O. As previously described [17], this ventilatory strategy was aimed at improving mucus clearance through the resulting expiratory flow bias [14], and hindering pulmonary aspiration of colonized subglottic secretions through PEEP. 2. Expiratory flow bias and PEEP (EFB + PEEP): Pigs were positioned as in the control group. The duty cycle (TI/TTOT) was adjusted daily to achieve a mean expiratory-inspiratory flow bias of 10 L/min and PEEP was set at 5 cm H2O. As previously described [17], this ventilatory strategy was aimed at improving mucus clearance through the resulting expiratory flow bias [14], and hindering pulmonary aspiration of colonized subglottic secretions through PEEP. Table 1 Clinical pulmonary infection score CPIS points 0 1 2 Tracheal secretions Rare Abundant Abundant and purulent Chest radiograph infiltrates No infiltrate Disseminated Localized Temperature (°C) ≥36.5 and ≤38.4 ≥38.5 and ≤38.9 ≥39 and ≤36 Leukocytes count (103/μl) ≥4 and ≤11 < 4 or > 11 PaO2/FIO2 (mmHg) ≥240 ≤240 Microbiology Negative Positive CPIS clinical pulmonary infection score. A CPIS score value ≥6 was considered suggestive of pneumonia. Chest radiographs were not collected. Nevertheless, given the initial healthy status of the animal and the macroscopic lung examination upon autopsy, we assumed in all pigs localized chest radiograph infiltrates in case of confirmed pulmonary infiltrates upon autopsy CPIS clinical pulmonary infection score. Autopsy, microbiological, histological studies, and VAP definitions Autopsy, microbiological, histological studies, and VAP definitions Tracheal secretions were collected before autopsy and P. aeruginosa concentration score was computed as follows: 0: < 3.0 log10 cfu/mL; 1: 3.0–3.9; 2: 4.0–4.9; 3: 5–6; 4: > 6 log cfu/mL. Seventy-six hours after tracheal intubation, the animal was euthanized. We took two samples from the most affected region of each of the five lobes for micro- biological assessments. Pulmonary infections were clinically suspected when two of the following clinical signs were present: white blood cell (WBC) ≥14,000 per mm3, puru- lent secretion, and body temperature ≥38.5 °C. Pulmonary biopsies were evaluated by pathologists and microbiologists blinded to the study treatments, and VAP was con- firmed according to a lobar histological injury score ≥3 (3 points = pneumonia, 4 points = confluent pneumonia, and 5 points = abscessed pneumonia), associated with a quantitative P. aeruginosa culture ≥3 log cfu/g [19, 28, 29]. Bacterial challenge Shortly after randomization, 5 mL of 107–108 ceftriaxone-resistant Pseudomonas aeru- ginosa suspension was instilled into the oropharynx to colonize the oropharynx and promote aspiration of P. aeruginosa-laden oropharyngeal secretions and VAP [13]. Randomization A CPIS score value ≥6 was considered suggestive of pneumonia. Chest radiographs were not collected. Nevertheless, given the initial healthy status of the animal and the macroscopic lung examination upon autopsy, we assumed in all pigs localized chest radiograph infiltrates in case of confirmed pulmonary infiltrates upon autopsy CPIS clinical pulmonary infection score. A CPIS score value ≥6 was considered suggestive of pneumonia. Chest radiographs were not collected. Nevertheless, given the initial healthy status of the animal and the macroscopic lung examination upon autopsy, we assumed in all pigs localized chest radiograph infiltrates in case of confirmed pulmonary infiltrates upon autopsy Page 4 of 15 Page 4 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental 3. Trendelenburg: Pigs were in prone position and ventilated as in the control group. The surgical bed was oriented 5° below horizontal To achieve aforementioned ventilatory endpoints, airway pressure was measured proximally to the endotracheal tube with a pressure transducer (MPX 2010 DP; Motorola, Phoenix, AZ, USA). Respiratory flow rates were measured with a heated pneumotachograph (Fleisch no. 2; Fleisch, Lausanne, Switzerland). Flow and pressure signals were recorded on a personal computer and assessed with dedicated software (Colligo; Elekton, Milan, Italy). Systemic biomarkers Prior to bacterial challenge, and at 24, 48, and 72 h thereafter, blood was drawn for meas- urement of serum inflammatory markers. Blood was centrifuged at 3000 rpm at 4 °C for 15 min, and serum aliquots were stored at −80 °C. Serum interferon (INF)-γ; interleukin (IL)-1α; IL-1β; IL-1 receptor antagonist (RA); IL-2; IL-4; IL-6; IL-8; IL-10; IL-12; IL-18; and tumor necrosis factor (TNF)-α were quantified by bead-based multiplex assays with Lumi- nex xMAP® technology (Millipore Iberica, S.A., Madrid, Spain). Whereas tissue factor, angiotensin-2, adrenomedullin, and protein C-reactive protein were quantified through enzyme-linked immunosorbent assay (ELISA) (Bionova cientifica S.L., Madrid, Spain). Ac- curacy in cytokine quantification by Luminex xMAP® technology is comparable to the ELISA assay [20–22]. Nevertheless, Luminex xMAP® assay allows measurement of multiple cytokines simultaneously providing additional benefits. All inflammatory markers data are reported as log pg/L. Aforementioned biomarkers were chosen based on previous clinical studies assessing systemic and pulmonary inflammation during pneumonia [10, 11, 23–27]. Results Inflammatory biomarkers of six, eight, and seven pigs—originally randomized in the control, EFB + PEEP, and Trendelenburg groups, respectively—were available for ana- lysis. One pig in the EFB + PEEP group was euthanized earlier for accidental extubation and colonization/histology of the lungs was not examined. Thus, we ultimately ana- lyzed data of six control pigs and seven animals in the EFB + PEEP and Trendelenburg groups. Overall, ten animals developed VAP (four controls, six EFB + PEEP, and zero Trendelenburg). In the control, EFB + PEEP, and Trendelenburg groups, lung P. aerugi- nosa burden was 2.4 ± 1.5, 1.9 ± 2.1, and 0.3 ± 0.6 log10 cfu/mL, respectively (p = 0.020). Whereas, lung bacterial burden was 2.4 ± 1.9 log10 cfu/g in animals with VAP, in com- parison with 0.6 ± 0.9 in animals without VAP (p < 0.001). Clinical and microbiology studies Statistical analysis A sample size of at least seven animals per group was calculated on the basis of the pri- mary outcome of the original study [18], which was powered to detect a difference in Page 5 of 15 Page 5 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental P. aeruginosa lung tissue concentration between control, EFB + PEEP, and Trendelen- burg groups of 3 ± 1.5 log cfu/g, 1 ± 1.5 log cfu/g, and 0 ± 1.5 log cfu/g, respectively, for an assumed effect size of 0.83, a fixed power of 0.85%, and an alpha error probability of 0.05. Restricted maximum likelihood (REML) analysis, based on repeated measures ap- proach, including type of infection, study treatments, and study times, were conducted to evaluate differences in cytokines concentrations. Post-hoc multiple comparisons among groups were computed through Bonferroni adjustment. The area under the re- ceiver operating curves (ROC-AUC) of clinical parameters were computed. Relation- ship between inflammatory biomarkers and P. aeruginosa lung tissue concentration was evaluated by linear regression analysis. Statistical analyses were performed using SAS software (version 9.4; SAS Institute, Cary, NC). Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score, EFB + PEEP expiratory flow bias and positive end expiratory pressure group The effects of study treatments on serum inflammatory markers As depicted in Table 4, study treatments changed significantly levels of INF-γ (p = 0.047), IL-1β (p = 0.021), IL-1RA (p < 0.001), IL-4 (p = 0.005), IL-8 (p = 0.008), IL-18 (p = 0.050), and angiotensin-2 (p = 0.048). In particular, as depicted in Fig. 1, at the end of the study, animals positioned in Trendelenburg presented lower levels of all aforementioned inflam- matory markers, but INF-γ and angiotensin-2. Clinical and microbiology studies VAP ventilator-associated pneumonia, PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. VAP ventilator-associated pneumonia, PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score following variables changed significantly: body temperature (37.0 ± 1.6, 38.4 ± 2.1, and 37.1 ± 1.3 °C in the control, EFB + PEEP, and Trendelenburg groups, respectively, p < 0.001) and CPIS calculated at 72 h, before autopsy (4.6 ± 0.9, 6.3 ± 0.5, and 4.6 ± 0.9, p = 0.035). Whereas, in animals with VAP, P. aeruginosa concentration in tracheal aspirates was 6.3 ± 0.6 log10 cfu/mL, in comparison with 5.4 ± 0.9 in animals without VAP (p = 0.024). As for others clinical parameters of organ injury, creatinine (pig reference value 06–1.2 mg/dL) was 1.16 ± 0.38, 1.42 ± 0.45, and 1.40 ± 0.28 mg/dL in the control, EFB + PEEP, and Trendelenburg groups, respectively, (p < 0.001); whereas in the animals with VAP was 1.41 ± 0.45 and 1.24 ± 0.31 without VAP was (p < 0.001). Finally, alanine amino- transferase (pig reference value 10–40 UI/L) was 41.7 ± 16.4, 34.4 ± 13.9, and 25.8 ± 9.6 U/L in the control, EFB + PEEP, and Trendelenburg groups, respectively, (p < 0.001); whereas in the animals with VAP was 31.2 ± 13.8 and 37.3 ± 16.2 U/L without VAP (p = 0.914). Clinical and microbiology studies Table 2 reports clinical and microbiology variables among study groups, whereas Table 3 report difference between animals with or without VAP. Among study treatments, the Table 2 Clinical and microbiology variables among study treatments Parameter Time of assessment Control (6) EFB + PEEP (7) Trendelenburg (7) p value Body temperature (°C) Throughout study time 37.0 ± 1.6 38.4 ± 2.1 37.1 ± 1.3 < 0.001 72 h 37.5 ± 0.9 39.6 ± 0.8 36.7 ± 1.3 White blood cells (× 109/L) Throughout study time 17.2 ± 6.8 13.4 ± 4.3 17.7 ± 5.9 0.133 72 h 17.3 ± 7.5 12.7 ± 6.1 18.1 ± 2.8 PaO2/FIO2 (mmHg) Throughout study time 424.8 ± 88.9 423.3 ± 76.5 443.6 ± 43.3 0.312 72 h 378.0 ± 85.0 339.1 ± 26.5 437.0 ± 40.6 CPIS 72 h 4.6 ± 0.9 6.3 ± 0.5 4.6 ± 0.9 0.035 Tracheal aspirate P. aeruginosa quantitative culture (log10 cfu/mL) 72 h 5.9 ± 1.1 6.0 ± 0.9 5.6 ± 1.0 0.487 Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score, EFB + PEEP expiratory flow bias and positive end expiratory pressure group Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. Clinical and microbiology studies PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score, EFB + PEEP expiratory flow bias and positive end expiratory pressure group Page 6 of 15 Page 6 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 (2018) 6:42 Table 3 Clinical and microbiology variables between animals with or without VAP Table 3 Clinical and microbiology variables between animals with or without VAP Parameter Time of assessment No-VAP (10) VAP (10) p value Body temperature (°C) Throughout study time 37.2 ± 1.6 37.9 ± 1.9 0.592 72 h 37.2 ± 1.6 38.8 ± 1.1 White blood cells (× 109/L) Throughout study time 16.8 ± 5.6 15.3 ± 6.4 0.420 72 h 16.5 ± 4.1 15.2 ± 7.5 PaO2/FIO2 (mmHg) Throughout study time 438.5 ± 58.5 390.5 ± 40.1 0.946 72 h 430.4 ± 40.5 334.7 ± 47.7 CPIS 72 h 4.6 ± 0.9 5.8 ± 0.9 0.260 Tracheal aspirate P. aeruginosa quantitative culture (log10 cfu/mL) 72 h 5.4 ± 1.0 6.3 ± 0.7 0.041 Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. VAP ventilator-associated pneumonia, PaO2/FIO2 arterial partial pressure of oxygen/inspiratory fraction of oxygen ratio, CPIS clinical pulmonary infection score Data are reported as mean ± standard deviation of various assessments throughout the study time or only at 72 h. Per each group, number of studied animals are reported between parenthesis. Of note, we report analyses p values of only the values of CPIS and tracheal aspirate P. aeruginosa quantitative culture at 72 h, whereas for the remaining parameters, we report p values of analysis of all assessed parameters throughout the study time (0, 24, 48, and 72 h). CPIS was computed as reported in the Table 1. Serum inflammatory markers to diagnose ventilator-associated pneumonia As depicted in Table 5, there were significant differences in the concentrations of IL-10 (p = 0.035) and TNF-α (p = 0.041) when comparing animals with or without VAP. We re- port in Fig. 2 dynamics of aforementioned cytokines, among animals with or without VAP. On the basis of aforementioned findings, the capacity for IL-10 and TNF-α and tra- cheal secretions P. aeruginosa burden to diagnose VAP were tested, and ROC curves computed (Table 6 and Fig. 3). We found that the best model, which provided moder- ate discrimination for the diagnosis of VAP, with a ROC-AUC of 0.82 (95% CI 0.61– 1.00) comprised all aforementioned parameters. Linear regression analyses showed that IL-10 (p = 0.995), TNF-α (p = 0.160), and tracheal secretions P. aeruginosa burden score Page 7 of 15 Page 7 of 15 (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Serum inflammatory markers to diagnose ventilator-associated pneumonia EFB + PEEP expiratory flow bias and positive end expiratory pressure group Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL- 12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. EFB + PEEP expiratory flow bias and positive end expiratory pressure group Fig. 1 Cytokines that significantly differed among study treatments, per times of assessment. a IFN-γ differed among study treatments (p = 0.043); no differences were found among times of assessment (p = 0.470) and study treatments × times of assessment (p = 0.847). b IL-1ß differed among study treatments (p = 0.038); whereas, we did not find differences among times of assessment (p = 0.869) and study treatments × times of assessment (p = 0.973). c IL-1RA differed among study treatments (p < 0.001); whereas, we did not find differences among times of assessment (p = 0.151) and study treatments × times of assessment (p = 0.618). d IL-4 differed among study treatments (p = 0.064); no differences among times of assessment (p = 0.861) and study treatments × times of assessment (p = 0.967) were found. e IL-8 differed among study treatments (p = 0.066) and no differences among times of assessment (p = 0.915) and study treatments × times of assessment (p = 0.978) were found. f IL-18 differed among study treatments (p = 0.005); whereas, among times of assessment (p = 0.879), and study treatments × times of assessment (p = 0.991) no differences were found. g Angiotensin-2 differed among study treatments (p = 0.048); whereas, among times of assessment (p = 0.552), and study treatments × times of assessment (p = 0.949) no differences were found. IFN interferon, IL interleukin, EFB + PEEP expiratory flow bias and positive end-expiratory pressure group Fig. 1 Cytokines that significantly differed among study treatments, per times of assessment. a IFN-γ differed among study treatments (p = 0.043); no differences were found among times of assessment (p = 0.470) and study treatments × times of assessment (p = 0.847). Serum inflammatory markers to diagnose ventilator-associated pneumonia Intensive Care Medicine Experimental Table 4 Inflammatory markers among study groups Inflammatory marker (log10 pg/L) Control (6) EFB + PEEP (7) Trendelenburg (7) p value INF-ɣ 1.70 ± 0.42 1.44 ± 0.33 1.76 ± 0.59 0.048 IL-1α −0.62 ± 0.47 −0.98 ± 0.54 −0.80 ± 0.64 0.709 IL-1β 0.35 ± 0.41 −0.06 ± 0.49 −0.03 ± 0.72 0.021 IL-1RA 1.33 ± 0.33 1.28 ± 0.31 0.82 ± 0.33 < 0.001 IL-2 −0.04 ± 0.37 −0.29 ± 0.51 −0.25 ± 0.79 0.558 IL-4 1.01 ± 0.52 0.25 ± 0.73 0.38 ± 0.83 0.005 IL-6 −0.01 ± 0.29 −0.07 ± 0.42 −0.15 ± 0.63 0.806 IL-8 0.16 ± 0.45 −0.03 ± 0.42 −0.08 ± 0.41 0.008 IL-10 −0.42 ± 0.45 0.13 ± 0.41 0.21 ± 0.53 0.450 IL-12 0.93 ± 0.13 0.77 ± 0.18 0.74 ± 0.23 0.058 IL-18 1.19 ± 0.31 0.94 ± 0.31 0.96 ± 0.40 0.050 TNF-alpha −0.16 ± 0.53 −0.08 ± 0.48 −0.31 ± 0.49 0.814 TF 1.95 ± 0.20 1.98 ± 0.16 2.04 ± 0.17 0.232 Angiotensin-2 1.73 ± 0.21 1.87 ± 0.17 1.62 ± 0.23 0.048 ADM 2.79 ± 0.80 2.99 ± 0.24 2.84 ± 0.65 0.515 CRP 4.15 ± 0.39 3.86 ± 0.55 3.55 ± 0.51 0.052 Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL- 12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. EFB + PEEP expiratory flow bias and positive end expiratory pressure group Table 4 Inflammatory markers among study groups Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL- 12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. Serum inflammatory markers to diagnose ventilator-associated pneumonia b IL-1ß differed among study treatments (p = 0.038); whereas, we did not find differences among times of assessment (p = 0.869) and study treatments × times of assessment (p = 0.973). c IL-1RA differed among study treatments (p < 0.001); whereas, we did not find differences among times of assessment (p = 0.151) and study treatments × times of assessment (p = 0.618). d IL-4 differed among study treatments (p = 0.064); no differences among times of assessment (p = 0.861) and study treatments × times of assessment (p = 0.967) were found. e IL-8 differed among study treatments (p = 0.066) and no differences among times of assessment (p = 0.915) and study treatments × times of assessment (p = 0.978) were found. f IL-18 differed among study treatments (p = 0.005); whereas, among times of assessment (p = 0.879), and study treatments × times of assessment (p = 0.991) no differences were found. g Angiotensin-2 differed among study treatments (p = 0.048); whereas, among times of assessment (p = 0.552), and study treatments × times of assessment (p = 0.949) no differences were found. IFN interferon, IL interleukin, EFB + PEEP expiratory flow bias and positive end-expiratory pressure group Page 8 of 15 Page 8 of 15 Li Bassi et al. Serum inflammatory markers to diagnose ventilator-associated pneumonia Of note, in animals that developed VAP, tracheal secretions P. aeruginosa burden was poorly associated with lung burden (p = 0.131). (p = 0.068) were not significantly associated with lung P. aeruginosa burden. In Fig. 4, we depicts tracheal secretions P. aeruginosa burden capability to predict lung burden, clustered by study groups and development of VAP. Of note, in animals that developed VAP, tracheal secretions P. aeruginosa burden was poorly associated with lung burden (p = 0.131). Serum inflammatory markers to diagnose ventilator-associated pneumonia Intensive Care Medicine Experimental (2018) 6:42 (2018) 6:42 Table 5 Inflammatory markers between animals with or without VAP Inflammatory marker (log10 pg/L) No-VAP (10) VAP (10) p value INF-ɣ 1.66 ± 0.58 1.60 ± 0.35 0.165 IL-1α −0.84 ± 0.58 −0.79 ± 0.57 0.339 IL-1β 0.04 ± 0.67 −0.12 ± 0.48 0.640 IL-1RA 0.99 ± 0.42 1.28 ± 0.31 0.663 IL-2 −0.30 ± 0.69 −0.10 ± 0.47 0.152 IL-4 −0.58 ± 0.80 −0.49 ± 0.75 0.130 IL-6 −0.14 ± 0.53 −0.01 ± 0.40 0.283 IL-8 0.05 ± 0.51 −0.01 ± 0.35 0.329 IL-10 0.15 ± 0.47 0.36 ± 0.48 0.035 IL-12 0.77 ± 0.20 0.86 ± 0.19 0.281 IL-18 1.03 ± 0.40 1.03 ± 0.31 0.707 TNF-alpha −0.33 ± 0.47 −0.04 ± 0.50 0.041 TF 2.01 ± 0.15 1.98 ± 0.21 0.517 Angiotensin-2 1.67 ± 0.21 1.81 ± 0.22 0.969 ADM 2.78 ± 0.71 2.99 ± 0.40 0.175 CRP 3.69 ± 0.46 4.04 ± 0.57 0.189 Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL-12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. VAP ventilator-associated pneumonia Table 5 Inflammatory markers between animals with or without VAP Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL-12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. VAP ventilator-associated pneumonia Determinations of interferon-γ (INF-γ), interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (RA), IL-2, IL-4, IL-6, IL-6, IL-8, IL-10, IL-12, IL-18, tumor necrosis factor (TNF)-α, tissue factor (TF), angiotensin-2, adrenomedullin (ADM), and C-reactive protein (CRP) in serum throughout the study are shown among study groups and in animals with or without VAP. Data are reported as mean ± standard deviation based on log10 transformation. VAP ventilator-associated pneumonia (p = 0.068) were not significantly associated with lung P. aeruginosa burden. In Fig. 4, we depicts tracheal secretions P. aeruginosa burden capability to predict lung burden, clustered by study groups and development of VAP. Discussion In this study, we observed that in pigs, challenged into the oropharynx with P. aerugi- nosa, the lateral-Trendelenburg position reduced systemic inflammation through the Fig. 2 Cytokines that significantly differed between animals with or without VAP, per times of assessment. a IL-10 differed among animals with or without VAP (p = 0.028) and for occurrence of VAP × study treatments (p = 0.029); whereas, among times of assessment (p = 0.984) and occurrence of VAP × times of assessment (p = 0.999) no differences were found. b TNF-α differed among types of pulmonary infection (p = 0.003) and study treatments (p = 0.008); whereas, among types of pulmonary infection × study treatments (p = 0.007); times of assessment (p = 0.984) and types of pulmonary infection × times of assessment (p = 0.995) no differences were found. IFN-γ interferon-γ, IL interleukin Fig. 2 Cytokines that significantly differed between animals with or without VAP, per times of assessment. a IL-10 differed among animals with or without VAP (p = 0.028) and for occurrence of VAP × study treatments (p = 0.029); whereas, among times of assessment (p = 0.984) and occurrence of VAP × times of assessment (p = 0.999) no differences were found. b TNF-α differed among types of pulmonary infection (p = 0.003) and study treatments (p = 0.008); whereas, among types of pulmonary infection × study treatments (p = 0.007); times of assessment (p = 0.984) and types of pulmonary infection × times of assessment (p = 0.995) no differences were found. IFN-γ interferon-γ, IL interleukin Page 9 of 15 Page 9 of 15 (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6 Table 6 Receiver operating curves parameters AU-ROC (95% CI) Best cut-off value* Sensitivity Specificity PPV NPV Single VAP diagnostic parameter IL-10 (log10 pg/L) 0.71 (0.47–0.96) 0.250 80% 70% 73% 78% TNF-α (log10 pg/L) 0.69 (0.44–0.96) −0.190 89% 56% 67% 83% Tracheal secretion P. aeruginosa concentration (cfu/mL) 0.80 (0.58–1.00) 6.34 70% 90% 88% 75% Tracheal secretion P. aeruginosa concentration scorea 0.71 (0.50–0.92) 4 80% 60% 67% 75% Combined VAP diagnostic parameterb IL-10 + tracheal secretion P. aeruginosa concentration scorea 0.78 (0.58–0.99) 5 70% 80% 78% 78% TNF-α + tracheal secretion P. aeruginosa concentration scorea 0.73 (0.51–0.95) 5 78% 67% 70% 75% IL-10 + TNF-α + tracheal secretion P. Discussion aeruginosa concentration scorea 0.82 (0.61–1.00) 6 67% 89% 86% 73% Receiver operating curves of ventilator-associated pneumonia diagnostic parameters and their combination. The optimal cut-off values were computed through the Youden’s index (J), which is the maximal vertical distance between the ROC curve and the first bisector (or chance line) aThe tracheal secretion P. aeruginosa concentration score was computed as follows: 0: < 3.0 log cfu/mL; 1: 3.0–3.9 log cfu/mL; 2: 4.0–5.9 log cfu/mL; 3: 5–6 log cfu/mL; 4: > 6 log cfu/mL. AU-ROC area under receiver operating curve, CI confidence interval, PPV positive predictive value, NPV negative predictive value, IL interleukin, TNF tumor necrosis factor bTo combine interleukins and tracheal secretion P. aeruginosa concentration score, we categorized IL-10 and TNF-α as 0–1 values, based on the best cut-off value Table 6 Receiver operating curves parameters Fig. 3 Analysis of the receiver operating characteristics curves. a Analysis of the receiver operating characteristic curve for IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score, which was computed as follows: 0 = < 3.0 log10 cfu/mL; 1 = 3.0–3.9 log10 cfu/mL; 2 = 4.0–4.9 log10 cfu/mL; 3 = 5–5.9 log10 cfu/mL; 4 = ≥6 log10 cfu/mL. The area under the receiver operating characteristics curves of IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score were 0.71, 0.69, and 0.81, respectively. b Analysis of the receiver operating characteristic curve for tracheal secretions P. aeruginosa concentration score with IL10, TNF-α, or IL10 and TNF-α. The area under the receiver operating characteristics curves of tracheal secretions P. aeruginosa concentration score with IL10 was 0.78, of tracheal secretions P. aeruginosa concentration score with TNF-α was 0.73, and of tracheal secretions P. aeruginosa concentration score with IL-10 and TNF-α was 0.82. We did not find any statistically significant differences among the tested receiver operating characteristics curves Fig. 3 Analysis of the receiver operating characteristics curves. a Analysis of the receiver operating Fig. 3 Analysis of the receiver operating characteristics curves. a Analysis of the receiver operating characteristic curve for IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score, which was computed as follows: 0 = < 3.0 log10 cfu/mL; 1 = 3.0–3.9 log10 cfu/mL; 2 = 4.0–4.9 log10 cfu/mL; 3 = 5–5.9 log10 cfu/mL; 4 = ≥6 log10 cfu/mL. The area under the receiver operating characteristics curves of IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score were 0.71, 0.69, and 0.81, respectively. Effects of study interventions on systemic inflammation We consistently demonstrated in previous studies in sheep [16, 30] and pigs [17, 31] that the Trendelenburg position avoided VAP, but to the best of our knowledge, this is the first comprehensive report regarding its effects on systemic inflammation. Our study adds to previous literature and suggests that during mechanical ventilation, the Trendelenburg position might limit systemic inflammation. In particular, we found that IL-1β, IL-1RA, IL-4, and IL-8 were consistently lower in the Trendelenburg group. In contrast, modifying duty cycle and PEEP did not have any effect on systemic inflamma- tion and, as previously reported [18], on VAP. Importantly, our study primarily focused on cytokines that might variate during the development of VAP, thus it is plausible that the association of aforementioned cytokines with the Trendelenburg position might have been related to the prevention of VAP. Indeed, previous findings confirmed higher levels of IL-1β, specifically in bronchoalveolar lavage fluids [24, 26], of patients with VAP, whereas an association between systemic and pulmonary IL-1RA and VAP [10, 26] has not been established. As for IL-4, this biomarker has not been tested in VAP and preliminary studies have found in IL-4-knockout mice resistance to P. aeruginosa pulmonary infection and increased TNF-α production [32]. Also in pediatric patients with pneumonia, IL-4 was a reliable marker of severity of the disease [23]. Finally, clin- ical studies have confirmed a surge in IL-8 with VAP [27]. Fig. 4 Lung P. aeruginosa burden as a function of tracheal secretions P. aeruginosa burden. a The linear regression equation was fitted to predict lung P. aeruginosa burden by tracheal secretions P. aeruginosa burden (log10 cfu/mL) and clustered by study groups. Regression equation control group: [lung P. aeruginosa burden (log10 cfu/g) = −3.06 + (0.85 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 6, R = 0.85, R2 = 0.73, Adjusted Rsqr = 0.67, p value = 0.029. Regression equation EFB + PEEP group: [lung P. aeruginosa burden (log10 cfu/g) = −3.68 + (0.94 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 7, R = 0.76, R2 = 0.57, Adjusted Rsqr = 0.49, p value = 0.048. Regression equation Trendelenburg group: [lung P. aeruginosa burden (log10 cfu/g) = −1.67 + (−0.25 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 7, R = 0.37, R2 = 0.14, Adjusted Rsqr = 0.00, p value = 0.411. Discussion b Analysis of the receiver operating characteristic curve for tracheal secretions P. aeruginosa concentration score with IL10, TNF-α, or IL10 and TNF-α. The area under the receiver operating characteristics curves of tracheal secretions P. aeruginosa concentration score with IL10 was 0.78, of tracheal secretions P. aeruginosa concentration score with TNF-α was 0.73, and of tracheal secretions P. aeruginosa concentration score with IL-10 and TNF-α was 0.82. We did not find any statistically significant differences among the tested receiver operating characteristics curves Fig. 3 Analysis of the receiver operating characteristics curves. a Analysis of the receiver operating characteristic curve for IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score, which was computed as follows: 0 = < 3.0 log10 cfu/mL; 1 = 3.0–3.9 log10 cfu/mL; 2 = 4.0–4.9 log10 cfu/mL; 3 = 5–5.9 log10 cfu/mL; 4 = ≥6 log10 cfu/mL. The area under the receiver operating characteristics curves of IL-10, TNF-α, and the tracheal secretions P. aeruginosa concentration score were 0.71, 0.69, and 0.81, respectively. b Analysis of the receiver operating characteristic curve for tracheal secretions P. aeruginosa concentration score with IL10, TNF-α, or IL10 and TNF-α. The area under the receiver operating characteristics curves of tracheal secretions P. aeruginosa concentration score with IL10 was 0.78, of tracheal secretions P. aeruginosa concentration score with TNF-α was 0.73, and of tracheal secretions P. aeruginosa concentration score with IL-10 and TNF-α was 0.82. We did not find any statistically significant differences among the tested receiver operating characteristics curves Page 10 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental prevention of VAP. Also, this study demonstrated that in a validated animal model of VAP, serum IL-10 and TNF-α were the only cytokines that varied during VAP development. Yet, culture of tracheal secretions still outperformed all evaluated diagnostic parameters. VAP diagnosis Considering that in clinical settings VAP still lacks of a diagnostic gold standard, an additional purpose of our study was assessing accuracy of several diagnostic parame- ters. In line with previous reports [29], clinical variables, such as body temperature, WBC, and PaO2/FIO2, were highly unspecific. As for systemic cytokines, previous clin- ical studies [10, 24, 25, 33–36] have appraised biomarkers in bloodstream, lungs, and pleural space to find the best diagnostic marker. Yet, discriminatory inflammatory markers that could reliably diagnose VAP are difficult to be identified in clinical set- tings, because at the time of VAP development, ICU patients often present other infec- tions. Furthermore, ICU patients might be in an immune-paralysis state [37–39], which increases the risk of developing VAP [25], while hindering patient’s inflammatory re- sponse. Given the abovementioned challenges, the use of a reliable animal model of VAP [28], developed in healthy animals without concomitant illnesses, could facilitate identification of diagnostic markers and redirect on the most promising. We found that only IL-10 and TNF-α were independently associated with the develop- ment of VAP. IL-10 is an anti-inflammatory cytokine that inhibits activation and effector function of T cells, monocytes, and macrophages [40]. Millo and collaborators [26] did not find any variation in plasma and BAL IL-10 in patients who developed VAP. Similarly, Conway Morris et al. confirmed that IL-10 did not have potential value for discriminating VAP from non-infected patients [34]. Whereas, Martin-Loeches and collaborators found significant differences in IL-10 concentration between VAP and no-VAP patients [10]; nevertheless, multivariate analyses failed to corroborate diagnostic value of IL-10. TNF-α is predominately produced by macrophages and exert various effects such as fever, cach- exia, and inhibition of tumorigenesis and viral replication. Millo et al. found higher levels of TNF-α in bronchoalveolar lavage fluids of VAP patients [26]. Of note, we found that P. aeruginosa endotracheal aspirate (ETA) concentration over- came diagnostic accuracy of all cytokines, yielding an AU-ROC higher than 80% (Fig. 3). A clinical trial [41] tested diagnostic value of culture of tracheal secretions vs. bronchoal- veolar lavage fluids and it did not find any difference between study groups. Thus, latest European [3] and American [2] guidelines for the management of patients with VAP rec- ommended obtaining samples of respiratory secretions to diagnose VAP. VAP diagnosis Our findings support this recommendation; yet, it is important to emphasize that tracheal secretions culture requires 1 to 3 days before definitive results, ultimately limiting initial therapeutic options. Also, as reported in Fig. 4, we found a marginal association between P. aerugi- nosa ETA concentration and lung colonization. This could be related to the limited num- ber of animals or to specific features of our model; indeed, following oropharyngeal challenge, the animals consistently developed colonization of the proximal airways, irre- spective of the subsequent colonization of the lungs and VAP development. Effects of study interventions on systemic inflammation EFB + PEEP expiratory flow bias and positive end expiratory pressure group Page 11 of 15 Page 11 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental Effects of study interventions on systemic inflammation b The linear regression equation was fitted to predict lung P. aeruginosa burden by tracheal secretions P. aeruginosa burden (log10 cfu/mL) and clustered by development of ventilator-associated pneumonia (VAP). Regression equation VAP: [lung P. aeruginosa burden (log10 cfu/g) = −1.69 + (0.64 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 10, R = 0.51, R2 = 0.26, Adjusted Rsqr = 0.17, p value = 0.130. Regression equation no VAP: [lung P. aeruginosa burden (log10 cfu/g) = 0.41 + (0.007 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 10, R = 0.01, R2 = 0.00, Adjusted Rsqr = 0.00, p value = 0.980. EFB + PEEP expiratory flow bias and positive end expiratory pressure group Fig. 4 Lung P. aeruginosa burden as a function of tracheal secretions P. aeruginosa burden. a The linear regression equation was fitted to predict lung P. aeruginosa burden by tracheal secretions P. aeruginosa burden (log10 cfu/mL) and clustered by study groups. Regression equation control group: [lung P. aeruginosa burden (log10 cfu/g) = −3.06 + (0.85 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 6, R = 0.85, R2 = 0.73, Adjusted Rsqr = 0.67, p value = 0.029. Regression equation EFB + PEEP group: [lung P. aeruginosa burden (log10 cfu/g) = −3.68 + (0.94 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 7, R = 0.76, R2 = 0.57, Adjusted Rsqr = 0.49, p value = 0.048. Regression equation Trendelenburg group: [lung P. aeruginosa burden (log10 cfu/g) = −1.67 + (−0.25 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 7, R = 0.37, R2 = 0.14, Adjusted Rsqr = 0.00, p value = 0.411. b The linear regression equation was fitted to predict lung P. aeruginosa burden by tracheal secretions P. aeruginosa burden (log10 cfu/mL) and clustered by development of ventilator-associated pneumonia (VAP). Regression equation VAP: [lung P. aeruginosa burden (log10 cfu/g) = −1.69 + (0.64 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 10, R = 0.51, R2 = 0.26, Adjusted Rsqr = 0.17, p value = 0.130. Regression equation no VAP: [lung P. aeruginosa burden (log10 cfu/g) = 0.41 + (0.007 × tracheal secretions P. aeruginosa burden (log10 cfu/mL)]. N = 10, R = 0.01, R2 = 0.00, Adjusted Rsqr = 0.00, p value = 0.980. Clinical implications Our preliminary findings should be interpreted in light of the potential clinical ap- plications. First, clinical feasibility of inverse-ratio ventilation is challenging, and given the marginal results reported in our latest analysis and previous studies [18], it should not be recommended in clinical settings. Second, in our studies, we failed to find efficacy of PEEP in reducing systemic inflammation or VAP, but these Page 12 of 15 Page 12 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental findings are in contrast with a previous clinical study that found lower incidence of VAP in patients ventilated with PEEP of 5 vs. 0 cm H2O [42]. This study was discontinued earlier for low recruitment rate, thus future clinical corroborations are essential to further explore the value of PEEP. Third, the recent results of the Gravity-VAP Trial [8] confirmed preventive benefits associated with the lateral-Trendelenburg position, but the study was discontinued earlier, due to a very low incidence of VAP and marginal effects in secondary outcomes. Of note, in the Gravity-VAP trial, the lateral-Trendelenburg position was applied for only 2 days following intubation, and higher safety was reported in patients who did not present pulmonary infiltrates. Considering the positive results from experimental studies [17, 30, 31], but the limitations of the latest randomized trial, risks and benefits of such intervention should be carefully pondered, carefully examining timing and duration of the intervention, which should exclusively be applied to pa- tients who are not intubated for pulmonary causes. Furthermore, pulmonary and systemic inflammation should be monitored. Finally, although our results confirm diagnostic accuracy of ETA, the delay for culture results often causes overtreat- ment or inappropriate treatment of multi-drug resistant pathogens. Thus, develop- ment of novel rapid molecular assays, custom-made for pathogen specific for VAP and for drug resistance genes, are needed. In addition, given the variability in bio- markers concentration among different patient populations and courses of treat- ment, a comprehensive evaluation of the dynamics of these markers, rather than the absolute cut-off values should be prioritized. Authors’ contributions G Li Bassi 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. Authors’ contributions He participated to the development of study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, and statistical analysis; RG Prats participated to the development of study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript; AA participated to the development of study concept and design, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; EA participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; J-DM participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; OTR participated to the acquisition of data, analysis and interpretation of data, statistical analysis, and critical revision of the manuscript for important intellectual content; MR participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; LF participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content and administrative and technical support; AM participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content and administrative and technical support; AM participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content and administrative and technical support; NL participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content and administrative and technical support; MF participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; IM-L participated to the acquisition of data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content; PP participated to the analysis and interpretation of data and critical revision of the manuscript for important intellectual content; DC participated to analysis and interpretation of data and critical revision of the manuscript for important intellectual content; PP participated to the analysis and interpretation of data and critical revision of the manuscript for important intellectual content; and AT participated to the development of study concept and design, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. Ethics approval and consent to participate The Institutional Ethics Committee evaluated and approved our study protocol: Dr. Jordi Alberch Vie; Álvaro Gimeno Sandig; Raquel Corral Vistué; Dr. Garikoitz Azkona Mendoza; Dr. Victor Fernández Dueñas; Dr. Jordi Guinea Mejías; Dr. Francesc López Soriano; Dr. Carmen Navarro Aragay; Dr. Francisco José Pérez Can; Dr. Montserrat Rigol Muixart; and Dr. Teresa Rodrigo Calduch. Authors’ contributions All authors read and approved the final manuscript. Study limitations First, although we conducted a 72-h study, in clinical settings, VAP may develop after several days of MV; therefore, in our model, some pathogenic mechanisms and the inflammatory response could somehow diverge in comparison with the critically ill, ventilated patient. Second, our animals were healthy at the beginning of the study and inflammatory changes were specifically related to the new iatro- genic infection. Nevertheless, in the early phase of the experiment, inflammatory markers could have been affected by the surgical interventions performed during animal preparation. Third, considering the complexity of cytokine signaling path- ways in critically ill patients and potential inter-species differences, our results re- quire further validation in humans. Fourth, this was an analysis of animals included in a previous trial [18] and inferences should primary assist for future confirmatory analyses. Fifth, our findings should be discussed critically, because pigs are quadruped and were maintained prone, due to inherent risks of lung dys- function when maintained in the supine position for prolonged period of times. Patients are normally maintained in the supine semirecumbent position, and the auto-regulation mechanisms in pulmonary and hemodynamic physiology, which may have played a role in our findings, could be different in pigs and humans. Finally, this study did not encompass the entire range of inflammatory biomarkers that could vary during the course of VAP. For instance, due to methodological lim- itations of the porcine assay, we did not measure procalcitonin, which was evalu- ated in previous clinical studies [43]. Page 13 of 15 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental Funding S Funding Support was provided by the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Ministerio de Ciencia e Innovación (PS09/01249); European Society of Intensive Care Medicine-ESICM (2009 Alain Harf Award on Applied Respiratory Physiology); Fundació Catalana de Pneumologıa (FUCAP); Sociedad Española de Neumología y Cirugía Torácica (SEPAR); Centro de Investigación Biomedica En Red-Enfermedades Respiratorias, (CIBERES). Availability of data and materials y The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conclusions In conclusion, this experimental study confirms that in an animal model of P. aeruginosa VAP, the Trendelenburg position hampers systemic inflammation through avoidance of VAP. In addition, in this model, culture of tracheal secretions is a precise method to diag- nose VAP, with marginal improvement in diagnostic accuracy when systemic IL-10 and TNF-α are assessed concurrently. Further clinical studies will be necessary to confirm these hypothesis-generating results. Abbreviations BAL: Bronchoalveolar lavage; CFU: Colony-forming unit; ETA: Endotracheal aspirate; ETT: Endotracheal tube; ICU: Intensive care unit; IL: Interleukin; MV: Mechanical ventilation; PEEP: Positive end-expiratory pressure; REML: Restricted maximum likelihood; ROC-AUC: Area under the receiver operating curves; TNF: Tumor necrosis factor; TP: Trendelenburg position; VAP: Ventilator-associated pneumonia; WBC: White blood cells Acknowledgements Acknowledgements We deeply thank Ignasi Roca and Jordi Vila from the Research and development in clinical molecular parasitology and microbiology, Barcelona Institute for Global Health, Hospital Clínic—Universitat de Barcelona for their support in the microbiology studies. Also, we acknowledge Jose Ramirez from the Department of Pathology, Hospital Clinic, Barcelona, Spain for his support in histology studies. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests Th h d l h Page 14 of 15 (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental (2018) 6:42 Li Bassi et al. Intensive Care Medicine Experimental Publisher’s Note S i N i 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. Author details 1 1Division of Animal Experimentation, Department of Pulmonary and Critical Care Medicine, Thorax Institute, Hospital Clinic, Calle Villarroel 170, Esc 6/8 Pl 2, Barcelona, Spain. 2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. 3Centro de Investigación Biomedica En Red- Enfermedades Respiratorias (CIBERES), Barcelona, Spain. 4University of Barcelona, Barcelona, Spain. 5Pathophysiological Laboratory, Institut de Investigacion Parc Tauli, Corporació Sanitaria Universitaria Parc Tauli, Autonomous University of Barcelona, Sabadell, Barcelona, Spain. 6Cardiology Department, Hospital Clinic, Barcelona, Spain. 7Dipartimento di Anestesia e Rianimazione, ASST Santi Paolo e Carlo, Dipartimento di Scienza e Salute, Universita degli Studi di Milano, Milan, Italy. 8Dipartimento Scienze Chirurgiche e Diagnostiche Integrate (DISC), Università degli Studi di Genova, Genova, Italy. 9Multidisciplinary Intensive Care Research Organization (MICRO), Department of Clinical Medicine, Trinity Centre for Health Sciences, St James’s University Hospital, Dublin, Ireland. 10Polyvalent Intensive Care Unit, São Francisco Xavier Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal. 11NOVA Medical School, CEDOC, New University of Lisbon, Lisbon, Portugal. 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https://openalex.org/W3177094233	https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/download/6210/3529	Arabic	null	Performance Evaluation of Intrusion Detection System using Selected Features and Machine Learning Classifiers	Mağallaẗ baġdād li-l-ʿulūm	2,021	cc-by	8,335	Abstract: Some of the main challenges in developing an effective network-based intrusion detection system (IDS) include analyzing large network traffic volumes and realizing the decision boundaries between normal and abnormal behaviors. Deploying feature selection together with efficient classifiers in the detection system can overcome these problems. Feature selection finds the most relevant features, thus reduces the dimensionality and complexity to analyze the network traffic. Moreover, using the most relevant features to build the predictive model, reduces the complexity of the developed model, thus reducing the building classifier model time and consequently improves the detection performance. In this study, two different sets of selected features have been adopted to train four machine-learning based classifiers. The two sets of selected features are based on Genetic Algorithm (GA) and Particle Swarm Optimization (PSO) approach respectively. These evolutionary-based algorithms are known to be effective in solving optimization problems. The classifiers used in this study are Naïve Bayes, k-Nearest Neighbor, Decision Tree and Support Vector Machine that have been trained and tested using the NSL-KDD dataset. The performance of the abovementioned classifiers using different features values was evaluated. The experimental results indicate that the detection accuracy improves by approximately 1.55% when implemented using the PSO- based selected features than that of using GA-based selected features. The Decision Tree classifier that was trained with PSO-based selected features outperformed other classifiers with accuracy, precision, recall, and f-score result of 99.38%, 99.36%, 99.32%, and 99.34% respectively. The results show that using optimal features coupling with a good classifier in a detection system able to reduce the classifier model building time, reduce the computational burden to analyze data, and consequently attain high detection rate. Key words: Intrusion detection system, Machine learning classifiers, Performance evaluation, Selected f Key words: Intrusion detection system, Machine learning classifiers, Performance evaluation, Selected features, Baghdad Science Journal Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) DOI: http://dx.doi.org/10.21123/bsj.2021.18.2(Suppl.).0884 Performance Evaluation of Intrusion Detection System using Selected Features and Machine Learning Classifiers versiti Putra Malaysia, Malaysia. rresponding author: raja_azlina@upm.edu.my, avinar1368@gmail.com, masnida@upm.edu.my RCID ID: https://orcid.org/0000-0002-1169-4226 , https://orcid.org/0000-0003-1063-8502 , https://orcid.org/0000- 1-7727-1739 y , y Corresponding author: raja_azlina@upm.edu.my, avinar1368@gmail.com, masnida@upm.edu.my ORCID ID: https://orcid.org/0000-0002-1169-4226 , https://orcid.org/0000-0003-1063-8502 , https://orcid.org/0000- 0001 7727 1739 Received 28/3/2021, Accepted 11/4/2021, Published 20/6/2021 This work is licensed under a Creative Commons Attribution 4.0 International License. Introduction: Intrusion detection system (IDS) is one of the protection methods against network attacks and threats in most organizations in addition to firewalls, authentication and encryption. IDS model was first proposed by (1), that is a software to monitor and detect any intrusion in a system or network. A modern effective network-based IDS should be able to automate the network surveillance, analysis process and attacks detection or classification with high accuracy percentage in short amount of time (2, 3). An IDS can be categorized into signature-based, anomaly-based or hybrid-based. Signature-based IDS only accurately detects known attacks while anomaly-based IDS able to detect unknown attacks by comparing the current profiles against the predefined normal behaviours. The later method is effective against zero-day attacks, but it still has high false positive rates (4, 5) and hence of recent, hybrid method has been developed to overcome these limitations (6). Due to the privacy and security issues, getting a reasonably large and complete real-world network traffic data with attacks footprints for IDS performance assessment has been made difficult. Alternatively, researchers use the publicly available benchmark datasets, namely KDD CUP 99 and NSL-KDD to evaluate the IDS performance. The NSL-KDD dataset has been used extensively, 884 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) including in this work, as it provides an improved version of the original KDD Cup 99 dataset that contains huge amount of redundant records (7). Nonetheless, NSK-KDD still consists of large network traffic volumes with 125,973 instances of 41 network-related features and an assigned label to classify each record instance as either normal or abnormal. Analyzing a huge dataset imposes a heavy computational burden and hence increases the processing time. Feature selection or reduction approach has then been proposed to solve such problem. Feature selection identifies and removes irrelevant features that do not contribute to the accuracy of a predictive model and has been widely used in machine learning, data mining and data analysis (8). Using reduced set of features, also known as the selected features, it reduces the complexity of the developed model, that is reduces the building classifier model time (9). presents the experimental setup including the dataset and performance metrics used in this study. The performance of classifier models using different sets of selected features are compared and discussed in Section 7. Final comments and conclusions are provided in Section 8. Machine Learning Classifiers: Machine learning (ML) enables the IDSes to detect new attacks without human intervene. ML allows the IDS to change its execution strategy based on the recently acquired data. In general, there are two types of learning techniques namely the supervised and unsupervised learning. Supervised learning involves algorithms that are ‘taught’ by examples, with the input and out-put labels are provided during training (13). The unsupervised learning algorithms are left to interpret the data without guidance as no labeled data are provided in training dataset. Unsupervised learning identifies similarities and differences in data by clustering and association techniques (14). This study investigates the performance of an IDS that uses only few selected features, as opposed to all 41 features using popular machine-learning based classifiers. Different features that have been selected using the evolutionary-based feature selection techniques from another research work have been adopted. In specific, 11 features selected using Genetic Algorithm (GA) by (10) and 20 features selected using Particle Swarm Optimization (PSO) by (11) have been used and hence, the feature selection implementation is not within the scope of this study. Machine learning (ML) techniques have been widely used for network intrusion detection as they are able to classify benign and attack patterns precisely. ML algorithms automate the improvement of their detection accuracy with subsequent trainings which may contain new and previously unseen data. However, building ML models are time consuming with the increase of data volumes (12). Hence, reducing the volumes of data to be processed using feature reduction method is critical to improve the detection performance. In this work, four state-of- the-art machine learning classifiers, namely Naïve Bayes, k-Nearest Neighbor, Decision Tree and Support Vector Machine have been implemented and evaluated. The detection accuracy of the abovementioned classifiers using different sets of features values were studied. The machine learning-based classifiers used in this study are the supervised probabilistic-based Naïve Bayes, k-Nearest Neighbors, Decision Tree and Support Vector Machine. All these classifiers are part of the state-of-the-art classifiers for they have been widely used for classification and regression problems due to their effectiveness. The theoretical background of these algorithms has been heavily discussed in many published works and hence not discuss in depth in the following subsections. The following subsections discuss the classifiers in general including their historical backgrounds, recent development and applications. (NB) Naïve Bayes (NB) classifier is a probabilistic-based classifier which uses Bayes’ theorem and assumes features are independent of each other and their weight are equally important (15). One of NB problems is the ‘Zero frequency or probability’ situation in which the model is not able to make prediction if it has not observed a certain category in the training data set, yet a new and unseen-before input variable appears in the test data set. Smoothing techniques such as Laplace estimation can be applied to avoid this undesirable situation (16). This paper is structured as follows. Section 2 presents an overview of the machine learning-based classifiers used in this. Section 3 discusses some of the IDS models using different machine learning classifiers. Existing feature selection approaches are covered in Section 4. Section 5 reviews some of the related works on IDS models using different feature selection methods and classifiers. Section 6 With some improvements made towards the traditional NB, it has been used extensively in text classification area, along with other classification areas as it is simple to implement, computationally fast and robust (17, 18). Moreover, Naïve Bayes are among the simplest Bayesian network models 885 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) that can achieve higher accuracy level if coupled with kernel density estimation (19, 20). generally balanced and hence traversing the tree from root to the leaf requires approximately O(log2 N). The tree-based algorithms include ID3 (Iterative Dichotomiser 3), C4.5 (successor of ID3), CART (Classification and Regression Tree), CHAID (Chi- Square Automatic Interaction Detection), MARS (Multivariate Adaptive Regression Splines) and cTree (Conditional Inference Trees). One of the main challenges in DT is to build a good decision tree, that is smallest decision tree possible. Nonetheless, DT is one of the most used techniques in IDS for its fast adaptation, simplicity, and accuracy (30). Support Vector Machine A Support Vector Machine (SVM) is based on statistical learning theory and was developed by Vapnik in 1995 (31). SVM finds the optimal hyperplane that differentiates any two classes efficiently. By using different types of kernel functions, the low dimensional input space is transformed to a high dimensional space. Hence these nonseparable classes can then be separated by adding more dimensions. Linear, sigmoid, polynomial and radial basis functions (RBF) are some of the commonly used kernel functions, which play a significant role in SVM (32). kNN requires no prior knowledge on the distribution of the data (24). However, kNN is biased by the selection of the k value. One way in choosing good k value is to run the algorithm many times and choose the one with the best performance. One of the disadvantages of this classifier is its computational cost is considerably high as it needs to compute distance the unlabeled data t to all training samples. One promising approach made to improve the kNN accuracy is by clustering technique (25, 26). kNN has been deployed in many domain areas including text mining, agriculture and medicine but has been heavily applied in finance-related areas such the stock market forecasting, bank customer profiling, managing financial risk as well as money laundering analyses (27). SVMs have performed well in multiple areas of biological analysis including analysing RNA- Sequencing and microarray gene expression data due to their capabilities to generalize well with high dimensional data (33, 34). However, SVM’s performance may degrade when data is not linearly separable and having large data sets to process, as the precompute of the kernel matrix might become infeasible (35). k-Nearest Neighbors The k-Nearest Neighbors (kNN) is a non- parametric classification method that has been widely used due to its simplicity and effectiveness (21). kNN was first described by Fix and Hodges in 1951 (22) in a USAF School of Aviation Medicine technical report and later expanded by Cover and Hart (23). kNN classifies each unlabeled data, t based on the k nearest neighbors, known as the neighborhood of t. Majority voting among the data label in the neighborhood is then used to decide the classification for t with or without consideration of distanced-based weighting. Decision Tree Decision Tree (DT) is a supervised learning method that maps from observations about a data to conclusions about its target value (28). The leaves represent the class or the label, the non-leaf nodes are the features and the branches represent conjunction of features that lead the specific a class. To create a DT, the training data or records are distributed recursively according to the attribute values (29). Intrusion Detection Learning Classifiers: Machine learning (ML) has been widely used in network intrusion detection for its ability to classify benign and attack patterns with high precision. Table 1 presents the performance evaluation of IDS models with different ML classifiers. DT is computationally fast even when dealing with large training sets since they are 886 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 2021, Vol. 18 No.2 (Suppl. June) E-ISSN: 2411-7986 Table 1. IDS Models with Different Machine Learning Classifiers. Authors / Year Dataset Classification Accuracy Precision Recall F-Score (Relan & Patil, 2015) (36) NSL_KDD Decision Tree 93.82% Naive Bayes 81.66% Random Forest 92.79% Multi-layer Perceptron 92.26% SVM 65.01% (Belavagi & Muniyal, 2016) (37) NSL_KDD Logistic Regression 84% 83% 85% 82% SVM 75% 76% 79% 77% Naive Bayes 79% 79% 81% 78% Random Forest 99% 99% 99% 99% (Amira et al., 2017) (38) NSL_KDD Naïve Bayes 84.41% 78.51% 81.35% BFTree 98.19% 68.32% 80.58% J48 98.59% 65.52% 78.72% Multi-layer Perceptron 98.24% 62.51% 76.41% NBTree 98.36% 62.64% 76.53% Random Forest 98.61% 62.58% 76.57% (Suleiman & Isaac, 2018) (39) NSL_KDD Random Forest 99.76% 99.9% 99.6% 99.7% Decision Tree (J48) 99.55% 99.5% 99.5% 99.5% k-Nearest Neighbor 99.44% 99.5% 99.3% 99.4% Naive Bayes 88.59% 89.7% 87.7% 88.7% SVM 97.32% 98.3% 95.9% 97.1% ANN 98.24% 98.9% 97.3% 98.1% (Devi & Abualkibash, 2019) (40) NSL_KDD Logistic Regression 79.7% Decision tree 81.05% k-Nearest Neighbor 94.17% SVM 83.09% Random Forest 99.0% Adaboost 90.73% Multi-layer Perceptron 80.5% Naïve Bayes 92.4% A decision tree-based intrusion detection system was presented by (36) and a comparison d h li d l ifi h h h Tree (J48), Multilayer Perceptron, NBTree and Random Forest (RF) classifiers and compare thei l Th l f h d i i b d Table 1. IDS Models with Different Machine Learning Classifiers. Tree (J48), Multilayer Perceptron, NBTree and Random Forest (RF) classifiers and compare their results. The results of the decision tree-based algorithms show high precision rate, which are above 98% while NB peformed the worst in this study. Suleiman and Isaac (39) evaluated six classiﬁers which are the Decision Tree (J48), Random Forest (RF), k-Nearest Neighbor (kNN), Naive Bayes (NB), Support Vector Machine (SVM) and Artiﬁcial Neural Networks (ANN). The experimental results show that RF and J48 classifiers outperformed others in accuracy and false positive rate. Feature Selection: IDS normally handle vast amounts of data traffic containing redundant and irrelevant features, which could negatively affect its detection performance. Many studies have shown that classifier that is developed with an efficient subset of relevant features provides higher predictive accuracy compared to a classifier developed from the complete set of features (41, 42). Feature Selection (FS) is a popular preprocessing technique aims to find the most relevant features, that is features that have high correlation with the respective results (43). Using only relevant features in building the predictive model, it reduces the complexity of the developed model, hence reduces the building classifier model time and improve the accuracy and efficiency. In general, FS approaches can be classified into three categories, which are the wrapper, filter and hybrid (44). Filter methods only consider the relevance between features and class labels, independent of the classifiers as depicted in Figure 1. It ranks the features using statistical techniques such as t-test or fisher discriminant ratio, information theory, correlation coefficient, variance threshold as well as using distance measurement (45). These methods require less computational resources and faster than wrapper methods as no cross-validation process is performed. Intrusion Detection Learning Classifiers: These tree-based classifiers A decision tree-based intrusion detection system was presented by (36) and a comparison study among the listed classifiers shows that the proposed model able to achieve high detection accuracy rate, at around 93.82%. Belavagi & Muniyal (37) presented classification and predictive models for intrusion detection by using machine learning classification algorithms namely Logistic Regression, Support Vector Machine, Naive Bayes and Random Forest (RF). Experimental results show that RF outperformed the other methods in all metrics with highest value of 99%. Amira et al. (38) implemented Naive Bayes, BFTree, Decision 887 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) managed to attain above 99% for accuracy and precision. Different machine learning algorithms been implemented by (40) that include Logistic Regression, Decision Tree (DT), Stochastic Gradient Descent (Adaboost), SVM, Random Forest(RF), Naive Bayes and Multilayer Perceptron and as expected, RF performed the best with achieved the highest accuracy rate of 99.0%. In summary, Decision Tree and Random Forest (which are composed of multiple decision trees) performed well in most studies in comparison to other classifiers for they are known to be efficient and accurate. generally achieve high accuracy than filter methods. Wrapper methods for feature selection can be categorized into step forward feature selection, step backwards feature selection and exhaustive feature selection. Meanwhile, the hybrid methods, also known as embedded methods combine both filtering and wrapping methods to obtain the best of both techniques. Figure 2. Wrapper Method. (Nolan & Lally, 2018)(47). Related Works: Multi-class SVM is used to classify the different types of attacks in the NSL-KDD dataset. Using the proposed model 31 features were selected out of 41. The proposed system achieved 98% in accuracy and 0.13% false positive rate. Al-Yaseen (41) suggested a wrapper feature selection method, based on the firefly algorithm and SVM. The SVM model was used to assess each of the subsets of features selected from the firefly approach. The key benefit of the proposed system is its ability to adjust the firefly algorithm to match the selection of features and 10 top ranked features are selected. Their solution achieved about 78.89% in accuracy, and only 75.81% when uses all 41 features. The results of the analysis show the effectiveness of proposed feature selection technique in improving the detection system. Chakir et al. (11) improved IDS efficiency by using the Information Gain (IG) feature selection method and SVM with Particle Swarm Optimization (PSO) for improved classification. PSO is a stochastic approach that performs searches using population or swarm of particles. Experiments were performed on the dataset NSL KDD and top ranked 20 features were selected. The experimental studies indicate that the proposed IG- Table 2. IDS Models with Feature Selection and Classifiers. Table 2. IDS Models with Feature Selection and Classifiers. Authors, Year Proposed IDS Models with Different Feature Selection Approaches and Classifiers Dataset (No. of Features) Accuracy (%) Precision (%) Recall (%) Sarvari et al., 2015 (10) Genetic Algorithm (GA) FS and Support Vector Machine (SVM) KDD Cup 99 (11 features) n/a 97.2 97.3 Ambusaid i et al., 2016 (48) Flexible Mutual Information FS and Least Square Support Vector Machine (LSSVM) NSL-KDD (17 features) 99.94 n/a n/a Thaseen and Kumar, 2017 (49) Chi-square FS and multi class SVM NSL-KDD (31 features) 98 n/a n/a Chakir et al., 2018 (11) Information Gain (IG) FS with Particle Swarm Optimization (PSO) and SVM NSL-KDD (20 features) 99.8 n/a 99.8 Al- Yaseen, 2019 (41) Firefly Algorithm (FA) FS and SVM NSL-KDD (10 features) 78.89 n/a n/a Table 2. IDS Models with Feature Selection and Classifiers. This study investigates the performance of the evolutionary-based feature selection methods when coupled with some of the state-of-the-art classifiers in detecting attacks in the NSL KDD data set. Therefore, the 11 GA-based selected features and the 20 PSO-based selected features by Sarvari et al. (10) and Chakir et al. Related Works: The following paragraphs discuss some of the existing IDS models with various feature selection techniques and classifiers and Table 2 presents the summarized information. Sarvari et al. (10) proposed an intrusion detection system using a hybrid SVM approach with Genetic Algorithm (GA) FS method. GA is a stochastic optimization algorithm, that is based on natural evolution aims to find the optimal solution. Hence, by implementing GA, the number of important features has been reduced from 41 to 11. These 11 significant features are categorized into three groups, ranked based on their importance. The 4 most important features are placed in the first priority, 5 features in the second priority and 2 least important featured in the third. The results demonstrate that the proposed algorithms, GA and SVM can attain true positive and false positive values of 97.3% and 0.17% respectively. Ambusaidi et al. (48) proposed a common information-based algorithm that choose the ideal element for grouping. This new filter-based feature selection method is an enhancement of Mutual Information Feature Selection (MIFS) and Modified Mutual Information-based Feature Selection (MMIFS) known as Flexible Mutual Information Feature (FMIFS). They employed Least Square SVM (LS-SVM) classifier to detect the attacks in NSL-KDD dataset with their proposed system is known as LSSVM-IDS-FMIFS. The FMIFS selected 17 most significant features, that are columns 1, 2, 3, 4, 8, 10, 11, 12, 19, 23, 24, 25, 29, 31, 32, 36, and 39. The proposed system achieved 99.94% in accuracy, 98.93% in detection rate, and 0.28% of false positive rate. Figure 1. Filter Methods(Khalid etal., 2017 )(46) Figure 1. Filter Methods(Khalid etal., 2017 )(46) In wrapper methods, the incremental learning sessions from the specific machine learning algorithm is integrated into the feature selection process as depicted in Figure 2. The prediction performance of the algorithm is tested using different feature subsets and finally, the subset with the best performance is selected. Wrapper methods which are based on greedy search algorithms Thaseen and Kumar (49) have proposed an intrusion detection model that uses rank-based chi- square feature selection technique and multi class SVM classifier. Chi-squared is a numerical test that measures deviation from the expected distribution 888 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) PSO-SVM detection model performed well with 0.9% false alarm rate and 99.8% accuracy as well as precision. considering the feature event is independent of the class value. Related Works: (11) respectively have been adopted in this work. As mentioned earlier, implementing evolutionary-based feature selections is not within the scope of the study. The authors use the features that have been selected from the abovementioned works and evaluate the performance of these two approaches. The following paragraph provides some background on the evolutionary computing that has gained increasing attention from researchers. the researchers. Among the popular algorithms include Genetic Algorithm (GA), Particle Swarm Optimization (PSO) and ant colony optimization that have been widely used (50-52). Genetic algorithms are randomized search algorithms that rely on biologically inspired operators such as mutation, crossover, selection and reproduction to provide optimization. GA is an iterative process that evolves in time and using the rule of survival of the fittest to arrive at the best solution. It operates on string structures like biological structures and in every generation, a new set of strings is created using parts of the fittest members of the old set. GA is computationally costly and can take a long time to converge due to its stochastic nature (53). PSO was inspired by the movement behavior exhibits by the flocks of birds and swarms of insects. Proposed by Elberhart and Kennedy (54). PSO consists of individuals or known as particles that have a the researchers. Among the popular algorithms include Genetic Algorithm (GA), Particle Swarm Optimization (PSO) and ant colony optimization that have been widely used (50-52). Genetic algorithms are randomized search algorithms that rely on biologically inspired operators such as mutation, crossover, selection and reproduction to provide optimization. GA is an iterative process that evolves in time and using the rule of survival of the fittest to arrive at the best solution. It operates on string structures like biological structures and in every generation, a new set of strings is created using parts of the fittest members of the old set. GA is computationally costly and can take a long time to converge due to its stochastic nature (53). PSO was inspired by the movement behavior exhibits by the flocks of birds and swarms of insects. Proposed by Elberhart and Kennedy (54). PSO consists of individuals or known as particles that have a Due to the optimization capabilities of the evolutionary-based feature selection techniques, these algorithms have gained much attention from 889 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. Related Works: June) the NSL-KDD dataset in their work. In this study, two sets of selected features, one with 11 features selected using GA and another set of 20 features selected using PSO, have been adopted to train the four different predictive models. position and a velocity. Using a mathematical formula, it iteratively improves the solution by moving these particles in the given search-space. The movement of each particle is influenced by its local best-known position but is also guided toward the best-known positions in the search-space, updated by other particles that have found better positions. This moves the swarm toward the best solutions. Methodology: The NSL-KDD dataset, proposed intrusion detection system and performance metrics used in this study are discussed in the following subsections. The NSL-KDD dataset, proposed intrusion detection system and performance metrics used in this study are discussed in the following subsections. PSO is easy to implement and computationally inexpensive compared to GA. However, with more features in the data set, the solution space increases rapidly. In addition, high number of uncorrelated or redundant features result in many local optima detected in a large solution space and thus, evolutionary-based methods still suffer from the local optimal stagnation problems (45). In this work the data dimension is limited to 41 and hypothetically, PSO should be able to converge fast and expected to have less selected features than GA. However, based on Table 2, the selected features of PSO derived by (11) is higher than those of GA derived by (10). This could due to the selection of Information Gain threshold value used in the experiments that led to 20 important features been selected. Similarly, another work that deploys a hybrid model that integrates Gini Index with PSO can be found in (55). The authors only consider features as important thus selected when the respective Gini Index’s scores are less than 0.4. Consequently, only 18 features are selected from Dataset NSL-KDD dataset (56), is an improved version of KDD-CUP 99 dataset that has been used in this study. It has no redundant and duplicate records and thus, better detection rate is expected. In this dataset, there are 125,973 instances with 41 attributes or features and one assigned label to indicate the record as normal or abnormal. Figure 3 depicts the 41 features of the NSL-KDD dataset. These features can be divided into three different categories as follows: 1) features extracted from the TCP/IP connection, 2) features to access TCP packet payload and 3) time-based traffic features and host-based traffic features. The attacks in this dataset can be classified into four different types of attacks, namely the DoS, Probe, U2R and R2L attacks. This public benchmark dataset has been widely used by many researchers to conduct different types of analyses and develop effective IDSes (57- 60). Figure 3. The 41 features of the NSL-KDD dataset (9). Figure 3. The 41 features of the NSL-KDD dataset (9). Design and Implementation selected features, building classification models, and evaluating performance are then elaborated in the following subsections. selected features, building classification models, and evaluating performance are then elaborated in the following subsections. Figure 4 shows the proposed IDS model used in this study and the processes involved. These processes include pre-processing data, using 890 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access Open Access 2021, Vol. 18 No.2 (Suppl. June) 2021, Vol. 18 No.2 (Suppl. June) Figure 4. Proposed IDS Model. Figure 4. Proposed IDS Model. scales the data features into a specific range without altering the feature’s statistical properties. The maximum and minimum values of the features were determined, and data is converted into a normalized form using the following equation: 𝑁𝑜𝑟𝑚𝑎𝑙 𝑑𝑎𝑡𝑎𝑖= (𝑑𝑎𝑡𝑎𝑖−𝑚𝑖𝑛𝑑𝑎𝑡𝑎) (𝑚𝑎𝑥𝑑𝑎𝑡𝑎−𝑑𝑎𝑡𝑎 ) scales the data features into a specific range without altering the feature’s statistical properties. The maximum and minimum values of the features were determined, and data is converted into a normalized form using the following equation: Pre-processing Data: Data Transformation and Normalization Figure 5 shows two records taken from the NSL-KDD dataset, in specific records for line 2 and 6 that contain mixed of numerical and string values. These strings or nominal feature values need to be transformed into numeric values with the affected columns are columns number 2 (Protocol_type), 3 (Services), 4 (Flag) and 42 (Attack or Normal). The data in column 42 for each record has been transformed, in particular the ‘normal’ value has been assigned to value 0 and the ‘anomaly’ value has been assigned to value 1. Figure 5. NSL-KDD Records. Figure 5. NSL-KDD Records. Using Selected Features: Adopting Two Sets of Selected Significant Features In this study, two sets of selected significant features have been applied. The 20 selected features by (11) obtained using Information Gain and Particle Swarm Optimization (PSO) and 11 selected features by (10) obtained using Genetic Algorithm (GA) are fed into the machine learning models. Both GA and PSO are evolutionary algorithms with their own advantages and limitations. Table 3 shows the selected features in these two sets. Most of the features selected by GA are also selected by PSO-based feature selection approach. However, PSO-based feature selection technique considers additional 9 features are also Building Classification Models: Training/Testing Data and Predictive Models 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑃+ 𝐹𝑁 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑃+ 𝐹𝑁 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑃+ 𝐹𝑁 The NSL-KDD data are split into training and testing sets for supervised learning. Following the previous works by Sarvari et al. (10) and Chakir et al. (11), 80% of the data has been randomly selected and used to train the machine learning models and the rest of 20% is used for the classifier’s performance evaluation. Table 4 shows the statistics of the data used in this study. (ii) Precision or the positive predictive value: refers to the ratio of correctly predicted positive observations to the total predicted positive observations. 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛= 𝑇𝑃 𝑇𝑃+ 𝐹𝑃 (iii) Recall known as sensitivity: refers to the true positive rate that is determined correctly. (iii) Recall known as sensitivity: refers to the true positive rate that is determined correctly. Table 4. Statistics of the NSL-KDD Dataset. Total Training (80%) Testing (20%) Normal 67,244 53,795 13,449 Attack 58,730 46,984 11,746 Total 125,974 100,779 25,195 Table 4. Statistics of the NSL-KDD Dataset. 𝑅𝑒𝑐𝑎𝑙𝑙= 𝑇𝑃 𝑇𝑃+ 𝐹𝑁 (iv) F-score: is the harmonic mean of the precision and recall. 𝐹−𝑠𝑐𝑜𝑟𝑒= 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛∗𝑅𝑒𝑐𝑎𝑙𝑙 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+ 𝑅𝑒𝑐𝑎𝑙𝑙 Figure 5. NSL-KDD Records. Due to the large variation among some of the feature values, for example values 146 and 0.08 as shown in line 2 of Figure 5, normalization is required for better performance. Normalization 891 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) relevant and important in classifying attacks accurately. The Decision Tree, Naïve Bayes, Support Vector Machine, and k-Nearest Neighbor algorithms are implemented using MATLAB version R2018b. Using the training data, four predictive models are then built and to be used for classifying the remaining 20% of the dataset. Table 3. Different Sets of Selected Features. N o Selected Significant Features Set 1: 20 Features Set 2: 11 Features 1 src_bytes protocol type 2 Service Service 3 dst_bytes Flag 4 Flag wrong fragment 5 diff_srv_rate logged_in 6 same_srv_rate numfile creations 7 dst_host_srv_count Count 8 dst_host_same_srv_rate dst_host_same_srv_rate 9 dst_host_diff_srv_rate dst_host_same_srv_port _rate 10 dst_host_serror_rate is_gus login 11 logged_in srv_diff_host_rate 12 dst_host_srv_serror_rat e 13 serror_rate 14 Count 15 srv_serror_rate 16 dst_host_srv_diff_host_ rate 17 dst_host_count 18 dst_host_same_src_port 19 srv_diff_host_rate 20 srv_count e 3. Different Sets of Selected Features. Performance Metrics Evaluation The accuracy, precision, recall and F-score performance measurements are used to evaluate the performance of the classifiers with different sets of selected features. The confusion matrix is the basis for calculating the abovementioned performance metrics of the classifiers. It includes true positive (TP) that specifies the normal instances that are correctly predicted, true negative (TN) that indicates the abnormal instances that are identified correctly, false positive (FP) that denotes the abnormal instances that are wrongly assumed as normal and false negative (FN) that specifies the abnormal instances detected as normal. The descriptions of the performance metrics are as follows: - (i) Classification rate or Accuracy: one of the most important performance measurements of a classification algorithm that shows the ability of the algorithm to accurately predict positive and negative instances, as shown in the following formula: Building Classification Models: Training/Testing Data and Predictive Models Building Classification Models: Training/Testing Data and Predictive Models Open Access 2021, Vol. 18 No.2 (Suppl. June) Open Access 2021, Vol. 18 No.2 (Suppl. June) Open Access 2021, Vol. 18 No.2 (Suppl. June) Figure 8 depicts the recall or sensitivity rate of the predictive models. The classifiers using PSO-based selected features outperformed the classifiers that are trained by the GA-based selected features except for SVM. Such problem is prominent in SVM and there has been published works discussing this phenomenon, known as the outlier sensitivity problem of standard SVM (61). Many have found SVMs do not perform well with certain noise intensities. The performance of SVM trained by the PSO-based selected features degraded with the presence of noise and even worse than that of DGA-based, by approximately 4.5%. The rest of the classifiers are consistent in their performance. The decision tree classifier again attained the highest recall percentages (of value 99.32%) compared to other classifiers. Figure 8. Classifiers’ Recall Percentage Comparison. Decision Tree SVM kNN Naïve Bayes PSO-based 99.32 91.54 98.82 86.84 GA-based 97.42 95.09 97.71 85.1980859095100 RECALL (%) Figure 8. Classifiers’ Recall Percentage Comparison. highest accuracy percentage, which is 99.34% with PSO selected features. In this experiment, the NB classifier performed the worst behind SVM and kNN with percentage of 87.6% using the GA-based features. Figure 9 shows the f-score or f-measure rate of the predictive models. In general, the classifiers’ f-score performs better by approximately 1.56% when implemented using the PSO-based selected features than that of using GA-based selected features. Decision Tree (DT) classifier attained the Figure 9. Classifiers’ F-score Percentage Comparison. Decision Tree SVM kNN Naïve Bayes PSO-based 99.34 93.01 98.82 89.2 GA-based 97.85 91.85 96.96 87.4680859095100 F-SCORE (%) Figure 9 Classifiers’ F-score Percentage Comparison Decision Tree SVM kNN Naïve Bayes PSO-based 99.34 93.01 98.82 89.2 GA-based 97.85 91.85 96.96 87.4680859095100 F-SCORE (%) Figure 9. Classifiers’ F-score Percentage Comparison. Results and Discussion: Accuracy is the most critical performance measurement in intrusion detection and Figure 6 shows all the classifiers’ performances using both PSO-based and GA-based selected features sets. Interestingly, even though PSO has greater number 892 Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Open Access 2021, Vol. 18 No.2 (Suppl. June) of selected features used to develop the predictive models, the overall performance of the models is superior than that of GA’s. This could due to the ability of the PSO, together with Information Gain to correctly anticipate the most relevant attack features in the dataset. In general, the accuracy improves by approximately 1.55% when implemented using the PSO-based selected features than that of using GA-based selected features. As expected, the Decision Tree (DT) classifier attained the highest accuracy percentage, which is 99.38% with PSO selected features. Meanwhile, decision tree classifier with GA-based selected features only able to detect up to 98% of accuracy. The results are consistent with the studies shown in Table 1, the decision tree’s performance. In this experiment, the NB classifier performed the worst behind SVM and kNN. In summary, the classifiers’ accuracy using significant features derived from PSO performed better than those with features obtained by GA. Figure 6. Classifiers’ Accuracy Percentage Comparison. Decision Tree SVM kNN Naïve Bayes PSO-based 99.38 93.55 98.89 90.13 GA-based 98 92.08 97.12 88.5580859095100 ACCURACY (%) Figure 6. Classifiers’ Accuracy Percentage Comparison. The precision results that show the classifier’s percentage of predicting instances correctly is one of the important indicators of good models, are shown in Figure 7. The classifiers using PSO-based selected features outperformed the classifiers that are trained by the GA-based selected features. Again, as expected, the decision tree classifier obtained the highest precision percentages (of value 99.36%) compared to other classifiers. Unlike previous results, in this experiment, SVM has the worst precision percentage with value of 88.81%, behind NB and kNN respectively. The performance difference rate shown by SVM in these two different features sets is huge, which is about 5.73%. Meanwhile the other three classifiers are considerably consistent in their performance. Figure 7. Classifiers’ Precision Percentage Comparison. Decision Tree SVM kNN Naïve Bayes PSO-based 99.36 94.54 98.82 91.69 GA-based 98.29 88.81 96.21 89.8780859095100 PRECISION (%) Figure 7. Classifiers’ Precision Percentage Comparison. 893 Baghdad Science Journal Baghdad Science Journal P-ISSN: 2078-8665 E-ISSN: 2411-7986 Conclusion: The performance of four supervised classifiers with different selected feature values on the NSL-KDD dataset were evaluated. The feature values were derived from Particle Swarm Optimization (PSO) and Genetic Algorithm (GA) feature selection approach respectively. The experimental results show that using smaller number of selected and relevant features may not necessarily improves the accuracy. Instead, using the appropriate number of relevant and significant features, even if the number is big, it could enhance the performance of the machine learning models. The 20 features selected by PSO outperformed the 11 features selected by GA in every performance metric except for recall due to existing SVM’s outlier sensitivity problem. The adopted PSO feature selection method with Information Gain selected the top 20 relevant features from the 41 features in NSL-KDD dataset and hence improves the complexity, time, and the accuracy of the predictive models. Decision Tree has proven to be an efficient classifier and outperformed Naïve Bayes, k-Nearest Neighbor and Support Vector Machine classifiers in every evaluation test. In this experimental study, a maximum accuracy of 99.38% and precision of 99.36% have been attained by the decision tree-based IDS using particle swarm optimization feature selection. In summary, combining a good feature selection with an efficient classifier in a detection system able to reduce to complexity of data analysis and consequently improve the detection performance. 3. 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Open Access 2021, Vol. 18 No.2 (Suppl. June) June) Open Access 2021, Vol. 18 No.2 (Suppl. June) تقييم أداء نظام كشف التسلل باستخدام الميزات ومصنفات مختارة في التعلم االلي رجاء ازلينا رجاء محمود عامر حسين عبدي مازنيدا حسين يزيا ، ماليزيا جامعة بوترا ماليزيا ، ماليزيا الخالصة الخالصة تتضمن ( بعض التحديات الرئيسية في تطوير نظام فعال للكشف عن التسلل المستند إلى الشبكةIDS ) تحليل أحجام حركة مرور الشبكة الكبيرة وإدراك حدود القرار بين السلوكيات العادية وغير الطبيعية. يمكن أن يؤدي نشر اختيار الميزات جنبًا إلى جنب مع المصن فات الفعالة في نظام الكشف إلى التغلب على هذه المشكالت. يجد اختيار الميزة أكثر الميزات ذات الصلة ، وبالتالي يقلل من األبعاد والتعقيد لتحليل حركة مرور الشبكة. عالوة على ذلك ، فإن استخدام الميزات األكثر صلة لبناء النموذج التنبئي ، يقلل من تعقيد النموذج المطور ، وبالت الي يقلل من وقت نم وذج مصنف المبنى والذي يؤدي الى تحسن أداء الكشف. في هذه الدراسة ، تم اعتماد مجموعتين مختلفتين من الميزات المختارة ( لتدريب أربعة مصنّفات قائمة على التعلم اآللي. تعتمد مجموعتا الميزات المحددة على الخوارزمية الجينيةGA ) ونهج تحسين حشد الجسيمات ( PSO) على التوال ي. من المعروف أن هذه الخوارزميات المستندة إلى التطور فعالة في حل مشاكل التحسين. المصنفات المستخدمة في هذه الدراسة هيNaïve Bayes وk-Nearest Neighbor وDecision Tree وSupport Vector Machine التي تم تدريبها واختبارها باستخدام مجموعة بياناتNSL-KDD. تم تقيي م أداء المصنفات المذكورة أعاله باستخدام قيم خصائص مختلفة. تشير النتائج التجريبية إلى أن دقة الكشف تتحسن بنسبة1.55 تقريبًا عند تنفيذها باستخدام الميزات المحددة المستندة إلى٪ PSO مقارنة باستخدام الميزات المحددة المستندة إلىGA. تفوق مصنف شجرة القرار الذي تم تدريبه باستخدام الميزات المحددة المستندة إلىPSO على المصنفات األخرى بدقة ودقة واستدعاء ونتائجf-Score بنسبة99.38 و٪ 99.36 و٪ 99.32 و٪ 99.34 على التوالي. أظهرت النتائج أن استخدام اقتران الميزات٪ المثلى مع المصنف الجيد في نظام الكشف قادر على تقليل وقت بنا ء نموذج المصنف ، وتقليل العبء الحسابي لتحليل البيانات ، وبالتالي تحقيق .معدل اكتشاف مرتفع الكلمات المفتاحية: نظام كشف التسلل ،مصنّفات التعلم اآللي، تقييم األداء، ميزات مختارة. 898 898
https://openalex.org/W4255239391	https://dro.deakin.edu.au/articles/journal_contribution/Accelerated_gravity_testing_of_aquitard_core_permeability_and_implications_at_formation_and_regional_scale/20788165/1/files/37039624.pdf	English	null	Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale	null	2,015	cc-by	14,003	Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale AUTHOR(S) Wendy Timms, R Crane, D J Anderson, S Bouzalakos, M Whelan, D McGeeney, P F Rahman, R I Acworth Wendy Timms, R Crane, D J Anderson, S Bouzalakos, M Whelan, D McGeeney, P F Rahman, R I Acworth 10536/DRO/DU:30115121 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale Citation: Timms, W.A., Crane, R., Anderson, D.J., Bouzalakos, S., Whelan, M., McGeeney, D., Rahman, P.F. and Acworth, R.I. 2016, Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale, Hydrology and earth system sciences, vol. 20, no. 1, pp. 39-54. DOI: http://www dx doi org/10 5194/hess 20 39 2016 Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale Citation: Timms, W.A., Crane, R., Anderson, D.J., Bouzalakos, S., Whelan, M., McGeeney, D., Rahman, P.F. and Acworth, R.I. 2016, Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale, Hydrology and earth system sciences, vol. 20, no. 1, pp. 39-54. DOI: http://www.dx.doi.org/10.5194/hess-20-39-2016 Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale Citation: Timms, W.A., Crane, R., Anderson, D.J., Bouzalakos, S., Whelan, M., McGeeney, D., Rahman, P.F. and Acworth, R.I. 2016, Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale, Hydrology and earth system sciences, vol. 20, no. 1, pp. 39-54. Citation: Timms, W.A., Crane, R., Anderson, D.J., Bouzalakos, S., Whelan, M., McGeeney, D., Rahman, P.F. and Acworth, R.I. 2016, Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale, Hydrology and earth system sciences, vol. 20, no. 1, pp. 39-54. Timms, W.A., Crane, R., Anderson, D.J., Bouzalakos, S., Whelan, M., McGeeney, D., Rahman, P.F. and Acworth, R.I. 2016, Accelerated gravity testing of aquitard core permeability and implications at formation and regional scale, Hydrology and earth system sciences, vol. 20, no. 1, pp. 39-54. DOI: http://www.dx.doi.org/10.5194/hess-20-39-2016 ©2016, The Authors ©2016, The Authors Reproduced by Deakin University under the terms of the Creative Commons Attribution Licence 1School of Mining Engineering, University of New South Wales, Sydney, Australia 2 Correspondence to: W. A. Timms (w.timms@unsw.edu.au) Correspondence to: W. A. Timms (w.timms@unsw.edu.au) Received: 11 November 2014 – Published in Hydrol. Earth Syst. Sci. Discuss.: 9 March 2015 Revised: 20 October 2015 – Accepted: 12 November 2015 – Published: 15 January 2016 Received: 11 November 2014 – Published in Hydrol. Earth Syst. Sci. Discuss.: 9 March 2015 Revised: 20 October 2015 – Accepted: 12 November 2015 – Published: 15 January 2016 Abstract. Evaluating the possibility of leakage through low- permeability geological strata is critically important for sus- tainable water supplies, the extraction of fuels from coal and other strata, and the conﬁnement of waste within the earth. The current work demonstrates that relatively rapid and real- istic vertical hydraulic conductivity (Kv) measurements of aquitard cores using accelerated gravity can constrain and compliment larger-scale assessments of hydraulic connectiv- ity. Steady-state ﬂuid velocity through a low-K porous sam- ple is linearly related to accelerated gravity (g level) in a cen- trifuge permeameter (CP) unless consolidation or geochemi- cal reactions occur. A CP module was custom designed to ﬁt a standard 2 m diameter geotechnical centrifuge (550 g max- imum) with a capacity for sample dimensions up to 100 mm diameter and 200 mm length, and a total stress of ∼2 MPa at the base of the core. Formation ﬂuids were used as inﬂuent to limit any shrink–swell phenomena, which may alter the per- meability. Kv results from CP testing of minimally disturbed cores from three sites within a clayey-silt formation varied from 10−10 to 10−7 m s−1 (number of samples, n = 18). Ad- ditional tests were focussed on the Cattle Lane (CL) site, where Kv within the 99 % conﬁdence interval (n = 9) was 1.1 × 10−9 to 2.0 × 10−9 m s−1. These Kv results were very similar to an independent in situ Kv method based on pore pressure propagation though the sequence. However, there was less certainty at two other core sites due to limited and variable Kv data. Blind standard 1 g column tests underes- timated Kv compared to CP and in situ Kv data, possibly due to deionised water interactions with clay, and were more time-consuming than CP tests. Our Kv results were com- pared with the set-up of a ﬂow model for the region, and considered in the context of heterogeneity and preferential ﬂow paths at site and formation scale. 1School of Mining Engineering, University of New South Wales, Sydney, Australia 2 Reasonable assess- ments of leakage and solute transport through aquitards over multi-decadal timescales can be achieved by accelerated core testing together with complimentary hydrogeological moni- toring, analysis, and modelling. Downloaded from DRO: Deakin University CRICOS Provider Code: 00113B Hydrol. Earth Syst. Sci., 20, 39–54, 2016 www.hydrol-earth-syst-sci.net/20/39/2016/ doi:10.5194/hess-20-39-2016 © Author(s) 2016. CC Attribution 3.0 License. 1 Introduction Clay or other low-permeability sediment and rock often dominate sedimentary sequences and can form important aquitards (Potter et al., 1980). These hydraulic barriers often overlie aquifers that yield strategically important fresh wa- ter resources and form important cap rocks or seals between shallow aquifers and deeper strata targeted for depressurisa- tion during gas or mineral extraction (Timms et al., 2012). The current work compares the results of steady-state cen- trifuge permeability testing of semi-consolidated drill core samples with column tests at standard gravity (1 g at earth’s surface, 9.8065 m s−2). Results of laboratory tests were also compared with in situ permeability, based on analysis of pore pressure propagation at formation scale. Thick, low hydraulic conductivity (K), un-oxidised, clay- rich aquitards represent important sites for waste conﬁne- W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 40 ment and disposal (including high-level radioactive waste and the sequestration of carbon dioxide and saline efﬂuents) and act as protective covers for regional aquifers (Cherry et al., 2004). Effective shale and claystone ﬂow barriers are re- quired to disconnect shallow aquifer systems from underly- ing coal seams that are depressurised to produce gas (Timms et al., 2012; APLNG, 2013). Fine-grained geologic media are also commonly used as engineered barriers to limit hor- izontal seepage of mine water (Bouzalakos et al., 2014), for containment of tailings (Znidarˇci´c et al., 2011), and disposal of municipal refuse and nuclear waste (Rowe et al., 1995). Low-permeability material is deﬁned by K of < 10−8 m s−1 (Neuzil, 1986). The US EPA requires low-permeability waste barriers for hazardous waste landﬁlls with K of < 10−9 m s−1 (US EPA, 1989). process, requiring relatively time-consuming and expensive ﬁeld and/or laboratory studies. Methods for measuring the in situ permeability of clay formations include: slug tests (piezometer tests, falling-head tests), aquifer pumping tests with piezometers in the aquitard, aquifer pumping tests with observation wells in the aquifer only, measurement of seasonal ﬂuctuations of pore pressure, measurement of pore pressure changes and settlement due to surface loading, and numerical analysis of local and re- gional groundwater ﬂow (van der Kamp, 2001). Neuman and Witherspoon (1968) developed generic analytical solutions for drawdown within an aquiclude, in which vertical ﬂow oc- curs, but is sufﬁciently small to have no effect on water levels within an overlying or underlying aquifer. Type curves were presented for analytical solutions applying to an inﬁnitely thick and a ﬁnite thickness aquiclude. In contrast, analysis of a leaky aquitard–aquifer system was presented by Neu- man and Witherspoon (1972). The ratio method compares drawdown within an aquitard with drawdown in an underly- ing aquifer from which extraction was occurring. Drawdown data are then used to calculate hydraulic diffusion of pres- sure transients, and Kv, assuming a uniform, homogeneous aquitard. Aquitards volumetrically constitute the bulk of sedimen- tary geologic deposits (Potter et al., 1980), and are typically assumed saturated if located below a water table (Cherry et al., 2004). Water-saturated K and diffusion coefﬁcients for aquitards are therefore not applicable to variably saturated or non-water-saturated low-permeability strata. Research is lacking for semi-consolidated clayey aquitards (e.g. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability alluvial, colluvial, and aeolian deposits), compared with aquitard re- search on glacial tills (Grisak and Cherry, 1975), claystones (Smith et al., 2013; Jougnot et al., 2010), and shale (Neuzil, 1994; Josh et al., 2012). Clay-bearing sediments formed via alluvial, colluvial, and aeolian processes frequently occur in the geosphere. For example clayey-silt aquitards account for 60 % of the ∼100 m thick alluvial sediment sequences in the Mooki catchment of Australia’s Murray–Darling Basin (Far- ley, 2011). The relative lack of information on the dominant type of sedimentary deposit represents a key gap in the cur- rent theoretical understanding of clay mineralogy and geo- chemistry. Deconvolution of the pressure response to depth through an aquitard can be analysed with a Fourier transform or har- monic analysis (Boldt-Leppin and Hendry, 2003). The hy- draulic diffusivity (hydraulic conductivity divided by speciﬁc storage) is expressed analytically, either based on the ampli- tude or phase shift of harmonic signals, assuming that the thickness of the aquitard is semi-inﬁnite. Jiang et al. (2013) further developed the harmonic analysis method for ﬁnite aquitards in a multi-layer system in the instance of wa- ter level monitoring within aquifers above and below an aquitard, but not monitoring within the aquitard. Coherence analysis of water level ﬂuctuations in bounding aquifers from indeterminate stresses (e.g. pumping, recharge, rainfall or earthquake) was used to derive Kv for deep rock aquitards on the basis of interpolated groundwater level data measured at irregular intervals of at least 10 days over a duration of several decades. Aquitard research on alluvial sediments is important be- cause recharge by slow seepage provides essential ground- water supplies for municipal water supply and crop irrigation in relatively dry inland settings (Acworth and Timms, 2009). Increased effective stress associated with aquifer drawdown for irrigation, may release saline water stored within shal- low aquitards with implications for the continuation of high yields of fresh water. Characterising the effects of variable chemical composition of formation water on the hydraulic conductivity of such sediments is therefore essential to de- termine the long-term changes to fresh water. A more direct method of determining in situ hydraulic pa- rameters is possible using fully grouted vibrating wire trans- ducers and high-frequency data recording within deep for- mations, as recently demonstrated by Smith et al. (2013) for a bedrock claystone at up to 325 m below ground (BG). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeabilit www.hydrol-earth-syst-sci.net/20/39/2016/ W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability depend on factors such as the preparation and size of core samples, conﬁguration of equipment and uncertainties of measurement, the inﬂuent water that is used and the stresses that are applied relative to in situ values, and whether perme- ability is directly measured from steady-state ﬂow, or sub- ject to additional parameters and assumptions with alterna- tive ﬂow regimes. Laboratory testing of clayey-silt cores by standard rigid and ﬂexible wall column techniques requires 1–2 weeks, compared with < 1 week for centrifuge perme- ameter (CP) methods in unsaturated samples (ASTM, 2010). Constant or falling-head tests in rigid-walled column per- meameters at natural gravity require a large water pressure gradient and/or long testing times for low-permeability sam- ples. They are subject to potential leakage, and may not repli- cate in situ conﬁning stresses. Column testing of core sam- ples is possible for some test conditions in triaxial cells on both Kh and Kv, for example those used in geotechnical and petroleum studies (Wright et al., 2002). However, stan- dard practice for testing ultra-low-permeability cores (e.g. Kv<1 × 10−10 m s−1) typically consists of applying a con- ﬁning pressure to a watertight system and measuring small transient pore pressures with high-resolution pressure trans- ducers (API, 1998). This paper demonstrates novel CP techniques and equip- ment that have been speciﬁcally developed for character- ising semi-consolidated clayey-silt cores. Kv results from CP methods are compared with standard 1 g column meth- ods and in situ measurements of permeability, based on har- monic analysis of the high-frequency pore pressure propaga- tion through a thick clayey sequence. The variability, conﬁ- dence limits and overall reliability of the Kv results to con- strain assessments of regional-scale vertical connectivity are considered in the context of sampling and ﬂow and stress conditions within the CP. This paper provides reasonable Kv for at least one local clayey-silt sequence and strategies for future testing that are important contributions towards evalu- ating ﬂow connectivity at a range of scales. These Kv results can be complimented with hydrogeological data such as pore pressure and tracer data to better constrain numerical ﬂow models. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability eral years under in situ conditions can be reproduced in a geotechnical centrifuge within hours or days, depending on test conditions. Table 1. Speciﬁcations and performance details of the Broad- bent GT-18 centrifuge permeameter (CP) system as constructed by Broadbent (2011). A CP, or a column mounted on a centrifuge strong box, is commonly used for hydraulic characterisation of porous me- dia. Accelerated gravity achieves a steady-state equilibrium for ﬂuid ﬂow through the CP within hours or days of instru- ment operation (for an unsaturated sample), while simulta- neously applying stresses to the solid matrix. A permeameter column, mounted on a geotechnical centrifuge is rotated suf- ﬁciently fast to accelerate ﬂow and approximate in situ total stresses, while the target g level is designed to ensure that the matrix is not consolidated and chemical equilibrium is maintained. Steady-state ﬂow can provide more reasonable K results than transient ﬂow techniques. Although transient tests are even more rapid than steady-state tests in the cen- trifuge, more complex instrumentation is required to ensure reliable results (Zornberg and McCartney, 2010). Dimensions/mass Diameter (lower rotary stack) 200.0 cm Radius to top sample chamber 45.0 cma Radius to base sample chamber 65.0 cmb Total mass 4800 kg Performance Rotational speed 10–875 rpm Maximum sample length 20.0 cm Maximum sample diameter 10.0 cm Maximum sample mass 4.7 kg Maximum sample density SG 3.0 Maximum efﬂuent reservoir capacity 1000 mL Maximum payload 18.11 kg a 385 G at 875 rpm; b 556 G at 875 rpm. The geotechnical centrifuge system described in this pa- per is moderately sized and relatively economical to operate, whilst able to perform both unsaturated and saturated testing of porous media with real-time measurement of various pa- rameters during ﬂight (Table 1). These attributes mean that CP testing of relatively large diameter cores (up to 100 mm diameter) in this facility is comparable in cost to testing of small cores (38 mm diameter) using alternative methods such as He-gas permeation. The system has been success- fully used for testing low-permeability rock cores (Bouza- lakos et al., 2013; Jiang et al., 2015). To date, there were no other direct Kv measurements on these deep shales available (APLNG, 2013) and alternative laboratory methods were not successful in obtaining a Kv value from these very low-K rocks (Bouzalakos et al., 2013). www.hydrol-earth-syst-sci.net/20/39/2016/ W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Pore pressure and barometric pressure were recorded at 30 min in- tervals and analysed, assuming no leakage in the grouted sys- tem, for barometric response, earth tides, and rainfall events. Core samples from the same drill holes were vacuum sealed on site for consolidation testing and triaxial permeameter testing. The in situ compressibility and speciﬁc storage cal- culated from barometric pressure responses were as much as an order of magnitude smaller than laboratory results. As an example, revised calculation of hydraulic parame- ters based on water level recovery from a bore pump test in glacial till (K = 10−11 m s−1) has been required to im- prove the ﬁt with the data emerging over ∼30 years (van der Kamp, 2011). Various ﬁeld and laboratory methods are available to directly measure or indirectly calculate hydraulic conductivity along the horizontal (Kh) or vertical (Kv), and saturated and unsaturated or multi-phase ﬂow (e.g. liquid and gas). Obtaining realistic measurements of groundwater ﬂow and solute transport within aquitards is by deﬁnition a slow A variety of laboratory testing techniques for low-K sam- ples are also available; however, the reliability of results may www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 41 Hydrol. Earth Syst. Sci., 20, 39–54, 2016 2 Geology of study sites with a diffusion dominated transport over thousands of years (Timms and Acworth, 2006). The saturated zone ﬂuctuates in response to rainfall events between the ground surface and approximately 2 m depth, while water levels in the conﬁned gravel aquifer at > 50 m depth display a delayed and damp- ened response to the same rainfall events. There is no ground- water extraction for irrigation from this aquifer in the vicinity of the site, and the valley has been artiﬁcially drained to pre- vent ponding of surface water and soil salinisation. Detailed geological studies and particle dating have identiﬁed that the clayey silt in the top ∼30 m at this site accumulated grad- ually at 0.2–0.3 mm year−1 by weathering of alkali basalts (Acworth et al., 2015). Flow testing of 100 mm diameter cores from the CL site, reported by Crane et al. (2015) has revealed evidence for dual porosity ﬂow when a hydraulic gradient is imposed on the low-permeability sediments, with further work in progress to identify the nature and signiﬁ- cance of these potential ﬂow paths. Semi-consolidated sediment cores were obtained from three sites in the Australia Murray–Darling Basin, in the Up- per Mooki subcatchment of the Namoi catchment (Fig. 1). Groundwater is extracted in this area for irrigation and town water supplies, with drawdowns of more than 10 m over 30 years. It can take years or decades for changing pore pres- sures to be transmitted through these mixed sediments that are heterogeneous, even though the effects of groundwater extraction were assumed to occur rapidly within homoge- neous, high-permeability sediments (Kelly et al., 2013). The alluvial sedimentary geology of the valley features signiﬁ- cant heterogeneity but a general ﬁning upwards that reﬂects climatic drivers of sedimentation (Kelly et al., 2014). This study found that the architectural features and the net (sand and gravel) to gross (total volume) line plot that identiﬁes low-permeability clays and silts of the valley-ﬁlling sequence are best represented by a distributive ﬂuvial system. In this type of ﬂuvial system, the avulsion frequency increases at a slower rate than the aggradation rate. Sediments at the Breeza farm and Norman’s Road site are relatively heterogeneous, with mixed sandy, clayey-sand, and clayey-silt alluvium overlying a semi-conﬁned aquifer. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Geotechnical centrifuges are used to subject porous sam- ples to high artiﬁcial gravities in order to characterise their hydraulic and/or consolidation properties (Conca and Wright, 1998; Nakajima and Stadler, 2006; Znidarˇci´c et al., 2011), and for physical modelling as part of geotechnical de- sign (Garnier et al., 2007; Parks et al., 2012). Accelerated gravity acts on both the solid particles and ﬂuids within the porous sample without use of a large ﬂuid pressure gradi- ent to drive ﬂow. The technique can be applied to investigate slow hydrogeological processes over shorter timescales; i.e. ﬂow through low-permeability layers that would take sev- Hydrol. Earth Syst. Sci., 20, 39–54, 2016 www.hydrol-earth-syst-sci.net/20/39/2016/ W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 42 Figure 1. Location of study sites in eastern Australia, state of New South Wales (NSW). The Norman’s Road (NR), Breeza Farm (BF) and Cattle Lane (CL) sites are shown within the Namoi catchment. Figure 1. Location of study sites in eastern Australia, state of New South Wales (NSW). The Norman’s Road (NR), Breeza Farm (BF) and Cattle Lane (CL) sites are shown within the Namoi catchment. 4.1 Preparation of cores To ensure that cores were tested under saturated realistic con- ditions, drill cores were adequately preserved, stored, pre- pared, and set on a vacuum plate prior to centrifuge testing. Cores from PET drill core liners were trimmed and inserted into an acrylic liner for the CP using a core extruder. The custom made core extruder had ﬁve precision cutting blades driven by a motorised piston suitable for a 100 mm diameter core. Cores for CP testing in this study were 100 mm diam- eter C size core, with a length of 50–100 mm. A close ﬁt between the clay core and the liner was achieved using this extruder. The cores contained within PET liners were transferred di- rectly from the core barrels to a cool room on site, and thence to a laboratory cool room, reducing the potential for moisture loss. Semi-consolidated clay cores were selected from below the saturated zone for CP tests, at depths up to 40 m BG. Sediment core samples of lengths between 50 and 100 mm were prepared for CP testing. The moisture content and bulk density of cores was measured using methods adapted from ASTM D7263-09 (2009). These measurements were com- pleted immediately on the drill site. A vacuum plate system for core samples was designed to ensure fully saturated cores, remove air at the base of the core, and ensure an effective seal between the CP liner prior to testing at accelerated gravity. The vacuum plate device was designed to ﬁt the CP liners containing the cores, drawing ponded inﬂuent from the top to the base of the cores using a standard laboratory vacuum pump at 100 kPa of negative pressure. After 12–48 h, or upon efﬂuent ﬂow from the base, the acrylic liners containing the prepared cores were then transferred directly to the CP module without disturbing the sample. The preferred method for preservation of drill core was double plastic bagging of sections of core within their PET liners using a food grade plastic sealing system (with brief application of a vacuum to extract air from the plastic bag). Alternatively, core within PET core barrel liners were trimmed of air or ﬂuid ﬁlled excess liner immediately after drilling, and then sealed with plastic tape. All cores were stored at 4 ◦C in a portable cool room on the drill site and then at the laboratory. 3.2 Groundwater sampling for inﬂuent Fluid for K testing (inﬂuent) should be taken from the for- mation at the same depth as the core. Formation water can be synthesised if it is not possible to sample directly from aquitard strata, by estimating the ionic strength, Na / Ca ra- tio, and pH. In this study, groundwater from piezometers at a similar depth to the core was obtained using standard ground- water quality sampling techniques (Sundaram et al., 2009). A 240 V electric submersible pump (GRUNDFOS MP1) and a surface ﬂow cell were used to obtain representative sam- ples after purging stagnant water to achieve constant ﬁeld measurements of electrical conductivity and other parame- ters (Acworth et al., 2015 and unpublished data). The non-rotating core barrel was forced into the formation, whilst a rotating device on the outside of the tube removes the cuttings as the barrel was advanced. The cutting edge of the non-rotating sample tube projects several millimetres beyond the rotary cutters. The thin-walled core barrel complied with the standard for undisturbed sampling, with an area ratio of less than 25 % for an open-drive sampler. The area ratio of 16 % was based on a core barrel design with an external di- ameter of 110 mm and internal diameter of 101 mm (C size). The 1.5 m length core barrel was a composite open sampling system with a core nose screwed onto the base with a bev- elled end to cut the core as the barrel pushed into the for- mation. After the core was extracted from the ground, an air supply was connected to the top of the core barrel to slide the core out of the barrel whilst it remained in the clear PET liner without rotation, distortion, or compression. 3 Study site characterisation and sampling were constructed of screwed sections of 50 mm PVC cas- ing with O-ring seals, with a 1.5 m machine slotted screen packed with pea-sized washed gravel. The annulus was then ﬁlled with a bentonite seal, backﬁlled to the surface and com- pleted with a steel casing monument and cement monument pad. 3.1 Drilling and core sampling Equipment and procedures for coring were compliant with ASTM D1587-08 (2008a) to obtain samples that were as undisturbed as possible. A rotary drilling rig equipped with Triefus triple core barrels, lined with seamless clear PET, was used in push coring mode. Local creek water was used as a drilling ﬂuid and casing was used to stabilise the hole behind the push core barrel such that drilling ﬂuid additives were not required. The holes were therefore fully cased to the maxi- mum depth of push core drilling at up to 40 m BG. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 2 Geology of study sites The saturated zone is approximately 18–20 m below surface and extraction for ﬂood irrigation of crops causes large ﬂuctua- tions in groundwater levels in the conﬁned aquifers at > 50 m depth. Hydrogeological and hydro-geochemical evidence in- dicate a leaky aquifer–aquitard system, with the variability in groundwater level responses controlled by a ﬁning upward alluvial sequence (Acworth and Timms, 2009). At the Nor- man’s Road site, highly saline porewater (15 mS cm−1) in the clayey silt in proximity to the surface (< 20 m) appears to have leached into the underlying aquifer, causing a signiﬁ- cant increase in salinity of the aquifer (Badenhop and Timms, 2012). Core drilling was completed at three research sites (Fig. 1) including Cattle Lane (CL), located south of the town of Caroona (31◦31′9′′ S, 150◦28′7′′ E), the Breeza farm (BF) operated by the New South Wales (NSW) Department of Primary Industries, south-east of Gunnedah (31◦10′32′′ S, 150◦25′15′′ E), and Norman’s Road (NR), east-southeast of Gunnedah (31◦2′48′′ S, 150◦26′7′′ E). Clayey-silt sediments at the Cattle Lane site are approx- imately 30 m thick (Timms and Acworth, 2005) and ex- tend throughout the valley (Wiesner and Acworth, 1999), as shown by numerous CCPT (conductivity cone penetrometer) proﬁles. The porewater salinity proﬁle at the site, increas- ing from 10 to 30 m depth through the clay, is consistent www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 43 Hydrol. Earth Syst. Sci., 20, 39–54, 2016 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 44 Figure 2. Cross sectional diagram of a core sample subjected to centrifugal force, with a free drainage boundary condition at the base of the core. content was not affected by the use of a vacuum to expel air from sealing bags or from the top or base of the cores ﬁtted into the CP liners. A self-seal was observed forming from material swelling at the interface with the liner within minutes of introducing the inﬂuent solution. Prior to the self-seal development, leak- age along the liner interface was identiﬁed by a ﬂow rate of several orders of magnitude higher than the steady-state ﬂow Kv value. The swelling that occurred to self-seal the core was estimated at less than 0.02 % of the cross sectional area of the core by comparing ﬂow rates through the CP drainage hole (described in Sect. S3 in the Supplement). It was calculated that this area of swelling was sufﬁcient to seal an annulus aperture of ∼0.01 mm between the clay core and the acrylic liner. Given the relatively shallow depth of these cores, and the semi-consolidated status, the maximum g level in the cen- trifuge was limited to prevent structural changes in the core matrix. To minimise changes in porosity of the core during testing, the g level and the weight of ponded ﬂuid on the cores were therefore designed to ensure that total stress was less than estimated in situ stress at the depth from which the core was drilled. Blind permeability tests were carried out by an inde- pendent laboratory, which adapted a constant/falling-head method (AS 1289 6.7.3/5.1.1, 1991) with methods from Head (1988). For these 1 g column tests, a sample diame- ter of 45.1 mm and length 61.83 mm was used, and a con- ﬁning pressure of 150 kPa and back pressure of 50 kPa was applied, providing a vertical uniaxial stress of 100 kPa. The test time was up to 100 h. These standard 1 g column tests used deionised water as the inﬂuent. Figure 2. Cross sectional diagram of a core sample subjected to centrifugal force, with a free drainage boundary condition at the base of the core. 4.2 Centrifuge permeameter testing The Broadbent CP module and some unique systems devel- oped as part of this study are described in this section, with further details in Sects. S1 and S3. A conceptual plan of a CP is shown in Fig. 2. The CP contains a cylindrical clay sample with length L and diameter D, and is spinning in a centrifuge around a central axis at an angular velocity ω. The perme- ameter has an inlet face at a radius r, and a drainage plate at a radius of r0. The co-ordinate z is deﬁned as positive from the base of the sample towards the central axis of rotation, consistent with deﬁnitions in 1 g column testing (McCartney and Zornberg, 2010). This frame of reference is in an oppo- site direction to that deﬁned by Nimmo and Mello (1991), but is convenient for interpretation and comparison of col- umn ﬂow tests. p The mass of two core samples were balanced to the near- est 100 g and tested simultaneously at either end of the cen- trifuge beam. The CP was operated at 10 g for 30 min, and if no rapid ﬂows due to leakage were detected, this was gradu- ally increased to 20, 40 g, and so on, until the maximum total stress on the core approached the estimated in situ stresses of the material at the given depth in the formation. The up- per permissible g level was designed to be less than the es- timated in situ stress from the depth at which the core was obtained. It was also important to ensure that effective stress (Sect. 4.4) was acceptable, as variable pore ﬂuid pressures during testing could cause consolidation of the core matrix. Inﬂuent volume was measured using both a calibrated contin- uous time record of pump rotations, and manual burette mea- surements, and efﬂuent volumes were measured by weight. steady-state ﬂow was deﬁned as ±10 % change in discharge over subsequent measurements in time, provided that inﬂu- ent ﬂow rate was within ±10 % of the efﬂuent ﬂow rate. Both of these conditions were required for the testing to be consid- Inﬂuent was fed from burettes located next to the cen- trifuge via a pair of custom designed low voltage peristaltic pumps mounted either on the centrifuge beam, or outside the centrifuge and through the low ﬂow rotary union. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability The K value is based on ﬂow rate, ﬂow area, radius, and revolutions per minute (RPM), although the method was adapted from a unsaturated/saturated ﬂow apparatus (UFA) centrifuge to this CP system (Sect. 4.3). Importantly, both testing systems are for steady-state ﬂow with free drainage due to zero pressure at the base of the core. 4.1 Preparation of cores Sections of cores, particularly at the nose end, that appeared to be damaged or disturbed were ex- cluded from permeability or bulk density testing. Additional steps that were taken in the laboratory to ensure core test- ing was representative of in situ conditions are described in Sect. 4.1. Furthermore, the moisture content and degree of saturation was monitored by measuring weight change of the perme- ameters during testing, and direct moisture tests of samples before and after CP testing. There was negligible difference observed between the moisture content of the core tests and in situ conditions, and the results were not associated with the time between sampling and testing of the core. Moisture After coring, the holes were completed as monitoring piezometers and the casing was jacked out. The piezometers www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 4.4 Fluid pressure and total stress calculations The total stress applied to the core, relative to stress, may affect the porosity of the core sample, depending on the stress history. In situ stress of the cores (Si) at the sampling depth below ground (D) was calculated using Eq. (3) Fluid pressures and hydraulic gradient through the centrifuge core were determined following the approach of Nimmo and Mello (1991). The total ﬂuid pressure P (kPa) was calculated in Eq. (1) Si = ρsDg . (3) (3) Si = ρsDg . (3) Si = ρsDg . P = ρw r′Z r0 rω2dr , (1) P = ρw r′Z r0 rω2dr , (1) It was assumed that the overlaying formations were fully saturated and of a similar bulk density to the supplied core samples. assuming a ﬂuid density ρw of 1.0 g cm−3 and where r is the radius of rotation (cm), and ω is the angular velocity (s−1). The total stress S (kPa) was determined through the centrifuge core, following Eq. (2) 4.3 Kv calculations and statistical analysis Hydraulic conductivity calculations for the CP in this study were based on ASTM D6527 (2008b) and ASTM D7664 (2010) with a form of Darcy’s Law that incorporates the additional driving force within a centrifuge. The gradi- ent in the centrifuge elevation potential (Nimmo and Mello, 1991), or the gradient in centrifuge “elevation head” (Zorn- berg and McCartney, 2010) due to the centrifuge inertial force driving was deﬁned as ﬂow away from the centre of rotation (or in the opposite direction to z in Fig. 2). The g level was deﬁned at the mid-point of the core. A ponded in- ﬂuent above the top of the core prevented loss of saturation along the core (Nimmo and Mello, 1991). The centrifuge in- ertial (elevation) head gradient and hydraulic head gradient (stationary centrifuge at 1 g) were calculated at 0.005 m in- crements through the core. Statistical analysis of the data followed a basic small- sampling theory using the Student’s t distribution, following the approach of Gill et al. (2005) and extending the approach of Timms and Anderson (2015) for estimating sample num- bers required for CP testing. Upper and lower conﬁdence in- tervals (UCI, LCI) were calculated from the mean ± t(n−1). sn/n1/2, where sn is the sample standard deviation and t(n−1) is the value of the Student’s t distribution at the selected con- ﬁdence limits (CL) of 90 and 99 %. The conﬁdence inter- vals were calculated for the increasing number (n) of Kv data from each core. Figure 3. Centrifuge permeameter testing at low stresses of a semi- consolidated clayey-silt core sample (CL 26.1 m depth; Test 39-1) showing variation of g level, Kv, and inﬂuent and efﬂuent ﬂow rate during the test (after Timms et al., 2014). assuming core bulk density ρs of 1.9 g cm−3. The total stress and ﬂuid pressure were calculated at 0.005 m increments through the core. The effective stress was then calculated as the difference between total stress and ﬂuid pressure. An in- crease in effective stress associated with decreased ﬂuid pres- sures near the base of the free draining core may cause con- solidation of the core matrix near the boundary. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 45 Figure 3. Centrifuge permeameter testing at low stresses of a semi- consolidated clayey-silt core sample (CL 26.1 m depth; Test 39-1) showing variation of g level, Kv, and inﬂuent and efﬂuent ﬂow rate during the test (after Timms et al., 2014). ered as a steady-state ﬂow condition. This protocol provided additional quantitative measures to the ASTM D7664 (2010), which states that steady-state conditions have been attained “if the outﬂow is approximately equal to the inﬂow”. Sec- tion S4 discusses the uncertainty of the measured data in more detail. ered as a steady-state ﬂow condition. This protocol provided additional quantitative measures to the ASTM D7664 (2010), which states that steady-state conditions have been attained “if the outﬂow is approximately equal to the inﬂow”. Sec- tion S4 discusses the uncertainty of the measured data in more detail. 4.2 Centrifuge permeameter testing In this study, the outlet face was a free drainage boundary, and is discussed further in Sects. S2 and S3. www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 Core properties and Kv results from CP testing (3) for in Site Depth Kv g level Estimated in situ Testing Inﬂuent (m BG) (m s−1) maximum stress (kPa) time (h) source NR 33.8 4 × 10−9 10 615 ∼144 NR P30 NR 33.90 2 × 10−9 10 615 ∼144 NR P30 NR 34.68 2.4 × 10−7 10 646 2.6 NR P30 CL 11.75 3.5 × 10−9 80 219 24 CL P15 CL 14.00 2.2 × 10−9 80 261 1 CL P15 CL 19.25 2.0 × 10−9 80 359 24 CL P20 CL 21.70 5.1 × 10−9 80 404 1 CL P20 CL 26.01 2.4 × 10−9 80 485 21 CL P40 CL 26.10 1.1 × 10−10 80 486 21 CL P40 CL 28.33 2.0 × 10−9 10 526 24 CL P40 CL 28.52 2.7 × 10−9 80 532 1 CL P25 CL 31.36 1.6 × 10−9 40 585 24 CL P40 BF 24.07 5.9 × 10−9 40 449 3 BF CP25 BF 24.14 3.4 × 10−8 40 450 3 BF CP25 BF 31.40 1.3 × 10−9 30 585 11 BF CP40 BF 36.46 3.5 × 10−7 10 680 2.5 BF CP40 BF 40.00 1.5 × 10−9 30 746 23 BF CP40 BF 40.10 4.3 × 10−8 30 746 23 BF CP40 limited to 30–40 g. Figure 3 shows the measured inﬂuent and efﬂuent rates and the calculated Kv values during a typ- ical CP test as the g level was gradually increased. Steady- state ﬂow (±10 % change over time with inﬂuent rate equal to efﬂuent rate) was achieved at ∼20 h (Fig. 3). However, a lower Kv value that was observed overnight (> 12 h interval between samples) than was observed during the day (∼1 h intervals between samples). The Kv values over shorter time periods, with minimal evaporative losses, were considered to be more reliable. Further experimentation and numerical modelling is required to adequately explain this anomalous data, which may be associated with evaporative losses over longer time periods of ﬂow measurement or other transient processes within the system. silt size boundary is deﬁned at 0.002 mm), except for one sandy clay core. The large proportion of silt relative to clay is an important characteristic of this formation, with clay min- eralogy dominated by smectite (Timms and Acworth, 2005; Acworth and Timms, 2009). Moisture content varied from 24.7 to 36.4 % by weight, and was consistent with site measured data on the core (Sect. Core properties and Kv results from CP testing S = ρs r′Z r0 rω2dr , (2) S = ρs r′Z r0 rω2dr , (2) Index properties for ﬁve representative cores are provided in Table 2. The cores were typically silty clay (where the clay– www.hydrol-earth-syst-sci.net/20/39/2016/ www.hydrol-earth-syst-sci.net/20/39/2016/ www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 46 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Table 2. Core descriptions and index properties. Core ID BF C2.8 BF C2.16 and C2.15 CL C4.8a CL C4.20a NR C3.23 Depth (m BG) 11.00–11.35/ 22.50–22.90/ 11.27–11.47 28.50–28.70 33.00–33.35/ 11.35–11.68 21.93–22.18 33.35–33.68 Description Sandy clay; Clayey silt; Silty clay; Silty clay; Clayey silt; brown brown brown pale brown brown Moisture (% wt.) 24.7 28.2 45.7 36.4 – D50 (mm) 0.025 0.0068 – – < 0.0013 Bulk wet density (g cm−3) 1.88 1.81 1.71 1.77 1.72 Particle density (g cm−3) 2.52 2.47 2.58 2.50 2.58 Initial void ratio 0.67 0.75 1.20 0.93 0.89 Initial degree of saturation (%) 93 95 96 99 74 46 Table 2. Core descriptions and index properties. Table 3. Kv results from CP tests indicating g level maximum and testing time. The inﬂuent source column identiﬁes the site (NR, CL, BF) and depth (P20 is piezometer screen at 20 m depth) of groundwater sampling. Calculations were based on Eq. (3) for in situ stress. and depth (P20 is piezometer screen at 20 m depth) of groundwater sampling. Calculations were based on Eq. Core properties and Kv results from CP testing Kv conﬁdence intervals (m s−1) Site n Kv geometric mean (m s−1) sn logKv CL % lower bound upper bound CL 5 1.3 × 10−9 0.21 99 4.8 × 10−10 2.4 × 10−9 CL 9 1.6 × 10−9 0.14 99 1.1 × 10−9 2.0 × 10−9 BF 6 1.3 × 10−8 0.19 99 6.5 × 10−9 2.1 × 10−8 NR 3 1.2 × 10−8 0.34 99 1.5 × 10−10 8.5 × 10−8 90 3.4 × 10−9 4.6 × 10−8 During centrifuge testing effective stress is maximum at the base of the free draining core, where ﬂuid pressure is zero, and thus effective stress is equal to total stress under hy- drostatic conditions (no ﬂow). In both testing methods in this study, the total stress was less than estimated in situ stress; however, the stress history of the core sample and effective stress dynamics were uncertain. It appears that the stresses during these tests were likely within an acceptable range to minimise structural changes including swelling and consoli- dation. There was no evidence of signiﬁcant changes in core length due to consolidation of the samples during spot checks of core length with a digital calliper. However, further atten- tion on these processes, including instrumentation to mea- sure ﬂuid pressures and core matrix changes during testing is required in future studies. A separate geotechnical study of these semi-consolidated sediments, including oedometer testing is in progress to better quantify the relationship be- tween stress and permeability in these semi-consolidated ma- terials. In future studies of semi-consolidated materials, mea- surement of consolidation state (over consolidation ratio) and pre-consolidation stress is recommended prior to centrifuge testing to ensure that an appropriate centrifuge stress is ap- plied. Anomalous ﬂow via preferential pathways could be read- ily identiﬁed by a ﬂow rate of several orders of magnitude greater than otherwise observed. Anomalous ﬂow was often observed along the interface of the cores and the liner during the early minutes of a test before sealing occurred and steady- state conditions were established. On one occasion a prefer- ential ﬂow path developed during the test, which caused very fast ﬂow at accelerated gravity that was easily detected. A test failure like this could be readily identiﬁed and excluded from further evaluation. A small uncertainty in Kv results for the CL site was cal- culated at a conﬁdence limit of 99 % using the methods de- scribed in Sect. 4.3. Core properties and Kv results from CP testing S5), although not all the cores were fully saturated as received by the external laboratory. Bulk wet density varied from 1.71 to 1.88 g cm−3 and particle density from 2.47 to 2.58 g cm−3. The Kv of cores tested in the CP module (Ta- ble 3) varied from 1.1×10−10 to 3.5×10−7 m s−1 (n = 18). Accelerations up to 100 g were applied during CP testing of semi-consolidated sediment cores and were more typically www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 47 Table 4. Geometric mean, standard deviation (sn) and conﬁdence limits (CL %) analysis for Kv data using the CP method to test core from the clayey-silt formation at the CL, BF, and NR sites. Kv conﬁdence intervals (m s−1) Site n Kv geometric mean (m s−1) sn logKv CL % lower bound upper bound CL 5 1.3 × 10−9 0.21 99 4.8 × 10−10 2.4 × 10−9 CL 9 1.6 × 10−9 0.14 99 1.1 × 10−9 2.0 × 10−9 BF 6 1.3 × 10−8 0.19 99 6.5 × 10−9 2.1 × 10−8 NR 3 1.2 × 10−8 0.34 99 1.5 × 10−10 8.5 × 10−8 90 3.4 × 10−9 4.6 × 10−8 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 47 Table 4. Geometric mean, standard deviation (sn) and conﬁdence limits (CL %) analysis for Kv data using the CP method to test core from the clayey-silt formation at the CL, BF, and NR sites. Table 4. Geometric mean, standard deviation (sn) and conﬁdence limits (CL %) analysis for Kv data using the CP method to test core from the clayey-silt formation at the CL, BF, and NR sites. 5.3 Comparison of in situ Kv and column testing methods at the CL site A comparison of Kv from in situ and column testing meth- ods are shown in Fig. 4 for the CL site. Results from the CP method (1.1 × 10−10 to 3.5 × 10−9 m s−1, n = 9) were sim- ilar to Kv values from the independent and in situ method (1.6×10−9 to 4.0×10−9 m s−1) conﬁrming that the sequence is of low-permeability at the CL site with a reasonable level of conﬁdence (Table 4). However, Kv from both in situ and CP methods were higher than the 1 g column tests of cores from 11.27 to 11.47 and 28.24 to 28.33 m BG from this site (1.4 × 10−9, 1.1 × 10−10, and 1.5 × 10−10 m s−1, n = 3). Core properties and Kv results from CP testing By increasing the number of samples, the conﬁdence bounds for Kv were narrowed from a range of 4.8 × 10−10 to 2.4 × 10−9 m s−1 (n = 5) to a range of 1.1×10−9 to 2.1×10−9 m s−1 (n = 9). Increasing the num- ber of samples from ﬁve to nine decreased the standard devi- ation, although a similar geometric mean occurred with the increased sample number (Table 4). However, there was less certainty at two other core sites (BF and NR) due to limited and more variable Kv data. At the BF site the 99 % con- ﬁdence interval had relatively wide Kv bounds for n = 6, while at NR site, a conﬁdence interval of 90 % results in similarly wide Kv bounds for n = 3. However, such a small number of samples is not considered sufﬁcient for statistical analysis. This evaluation of the results highlights the relative Kv variability and small sample set for the BF and NR sites, and the need for further testing, particularly at the NR site. This issue will be expanded in the following discussion. www.hydrol-earth-syst-sci.net/20/39/2016/ W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 48 Figure 4. Vertical hydraulic conductivity (Kv) measurements by centrifuge permeameter and column permeameter compared with in situ Kv derived from pore pressure data at 6 h intervals over 4 years interpreted with harmonic analysis (after Timms and Ac- worth, 2005) for the Cattle Lane site with massive clayey-silt from the surface to 35 m depth. in test set-up (e.g. 45 vs. 100 mm diameter core) and stress changes that occur as discussed in Sects. 5.2 and S2. CP testing was relatively rapid, typically with a few hours up to 24 h required for steady-state ﬂow CP, compared with an average of 90 h (73, 96, and 100 h for the tests reported here) for 1 g column testing. In addition, an extended test of 830 h in the CP (unpublished data) veriﬁed that no sig- niﬁcant changes occurred over extended testing periods. The CP technique can therefore reduce average testing time to ∼20 % of the time that would be required in 1 g labora- tory testing systems, similar to the reduced time requirement of centrifuge methods for unsaturated hydraulic conductiv- ity functions compared with 1 g column tests ASTM (2010). The relative time advantage of testing cores at accelerated gravity may be greater at lower Kv, due to the increased time required to establish steady-state ﬂow conditions. The relative time advantage could be signiﬁcant for contami- nant transport testing, which requires several pore volumes of steady-state ﬂow, compared to permeability testing where steady-state ﬂow is established before one pore volume. The similarity of Kv measurements with different scales at the CL site (Fig. 4) indicates that in this part of the allu- vial deposit K is independent of vertical scale from centime- tres to several metres. These Kv results from both in situ and laboratory methods provide an important constraint for eval- uations of hydraulic connectivity, particularly as there is a general lack of Kv data for these sediments. Complimentary studies of hydraulic connectivity to quantify leakage rates include pore pressure monitoring and piezometer slug test- ing at various depth intervals along with hydrogeochemical and isotope tracer data. Recent geological studies of the allu- vial sequence (Acworth et al., 2015) outlined in Sect. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 2, and identiﬁcation of dual porosity structures in the large diameter cores (Crane et al., 2015) indicate that it may be possible for vertical leakage to occur through clayey silts if a vertical hy- draulic gradient were to be imposed. A diffusion dominated salt proﬁle through the sequence suggest negligible vertical ﬂow (Timms and Acworth, 2006); however, a proper assess- ment of ﬂow connectivity requires vertical hydraulic gradi- ents to also taken into account any salinity variations with depth and pore pressure variations that have occurred over at least the past decade. Figure 4. Vertical hydraulic conductivity (Kv) measurements by centrifuge permeameter and column permeameter compared with in situ Kv derived from pore pressure data at 6 h intervals over 4 years interpreted with harmonic analysis (after Timms and Ac- worth, 2005) for the Cattle Lane site with massive clayey-silt from the surface to 35 m depth. lag pore pressure analysis resulted in a Kv value of 1.6 × 10−9 m s−1, while the change in amplitude over a vertical clay sequence of 18 m (from a 17 m depth piezometer to the inferred base of the aquitard at 35 m depth) resulted in a Kv value of 4.0 × 10−9 m s−1. It is noted that the reliability of harmonic analysis related methods may be compromised by speciﬁc storage measure- ments. Jiang et al. (2013) relied on indirect speciﬁc stor- age values derived from downhole sonic and density log data from boreholes in the region, while Timms and Ac- worth (2005) calculated speciﬁc storage from barometric and loading responses that were recorded in the same groundwa- ter level data set and boreholes used for harmonic analysis. The reduced test times of CP testing may be attributed to the reduced time required to achieve steady-state ﬂow with centrifugal forces driving ﬂow. Alternatively, the relatively longer time required for 1g column testing may be attributed to deionised water interaction with clay that reduced inﬁltra- tion rates into the cores (10–100 lower Kv result for 1 g col- umn tests compared with CP tests). It is known that decreased ionic strength of inﬂuent (e.g. deionised water) causes a lin- ear decrease in permeability, and that the relative concentra- tions of sodium and calcium can affect permeability due to swelling and inter-layer interactions (e.g. Shackelford et al., 2010; Ahn and Jo, 2009). W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Differences in Kv values from the two laboratory testing methods could be due to differences 5.2 Pore ﬂuid pressure and stress conditions at accelerated gravity How realistic the Kv measured by CP testing is of in situ conditions depends in part on the magnitude of stress and any structural changes that occur within the core matrix. Sec- tion S2 provides background on the deﬁnition and signiﬁ- cance of hydrostatic pore pressure, centrifuge inertial (eleva- tion) head, and gradients driving ﬂuid ﬂow. Section S2 also discusses the possibility that K values reported in this study could be biased on the high side, considering total stress at the base of the core under steady-state conditions. In situ Kv of the clayey-silt at the CL site was based on observed amplitude and phase changes of pore pressures (at hourly or 6-hourly intervals) due to ﬁve major rainfall events over 4 years (Timms and Acworth, 2005). The phase lag at the base of the clay varied between 49 and 72 days. The phase www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 49 The calibrated leakance values were found to vary over 3 orders of magnitude across the Breeza area, with relatively high values in isolated areas in the south, centre, and north. In comparison, the Kv results on clayey-silt cores appear to be higher than the apparent Kv of the regional groundwa- ter model, but with a similar degree of heterogeneity. The reasons for this discrepancy are not yet clear, but may be at- tributed to non-unique calibration of the regional ﬂow model (e.g. underestimation of inter-aquifer leakance) or the lack of representative Kv values for this aquitard at a scale that accounts for heterogeneities and preferential ﬂow paths. vial sequence extends to over 100 m depth, we focussed this study on sediments deﬁned by a low net-to-gross ratio (Larue and Hovadik, 2006) of < 0.4 that reﬂects that clay-rich part of the sequence (Timms et al., 2011). We assumed a log-normal distribution of Kv within this formation, which as noted by Fogg et al. (1998) might be justiﬁed within individual facies, but not over the full stratigraphic section. It was also assumed that the standard deviation of the samples tested is similar to the standard deviation of the total population of Kv results from the formation, which may only be known if a signiﬁ- cantly large number of samples are tested. vial sequence extends to over 100 m depth, we focussed this study on sediments deﬁned by a low net-to-gross ratio (Larue and Hovadik, 2006) of < 0.4 that reﬂects that clay-rich part of the sequence (Timms et al., 2011). We assumed a log-normal distribution of Kv within this formation, which as noted by Fogg et al. (1998) might be justiﬁed within individual facies, but not over the full stratigraphic section. It was also assumed that the standard deviation of the samples tested is similar to the standard deviation of the total population of Kv results from the formation, which may only be known if a signiﬁ- cantly large number of samples are tested. g p p The Kv results in this study are within the range of values reported elsewhere for semi-consolidated clay-silt sediments. For example, Neuzil (1994) reviewed aquitard Kv values for intact muds and lacustrine clays (10−8–10−11 m s−1) com- pared to consolidated materials such as shale with values as low as 10−16 m s−1 for argillite. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability A detailed study of a clayey- marl and limestone aquitard in France (Larroque et al., 2013) found a quasi-systematic bias of 1 order of magnitude be- tween petrophysical Kv estimates (10−8–10−10 m s−1), com- pared with values (10−9–10−11 m s−1) from hydraulic diffu- sivity monitoring between 30 and 70 m BG. However, the empirical petrophysical relationships between porosity, pore size and intrinsic permeability do not adequately account for structural effects of clay materials. Field piezometer rising head tests (n = 225) indicated that Kv of a lacustrine clay aquitard around Mexico City was 10−8–10−9 m s−1 in two areas, 100 times greater than matrix-scale permeability (Var- gas and Ortega-Guerrero, 2004). However, in a third area of the Mexico City aquitard, the ﬁeld tests were 10−10 m s−1 in- dicating the regional variability that can occur within clayey deposits. Kv values for cores from the NR and BF sites were signif- icantly larger than for the CL site, although additional data from the NR site are required to increased conﬁdence inter- vals (Tables 3 and 4). Based on the data set currently avail- able for each site there did not appear to be any signiﬁcant Kv trend with depth, except at the CL site, with a possible decrease of Kv by a factor of 3 with depth increasing from 11 to 28 m BG. Further testing is in progress to better identify any spatially signiﬁcant trends in Kv. Kv results obtained from the CP for these clayey-silt aquitards were signiﬁcantly larger than Kv for consolidated rock cores tested in this system (Bouzalakos et al., 2013). The relatively low g levels in this study (up to 80 g), com- pared to rock core testing (up to 520 g; Bouzalakos et al., 2013) were necessary for the shallow and semi-consolidated nature of the clayey-silt cores. In fact, steady-state ﬂow was achieved at low g levels for Kv values that were at least 100 times higher than the current detection limit and uncertainty of the CP system (Sect. S4). The vertical permeability of the clayey-silt aquitards in this region, and the relative importance of matrix ﬂow and preferential ﬂow through fractures and heterogeneities are critical to the sustainability of the groundwater resource. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability The Kv data reported in this study for these silty and semi- consolidated sediments are higher than reported for regional ﬂow modelling in this area (McNeilage, 2006), indicating that the aquitards allow for signiﬁcant recharge to underly- ing aquifers. Studies of glacial till aquitards in Canada, the US, and Denmark ﬁnd that regional permeability is typically at least 2 orders magnitude greater than laboratory tests (van der Kamp, 2001; Fredericia 1990; Bradbury and Muldoon, 1990; Gerber and Howard, 2000), although one study (Husain et al., 1998) showed that for a thick glacial till aquitard in southern Ontario, Canada, the regional permeability is similar to the laboratory-obtained measurements, indicating the absence of signiﬁcant permeable structures. A regional groundwater ﬂow model developed by Mc- Neilage (2006), with a two-layer MODFLOW code, deter- mined the dominant source of recharge to be diffuse leak- age through the soil (and aquitards) in the Breeza groundwa- ter management area. As in typical groundwater modelling practice (Barnett et al., 2012), the aquitard was not explic- itly modelled, with water instead transferred from a shallow to a deeper aquifer using a vertical leakance value (units in day−1). There is evidence of fracturing near the surface of the clayey aquitards that are the focus of this study. Fracture ﬂow to a shallow pit and the freshening of porewater in the aquifers at 16 and 34 m depth during the irrigation season in- dicated rapid leakage had occurred at the BF site (Acworth and Timms, 2009). The dynamics of fracturing within ∼2 m of the ground surface in these sediments was described by Greve et al. (2012). However, beyond the zone of fractur- ing near the ground surface, there appears to be insigniﬁ- cant groundwater ﬂow. Solute proﬁles through the 30 m thick clayey deposit at the CL site indicate that downwards migra- tion of saline water is limited to diffusion and that ﬂow is insigniﬁcant (Timms and Acworth, 2006). On the basis of available evidence, the clayey sediments in this region may The calibrated groundwater model indicated that approxi- mately 70 % of the long-term average groundwater recharge (11 GL year−1) was attributed to diffuse leakage in this area that included the CL and NR sites. This volume is equivalent to 20 mm year−1, or a Kv of ∼6 × 10−10 m s−1 assuming a unit vertical hydraulic gradient over an area of approximately 500 km2. 5.4 Geological and regional context for permeability of a clayey-silt aquitard The Kv measurements reported in this paper are important because there is a lack of aquitard data for alluvial groundwa- ter systems globally. Even where many groundwater inves- tigations have been completed (e.g. Murray–Darling Basin) there continues to be a lack of information on the thick clayey-silt sediments at various spatial scales. The core samples for testing were randomly selected from the same lithostratigraphic formation, the upper 30 m of the alluvial sequence as described in Sect. 2. Although the allu- www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 50 at natural gradient, unit gradient, and for various centrifuge permeameter set-ups (assuming a Kv of Table 5. Linear ﬂow velocity at natural gradient, unit gradient, and for various centrifuge permeameter set-ups (assuming a Kv of 10−8 ms−1). Table 5. Linear ﬂow velocity at natural gradient, unit gradient, and for various centrifuge permeameter set-ups (assuming a Kv of 10−8 ms−1). Natural Unit Centrifuge gradient gradient permeameter Core length × diameter (mm) 200 × 100 200 × 100 200 × 100 30 × 100 30 × 65 RPM n/a n/a 202 202 310 g level 1 1 30 30 70 Vertical ﬂuid head gradient (mm−1) 0.5 1 ∼0.2∗ ∼0.5∗ ∼0.5∗ Flow (mLh−1) 0.3 0.6 8.5 8.5 8.5 Time for 1 pore volume (h) 3333 1667 55.4 8.3 3.5 Normalised Increased linear ﬂow velocity 30 30 71 Reduced time for 1 PV 30 200 474 ∗Fluid head gradient depends on the depth of inﬂuent on the core and the length of the core. ∗Fluid head gradient depends on the depth of inﬂuent on the core and the length of the core. laws for physical modelling (Tan and Scott, 1987) providing further evidence that the Kv results are realistic. lack preferential ﬂow paths at some sites, and in other areas preferential ﬂow may occur through features such as frac- tures and heterogeneity at a range of scales (Crane et al., 2015). Further work is required to determine permeability at a range of scales, and to better understand preferential ﬂow paths. The current conceptual model on which the numerical models in this region are based (simple layered aquitard over- lying an aquifer) do not allow for spatial variability in con- nectivity mechanisms that could be important across a large valley alluvial ﬁll sequence. Multiple mechanisms for ver- tical connectivity, including matrix ﬂow, fracture ﬂow, and sedimentary heterogeneity, could be important in aquitards. The relative importance of each of these pathways for verti- cal ﬂow would depend on the spatial scale and local setting in each aquitard. The accelerated ﬂow conditions, whilst realistic for hy- drogeological environments, can also be an advantage for experimental studies of solute transport. Kv results of the order of 10−9 m s−1 were obtained in ∼20 % of the time required for 1 g column permeameter tests. Solute break- through experiments require longer testing periods of steady- state ﬂow than for permeability testing. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability For example, Timms and Hendry (2008) and Timms et al. (2009) described con- tinuous CP experiments over 90 days to quantify reactive so- lute transport during several pore volumes (PV) of ﬂow. The comparisons of time required for 1 PV provided in Table 5 illustrate the possible advantages of CP for contaminant ﬂow that may affect the structural integrity of the material. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability The actual Kv or leakance values were not reported. www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 50 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Table 5. Linear ﬂow velocity at natural gradient, unit gradient, and for various centrifuge permeameter set-ups (assuming a Kv of 10−8 ms−1). Natural Unit Centrifuge gradient gradient permeameter Core length × diameter (mm) 200 × 100 200 × 100 200 × 100 30 × 100 30 × 65 RPM n/a n/a 202 202 310 g level 1 1 30 30 70 Vertical ﬂuid head gradient (mm−1) 0.5 1 ∼0.2∗ ∼0.5∗ ∼0.5∗ Flow (mLh−1) 0.3 0.6 8.5 8.5 8.5 Time for 1 pore volume (h) 3333 1667 55.4 8.3 3.5 Normalised Increased linear ﬂow velocity 30 30 71 Reduced time for 1 PV 30 200 474 ∗Fluid head gradient depends on the depth of inﬂuent on the core and the length of the core. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 51 The Supplement related to this article is available online at doi:10.5194/hess-20-39-2016-supplement. down, the associated release of water into adjacent aquifers over long time periods, and the possibility of preferential ﬂow (Jiang et al., 2015). Realistic information on aquitard hydraulic properties could improve conﬁdence in conceptual and numerical hydrogeological models of aquifer–aquitard systems. Acknowledgements. Funding from the Australian Research Coun- cil and National Water Commission, through the National Centre for Groundwater Research and Training Program 1B is gratefully acknowledged. The contributions of N Baker and A Ainsworth of Broadbent and Sons, Huddersﬁeld, UK, are acknowledged and J McCartney for a helpful discussion on the theory of ﬂuid ﬂow during centrifuge testing. We appreciated research support at the Breeza farm provided by M McLeod and S Goodworth of the NSW Department of Primary Industries. Clayey-silt cores were drilled by New South Wales Ofﬁce of Water, with S McCulloch, H Studhome, and G Regmi. Experimental testing was assisted at UNSW by A Hartland, B Bambrook, M Aitkins, P King, S May, and T Meyers. Five reviewers and an editor are thanked for their constructive comments on earlier drafts of the paper. In the absence of direct measurement of aquitard perme- ability there is a real risk that aquitard parameters may be ignored or misrepresented in analyses resulting in a corre- sponding underprediction of vertical connectivity via pref- erential ﬂow paths and/or overprediction of aquifer storage and transmissivity. This is an especially important consider- ation in the analysis of aquifer tests that may not have been conducted for sufﬁcient periods of time to identify distant boundary conditions or the characteristic effects of aquitard leakage and/or storage (Neuman and Witherspoon, 1968). In very low-permeability strata, however, there are practi- cal limitations to pump tests and packer testing below about 10−8 m s−1, depending on the equipment and the thickness of strata that is subject to testing. It is recognised that in many heterogeneous systems time lags for the propagation of draw- down responses through an aquitard can be signiﬁcant (Kelly et al., 2013). Edited by: M. Riva Core-scale measures of aquitard hydraulic conductivity are an integral component of hydrogeological studies con- cerning aquifer connectivity. The availability of core-scale facies measurements enables the up-scaling of bore log and geophysical data to determine upper and lower hydraulic conductivity bounds for regionally up-scaled aquitard units. Hydrol. Earth Syst. Sci., 20, 39–54, 2016 6 Conclusions An accurate and reasonable measurement of the vertical hy- draulic conductivity (Kv) of aquitards is a critical concern for many applications. For example, following an empirical analysis of selected case studies, Bredehoeft (2005) reported that the collection of new ﬁeld data may render the prevail- ing conceptual hydrogeological models invalid in 20–30 % of model analyses. Bredehoeft (2005) coined the term “con- ceptual model surprise” to explain this phenomenon. He then went on to explain that “often one does not have hydraulic conductivity values for conﬁning layers because of the difﬁ- culties associated with acquiring such data”. To determine if accelerated ﬂow conditions are realistic for hydrogeological environments, the linear ﬂow velocity for various CP set-ups was compared with other ﬂow scenar- ios. The rationale behind this comparison was that if the ﬂow rate was consistent with scaling laws for physical modelling, with a unit gradient as a point of reference, then the results could be consider realistic for atypical in situ vertical hy- draulic gradient. In Table 5, an in situ hydraulic gradient of 0.5 is compared with CP set-ups for 100 and 65 mm diame- ter cores of various lengths for an aquitard material with a Kv of 10−8 m s−1. The vertical ﬂow rate varies from 0.3 mL h−1 under in situ conditions to 8.5 mL h−1 in the CP, such that linear ﬂow velocities remain very low at 10−8–10−6 m s−1. The ﬂow rate during centrifugation was N times greater than if a hydraulic gradient of 1 was applied to the core samples at 1 g. This increase in ﬂow rate is consistent with scaling The centrifuge technology described within this paper helps investigators to overcome some of the modelling limi- tations identiﬁed by Bredehoeft (2005). With centrifuge tech- nology, realistic point-scale measures of hydraulic property data can be collected to develop more realistic numerical ﬂow models to quantify the signiﬁcance of transient draw- www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. Sci., 20, 39–54, 2016 www.hydrol-earth-syst-sci.net/20/39/2016/ Hydrol. Earth Syst. 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J. and Hendry, J. W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability Any differences between K values at various scales are important for indicating the possibility of preferential ﬂow through heterogeneous strata or aquitard defects (e.g. faults and fractures). The availability of these bounded estimates helps to constrain the uncertainty analyses conducted on re- gional groundwater ﬂow models to yield more conﬁdent pre- dictions (Gerber and Howard, 2000). Nevertheless, regional groundwater ﬂow models generally use hydraulic resistance (leakance) values to transfer water vertically between aquifers (Barnett et al., 2012) rather than spatial discretisation of aquitards that control this transfer. While this simpliﬁcation is justiﬁed in many models, such an approach is not capable of identifying rapid ﬂow pathways through defects in the aquitards or the release of stored water from an aquitard to an aquifer and cannot resolve the ver- tical hydraulic head distribution across the aquitard to ver- ify drawdown responses. An aquitard should be subdivided into at least three thinner layers to effectively model tran- sient pressure responses (Barnett et al., 2012). Rather than assigning constant theoretical values for aquitard properties through these multiple layers a combination of realistic and rapid laboratory measurement and direct in situ measure- ments could improve conﬁdence in conceptual understanding and model predictions. www.hydrol-earth-syst-sci.net/20/39/2016/ W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 52 W. A. Timms et al.: Accelerated gravity testing of aquitard core permeability 53 Potter, P. E., Maynard, J. B., and Pryor, W. 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https://openalex.org/W2986164387	http://addi.ehu.es/bitstream/10810/41860/1/s41398-019-0647-7.pdf	English	null	Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics	Translational psychiatry	2,019	cc-by	9,880	The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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/. Abstract A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at ﬁrst episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we conﬁrmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efﬁcacy. classiﬁed into typical (i.e., haloperidol) and atypical (i.e., aripiprazole, risperidone or clozapine), the latter pre- scribed as ﬁrst-line drugs and/or in refractory patients4. Despite the effectiveness of APDs in the clinical realm, there is a marked disparity among patients with respect to symptoms, responses and side effects5,6. Whereas 50–60% of patients achieve an optimal degree of clinical improvement of positive symptoms, little to no improvement of negative symptoms or cognitive deﬁcits is common5. Overall, estimates suggest that one-ﬁfth to one-half of patients have treatment-resistant SCZ and about 30–60% of these respond to clozapine6–8. These Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics Fulgencio Ruso-Julve 1,2,3, Ana Pombero3,4, Fuencisla Pilar-Cuéllar3,5,6, Nuria García-Díaz 2,7, Raquel Garcia-Lopez3,4, María Juncal-Ruiz3,8, Elena Castro3,5,6, Álvaro Díaz3,5,6, Javier Vazquez-Bourgón1,3, Agustín García-Blanco 2,7, Emilio Garro-Martinez3,5,6, Helena Pisonero2,7, Alicia Estirado3,4, Rosa Ayesa-Arriola1,3, Juan López-Giménez9, Federico Mayor Jr.10,11, Elsa Valdizán3,5,6, Javier Meana 3,12, Javier Gonzalez-Maeso13, Salvador Martínez3,4, José Pedro Vaqué2,7 and Benedicto Crespo-Facorro 1,3,14 A R T I C L E O p e n A c c e s s Ruso-Julve et al. Translational Psychiatry (2019) 9:306 https://doi.org/10.1038/s41398-019-0647-7 Ruso-Julve et al. Translational Psychiatry (2019) 9:306 https://doi.org/10.1038/s41398-019-0647-7 Translational Psychiatry Translational Psychiatry Correspondence: José Pedro Vaqué (jose.vaque@unican.es) or Benedicto Crespo-Facorro (crespob@unican.es) 1Department of Psychiatry, University Hospital Marqués de Valdecilla-IDIVAL, Santander 39011 Cantabria, Spain 2Department of Molecular Biology, School of Medicine, University of Cantabria, Santander 39011 Cantabria, Spain Full list of author information is available at the end of the article. These authors contributed equally: José Pedro Vaqué, Benedicto Crespo-Facorro Human samples and study setting Human samples for this study were obtained from an ongoing epidemiological and 3-year longitudinal inter- vention programme of ﬁrst-episode psychosis (PAFIP) conducted at the University Hospital Marques de Valde- cilla (Cantabria, Spain) and biological samples were pro- vided by the IDIVAL biobank. The study was approved by the Cantabria Ethics Institutional Review Board, con- forming to international standards for research ethics. Patients meeting inclusion criteria and their families provided written informed consent to be included in the PAFIP. A new independent set of 30 APD-naïve, ﬁrst-episode non-affective individuals and 10 healthy individuals (without a history of neuropsychiatric disorders) were used to validate gene expression proﬁles related to clinical response (Table 1). A new paradigm has emerged based on the investigation of transcriptional patterns associated with clinical responses in SCZ, raising the likelihood of revealing unknown molecular mechanisms associated with anti- psychotic action that may be crucial in accomplishing optimal clinical responses16,17. Using an unbiased tran- scriptome approach, we have previously described a set of genes that are differentially expressed at the onset of SCZ16. In addition to this, we also showed signiﬁcant differential gene expression elicited after a 3-month APD treatment in responder patients17. Among the above- mentioned results, ADAMTS2 was the highest upregu- lated gene in SCZ patients at the onset of the disease and also was the most signiﬁcant downregulated gene, back to ‘healthy levels’, in responder patients to treatment with APDs. The observation that the expression of ADAMTS2 can be modulated by APDs and its association with clinical response, makes it an appealing candidate to investigate its interface with illness pathology and clinical efﬁcacy17. Brieﬂy, all patients underwent a head-to-head risper- idone and aripiprazole randomized (simple randomization procedure), ﬂexible-dose, open-label study (EudraCT number 2013-005399-16). More detailed information about PAFIP and treatment protocol has been published elsewhere17,18 and also is brieﬂy available in Supplemen- tary Information. RNA extraction Peripheral blood from patients and controls was extracted using the TempusTM Blood RNA Tube (Applied Biosystems, UK). Peripheral blood mononuclear cell (PBMC) isolation was performed using a Ficoll-Paque Premium reagent (Sigma) and total mRNA isolation using a TempusTM Spin RNA Isolation Kit (Invitrogen, CA, USA), following the manufacturer’s instructions. RNA Integrity Number (RIN) was characterized with a Bioa- nalyzer (Agilent Technologies) and samples with a RIN > 8 were selected. Total mRNA from culture cells was extracted using Trizol reagent (Invitrogen) and its con- centration was determined using a Nanodrop 2000 spec- trophotometer (Thermo Scientiﬁc, IL, USA). In the present work we initially performed an inde- pendent validation using a new cohort of drug-naïve non- affective psychosis patients. We conﬁrmed that ADAMTS2 expression was signiﬁcatively overexpressed at diagnosis, and that treatment with APDs reduced their expression levels back to ‘healthy’ values, which, in turn, was associated with clinical responses (positive symp- toms). However, the molecular or cellular mechanisms exerted by APDs to regulate ADAMTS2 expression are unknown. Following a bottom-up strategy starting from the transcriptional data obtained in SCZ cases and taking advantage of neuronal-like cells towards the identiﬁcation Introduction Antipsychotic drugs (APDs) remain the standard pharmacological treatment for schizophrenia (SCZ) and psychotic disorders, mainly by targeting dopamine neu- rotransmission (primarily D2 receptors)1–3. APDs can be e ut o (s) 0 9 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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/. Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 2 of 12 evidences suggest that although D2 receptors is a direct target for all drugs used to treating SCZ, its blockade may not tackle the primary biological anomaly in a signiﬁcant percentage of patients9. In this respect, the combined D1 and D2 receptors antagonism has been proposed to have synergic effects, which could account for the atypical clinical effectiveness of clozapine10. From a molecular perspective, the effects of APDs may also include the modulation of D2 receptor-independent mechanisms through indirect effects, such as, e.g., those related to other metabotropic receptors implicated in the control cAMP-dependent signalling11 and/or those related with receptor-biased agonism4,12. Downstream of cAMP, the activation of protein kinase A (PKA) promotes phos- phorylation of DARPP-32 (a cytosolic protein), which has been associated with the pathophysiology of SCZ13,14. In addition, the glutamate hypothesis of SCZ led to explore its potential clinical application as targets for therapy15. The investigation of the mechanisms downstream of the receptors targeted by APDs will certainly guide to ﬁnding out new explanations for pathophysiological mechanisms and novel approaches for therapy4. of membrane receptors and the intracellular mechanism controlled by them, we herein provide new evidence that ADAMTS2 expression is regulated by dopaminergic sig- nalling cascade (D1-class receptors) through ERK and cAMP/cAMP response element-binding protein (CREB) activities. RT-PCR and quantitative PCR aracteristics at baseline, at 3 months and clinical changes during the follow-up period. le and risperidone. BPRS Brief Psychiatric Rating Scale, CDSS Calgary Depression Rating Scale for Schizophrenia, CGI Clinical Global Impression, SANS Scale for the Assessment of Negative Symptoms, SAPS Scale for the Assessment of Positive Symptoms, YMRS Young Mania Rating Scale aWilcoxon matched-pairs signed-rank test bPaired Student’s t-test cComparison between aripiprazole and risperidone at baseline; unpaired Student’s t-test dComparison between aripiprazole and risperidone following the antipsychotic treatment, using the total score of the clinical scales at baseline as covariate; analysis f i (ANCOVA) ADAMTS2, CREB1, and C-FOS were designed using Primer-Blast (NCBI; see sequences in Supplementary Table S1). ACTB expression was used to normalize values. Gene expression changes were determined using 2(–ΔΔCt) formula. A melting curve was generated for every run to conﬁrm assay speciﬁcity. were cultured in modiﬁed Eagle’s medium and Dulbecco’s Modiﬁed Eagle’s Medium, respectively (Corning, VA, USA). Culture medium was supplemented with 10% dia- lysed fetal bovine serum (dFBS; HyClone, UT, USA), glucose, L-glutamine, streptomycin sulphate and potas- sium penicillin (10,000 U/L) (Lonza, Belgium). RT-PCR and quantitative PCR cDNA synthesis was performed using the SuperScript IV Reverse Transcriptase (Invitrogen). cDNA was ampli- ﬁed using the Power SYBR™Green PCR Master Mix (Applied Biosystems) in a 7300 Fast Real-Time PCR System (Applied Biosystems). Speciﬁc oligos for human Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 3 of 12 Table 1 Psychopathological characteristics at baseline, at 3 months and clinical changes during the follow-up period. Comparison between aripiprazole and risperidone. Total (N = 30) Statistics p Aripiprazole (n = 16) Risperidone (n = 14) Statistics p Mean SD Mean SD Mean SD CGI Baseline 6.5 0.6 6.56 0.51 6.43 0.65 t = 0.63c 0.532 3 Months 1.8 1.2 1.63 1.20 2.00 1.24 3-Month change from baseline −4.7 1.2 z = 4.83a 0.000 −4.90 0.30 −4.47 0.33 F = 0.96d 0.336 YMRS Baseline 13.8 6.2 14.25 7.24 13.29 5.01 t = 0.42c 0.679 3 Months 1.2 2.3 1.56 2.63 0.79 1.93 3-Month change from baseline −12.6 6.5 z = 4.78a 0.000 −12.25 0.59 −13.00 0.63 F = 0.75d 0.394 CDSS Baseline 1.5 3.0 2.06 3.82 0.86 1.75 t = 1.08c 0.288 3 Months 1.2 2.3 1.19 2.34 1.14 2.25 3-Month change from baseline −0.3 3.7 z = 0.35a 0.724 −0.34 0.59 −0.32 0.63 F = 0.00d 0.985 BPRS Baseline 70.3 15.0 69.06 16.08 71.85 13.95 t = −0.49c 0.627 3 Months 29.9 7.1 29.06 7.07 30.92 7.48 3-Month change from baseline −40.4 15.2 t = 14.32b 0.000 −41.13 1.81 −39.53 2.01 F = 0.35d 0.561 SAPS Baseline 15.9 4.2 15.63 3.50 16.07 4.94 t = −0.29c 0.775 3 Months 0.6 1.7 0.69 1.89 0.71 1.49 3-Month change from baseline −15.4 4.3 t = 19.28b 0.000 −15.14 0.44 −15.12 0.47 F = 0.00d 0.975 SANS Baseline 4.8 6.9 3.47 6.56 5.93 7.43 t = −0.95c 0.352 3 Months 3.9 6.6 4.00 6.47 4.93 7.04 3-Month change from baseline −0.9 5.6 z = 1.14a 0.255 −0.07 1.54 −0.36 1.59 F = 0.02d 0.899 BPRS Brief Psychiatric Rating Scale, CDSS Calgary Depression Rating Scale for Schizophrenia, CGI Clinical Global Impression, SANS Scale for the Assessment of Negative Symptoms, SAPS Scale for the Assessment of Positive Symptoms, YMRS Young Mania Rating Scale aWilcoxon matched-pairs signed-rank test bPaired Student’s t-test cComparison between aripiprazole and risperidone at baseline; unpaired Student’s t-test d Table 1 Psychopathological characteristics at baseline, at 3 months and clinical changes during the follow-up period. Comparison between aripiprazole and risperidone. Cell culture and treatments Human neuroblastoma (SK-N-SH, ATCC HTB-11) and 293T (ATCC CRL-3216) cells were obtained from the American Type Cell Collection (Rockville, MD). Cells Aripiprazole, clozapine, haloperidol hydrochloride, H89 dihydrochloride, L 741,626, MDL 100907, paliperidone, risperidone, SKF 83822, SCH 39166, TCB-2, WAY Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 4 of 12 CREB1 shRNA expression vectors or scrambled control (Origene, MD, USA). Transfected cells were selected with puromycin (1 μg/mL) at least 7 days. 100635, 7-OH-DPAT and 8-OH-DPAT were purchased from Tocris Bioscience (Spain). Forskolin and selumetinib (AZD6244) were purchased from Selleckchem (Spain). Cholera toxin (CTX), pertussis toxin (PTX) and 12-O- tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma-Aldrich (MO, USA). YM-254890 was pur- chased from Adipogen Life Sciences (CA, USA). All were dissolved in dimethyl sulfoxide, except CTX and WAY 100635 that were dissolved in water. Luciferase report assays Luciferase report assays were performed by transfection with 0.5 µg DNA of the following plasmids mix (ratio 3:1): pGL4.29[luc2P/CRE/Hygro] containing ﬁreﬂy luciferase reporter, alongside pRL-Null containing Renilla luciferase used as control (Promega, WI, USA). Cells were trans- fected with Lipofectamine LTX with PLUS reagents (Invitrogen) in transient conditions. Fireﬂy and Renilla levels were detected using Dual-Luciferase Reporter Assay System kit and quantiﬁed using a GloMax-Multi appara- tus (Promega). Wilcoxon’s signed-rank test for independent data was used to compare the ADAMTS2 mRNA expression level among healthy volunteers and drug-naïve patients at baseline. Wilcoxon’s matched-pairs signed-rank test for paired data was performed to compare the change in the mRNA expression level from baseline to 3 months fol- lowing the antipsychotic treatment (α = 0.05). STATA 15.1 was used for statistical analysis. Statistical analyses Kolmogorov–Smirnov test and Levene test were used to test normality and equality of variances, respectively. For patients’ studies, to ensure group comparability between healthy volunteers and patients, socio- demographic and clinical characteristics at baseline were tested by unpaired Student’s t-test or one-way analysis of variance (ANOVA) for continuous variables as necessary, and by Fisher’s exact test for qualitative variables (Table 1 and Supplementary Table S2). ADAMTS2 expression is controlled by APD treatment in SCZ patients To better evaluate the mechanistic effects of atypical APDs in SCZ patients, we prepared and studied a new cohort of 30 drug-naïve SCZ patients with baseline quantitative data (sociodemographic and clinical char- acteristics) and clinical follow-up data, after 3 months of treatment with APDs (Table 1). We found signiﬁcant within-subject changes between baseline data and after 3 months of treatment in both risperidone and aripipra- zole groups. These changes were observed in CGI (Clin- ical Global Impression, p < 0.000), YMRS (Young Mania Rating Scale, p < 0.000), BPRS (Brief Psychiatric Rating Scale, p < 0.000) and SAPS (Scale for the Assessment of Positive Symptoms, p < 0.000) total scores. However, as it is shown in Table 1, no differences were found in total scores of the clinical scales when the two APD groups were compared at baseline and after 3 months of treatment. Western blotting For in vitro studies, unless otherwise speciﬁed, all experiments were independent and numerical data were summarized as the mean ± SEM using GraphPad Prism6 software. Each global mean was compared using unpaired Student’s t-test (two tailed; α = 0.05) or one-way ANOVA followed by post-hoc test where appropriated, as described in each ﬁgure legend. Cells were starved overnight before treatment. Whole cell lysates were obtained using RIPA buffer (Sigma) supplemented with phosphatase and protease inhibitors (Roche, Germany). Protein expression was analysed by western blotting as described previously19. Brieﬂy, anti- bodies used were as follows: phospho-CREB, CREB, phospho-ERK1/2, ERK1/2, phospho-p38, p38, and phospho-PKA substrates (Cell Signaling, MA, USA), and β-Tubulin (Santa Cruz Biotechnology). Fluorophore conjugate antibodies were obtained from Invitrogen. Signals were visualized and recorded with an Odyssey Infrared Imaging scanner (LI-COR Biosciences, NE, USA). Immunoblot densitometry analysis on every band was calculated using Image Studio Software (LI-COR Biosciences). Phosphorylation and total protein densito- metry values were normalized to β-Tubulin signal. CREB knocked down reagents and procedures Relative ADAMTS2 mRNA expression level in SK-N-SH cells incubated with clozapine (1 µM) (N = 4), haloperidol (1 µM) (N = 5), paliperidone (1 µM) (N = 3) and aripiprazole (1 µM) (N = 5) for the indicated times. Data are mean ± SEM; one-way ANOVA for multiple comparations: p < 0.05, p < 0.01, p < 0.001 shows signiﬁcance with respect to control (C; vehicle, grey bars). context, using quantitative reverse transcriptase PCR (RT- qPCR), we performed an independent validation of tran- scriptional changes by focusing on ADAMTS2. Thus, we conﬁrmed ADAMTS2 as a signiﬁcant gene that was highly overexpressed at the onset, with respect to healthy con- trols, and downregulated by APDs after 3 months of treatment (Fig. 1). fast and sustained (up to 24 h) inhibition of ADAMTS2 expression (Fig. 2). fast and sustained (up to 24 h) inhibition of ADAMTS2 expression (Fig. 2). CREB knocked down reagents and procedures Relative mRNA expression level of ADAMTS2 gene in PBMCs from peripheral blood samples of an independent cohort of SCZ patients: healthy controls (basal; grey bar), untreated FEP patients (basal drug-naïve; black bar) and 3-month treated patients (striped bar). FEP: ﬁrst episode of psychosis. All patients were treated with risperidone or aripiprazole drugs and showed clinical response to treatment (Cohort: patients N = 30 and controls N = 10). Data are mean ± SEM; unpaired Wilcoxon’s signed-rank test: p < 0.001 compares patients at baseline vs. healthy controls; paired Wilcoxon’s signed-rank test: ###p < 0.001 compares patients at baseline vs. those after 3-month AP treatment. Fig. 1 Transcriptional control of ADAMTS2 in SCZ patients at onset and after 3-month antipsychotic treatment. Relative mRNA expression level of ADAMTS2 gene in PBMCs from peripheral blood samples of an independent cohort of SCZ patients: healthy controls (basal; grey bar), untreated FEP patients (basal drug-naïve; black bar) and 3-month treated patients (striped bar). FEP: ﬁrst episode of psychosis. All patients were treated with risperidone or aripiprazole drugs and showed clinical response to treatment (Cohort: patients N = 30 and controls N = 10). Data are mean ± SEM; unpaired Wilcoxon’s signed-rank test: p < 0.001 compares patients at baseline vs. healthy controls; paired Wilcoxon’s signed-rank test: ###p < 0.001 compares patients at baseline vs. those after 3-month AP treatment. g y p g and 3-month treated patients (striped bar). FEP: ﬁrst episode of psychosis. All patients were treated with risperidone or aripiprazole drugs and showed clinical response to treatment (Cohort: patients N = 30 and controls N = 10). Data are mean ± SEM; unpaired Wilcoxon’s signed-rank test: p < 0.001 compares patients at baseline vs. healthy controls; paired Wilcoxon’s signed-rank test: ###p < 0.001 compares patients at baseline vs. those after 3-month AP treatment. Fig. 2 Transcriptional control of ADAMTS2 using antipsychotic Fig. 2 Transcriptional control of ADAMTS2 using antipsychotic drugs. Relative ADAMTS2 mRNA expression level in SK-N-SH cells incubated with clozapine (1 µM) (N = 4), haloperidol (1 µM) (N = 5), paliperidone (1 µM) (N = 3) and aripiprazole (1 µM) (N = 5) for the indicated times. Data are mean ± SEM; one-way ANOVA for multiple comparations: p < 0.05, p < 0.01, p < 0.001 shows signiﬁcance with respect to control (C; vehicle, grey bars). Fig. 2 Transcriptional control of ADAMTS2 using antipsychotic drugs. CREB knocked down reagents and procedures ShCREB1-inducible SK-N-SH cells were generated by lentiviral infection of SK-N-SH cells with SMARTvector carrying tGFP and human inducible CREB1 short hairpin RNA (shRNA) mCMV constructs or non-target control shRNA (Dharmacon, CO, USA). Lentiviral particles were produced by co-transfection of 293T cells using Trans- Lentiviral shRNA Packaging System (Dharmacon), according to the manufacturer’s protocol. Cells were incubated with doxycycline (1 μg/ml) (Sigma) for 72 h to induce green ﬂuorescent protein and shRNA expression. SK-N-SH cells expressing stable shCREB1 constructs were generated by direct transfection by Lipofectamine LTX with PLUS reagent (Invitrogen) using pGFP-V-RS- To analyse transcriptomic changes, we obtained PBMCs isolated from blood samples from SCZ patients, which were obtained at the ﬁrst episode of psychosis (onset) and after a 3-month period of treatment with APDs. In this Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 5 of 12 Fig. 1 Transcriptional control of ADAMTS2 in SCZ patients at onset and after 3-month antipsychotic treatment. Relative mRNA expression level of ADAMTS2 gene in PBMCs from peripheral blood samples of an independent cohort of SCZ patients: healthy controls (basal; grey bar), untreated FEP patients (basal drug-naïve; black bar) and 3-month treated patients (striped bar). FEP: ﬁrst episode of psychosis. All patients were treated with risperidone or aripiprazole drugs and showed clinical response to treatment (Cohort: patients N = 30 and controls N = 10). Data are mean ± SEM; unpaired Wilcoxon’s signed-rank test: *p < 0.001 compares patients at baseline vs. healthy controls; paired Wilcoxon’s signed-rank test: ###p < 0.001 compares patients at baseline vs. those after 3-month AP treatment. fast and sustained (up to 24 h) inhibition of ADAMTS2 expression (Fig. 2). Fig. 2 Transcriptional control of ADAMTS2 using antipsychotic drugs. Relative ADAMTS2 mRNA expression level in SK-N-SH cells incubated with clozapine (1 µM) (N = 4), haloperidol (1 µM) (N = 5), paliperidone (1 µM) (N = 3) and aripiprazole (1 µM) (N = 5) for the indicated times. Data are mean ± SEM; one-way ANOVA for multiple comparations: p < 0.05, p < 0.01, p < 0.001 shows signiﬁcance with respect to control (C; vehicle, grey bars). Fig. 1 Transcriptional control of ADAMTS2 in SCZ patients at Fig. 1 Transcriptional control of ADAMTS2 in SCZ patients at onset and after 3-month antipsychotic treatment. Relative mRNA Fig. 1 Transcriptional control of ADAMTS2 in SCZ patients at onset and after 3-month antipsychotic treatment. Dynamic control of ADAMTS2 transcription by APDs in neuronal-like cells b ADAMTS2 mRNA levels by RT-qPCR in cells incubated for 1 h with SKF 83822 (N = 4) and pre- incubated also for 30 min with SCH 39166 (N = 4), clozapine (N = 3), haloperidol (N = 4), paliperidone (N = 4) or aripiprazole (N = 4) (Drug concentration 1 µM). c CREB activity in cells transfected with CRE-Luc alongside pRL-Null: cells were pre-incubated for 1 h with the indicated APDs and then, incubated for 24 h with SKF 82833 (10 µM) (N = 4). d ADAMTS2 mRNA levels by RT-qPCR: SK-N-SH cells were pre-incubated for 30 min with MAPK/ERK and cAMP-PKA inhibitors (selumetinib 1 µM N = 6 and H89 10 µM N = 4, respectively) and then, incubated for 1 h with SKF 82833 (1 µM) (N = 5). e CREB activity in cells transfected with CRE-Luc alongside pRL-Null (N = 4): SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 24 h with SKF 82833 (10 µM). f Western blottings showing relative phosphorylation levels of CREB and ERK1/2: SK-N- SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 15 min with SKF 82833 (1 µM) (N = 3). Blots are representative images of each western-blot. Data are mean ± SEM; Student’s t-test: p < 0.05 and **p < 0.001 vs. control condition (vehicle), and #p < 0.05, ##p < 0.01, ###p < 0.001 vs. SKF 83822 condition. cells, ADAMTS2 was speciﬁcally localized in brain regions that are part of the mesolimbic and mesocortical dopamine systems (Supplementary Fig. S2). demonstrate that preincubation with clozapine, but not haloperidol, paliperidone, or aripiprazole, prevented SKF 83822-induced CREB activation using a speciﬁc reporter assay. Next, we studied whether treatment with APDs could modulate ADAMTS2 gene expression activated down- stream of the D1 receptor. Our results show that haloper- idol and clozapine could signiﬁcantly block SKF 83822- mediated transcriptional activation of ADAMTS2. As expected, SCH 39166, a selective D1 receptor antagonist, also prevented D1 receptor-mediated activation of ADAMTS2 (Fig. 3b). In addition, the reduction in ADAMTS2 expression induced by clozapine was prevented by L 741,626 and MDL 100907 (a D2 and 5-HT2A receptor antagonists, respectively) (Supplementary Fig. S3). Dynamic control of ADAMTS2 transcription by APDs in neuronal-like cells To gain mechanistic insight into the transcriptional control of ADAMTS2, we analysed the effects of APDs in SK-N-SH cells using dFBS. No signiﬁcant differences in ADAMTS2 expression were observed when dFBS was not present in the culture medium (data not shown). These cells express cell surface receptors that are targets of APDs (i.e., D1, D2, 5-HT1A and 5-HT2A receptors) as well as detectable basal expression of ADAMTS2 mRNA. Our data showed that incubation with APDs in these cells induced dynamic changes in ADAMTS2 mRNA expres- sion along time. Incubation with atypical APDs such as paliperidone, aripiprazole (Fig. 2) and risperidone (Sup- plementary Fig. S1), provoked a dynamic inhibition of ADAMTS2 basal mRNA expression in these cells with fast (<1 h) but transitory responses. Interestingly, clozapine (atypical APD) and haloperidol (typical APD) induced a Using selective agonists for D1, D2, 5-HT1A and 5-HT2A receptors in SK-N-SH cells, we observed that SKF 83822 (a D1-class receptor agonist) signiﬁcantly triggered ADAMTS2 mRNA expression compared with the lack of effect of 7-OH-DPAT, 8-OH-DPAT and TCB-2 (agonists of D2, 5-HT1A and 5-HT2A receptors, respectively) (Fig. 3a). In addition, using selective antagonists for these receptors, we observed that only SCH 39166 (a D1-class receptor antagonist) signiﬁcantly downregulated ADAMTS2 mRNA expression, whereas L 741,626, WAY 100635 and MDL 100907 (antagonists of D2, 5-HT1A and 5-HT2A receptors, respectively) were devoid of this effect (Fig. 3a). Furthermore, we analysed ADAMTS2 mRNA and protein expression proﬁles in the brain of developing and early postnatal mice (see Supplementary Methods). Interestingly and in support of our previous results in Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 6 of 12 Fig. 3 (See legend on next page.) Page 7 of 12 Ruso-Julve et al. Translational Psychiatry (2019) 9:306 (see ﬁgure on previous page) Fig. 3 Neurotransmitter receptors and associated signalling pathways involved in the control of ADAMTS2 gene expression. a ADAMTS2 mRNA levels by RT-qPCR in SK-N-SH cells incubated 1 h with the indicated selective receptor agonist (red bars): SKF 83822 (D1-class receptors) (N = 5), 7-OH-DPAT (D2-class receptors) (N = 3), 8-OH-DPAT (5-HT1A receptor) (N = 3) and TCB-2 (5-HT2A/2C receptors) (N = 5); and selective antagonist (blue bars): SCH 39165 (D1-class receptors) (N = 3), L 741,626 (D2-class receptors) (N = 4), WAY 100635 (5-HT1A receptors) (N = 3) and MDL 100907 (5-HT2A receptors) (N = 3) (Drug concentration 1 µM). Dynamic control of ADAMTS2 transcription by APDs in neuronal-like cells To evaluate the potential contribution of PKA and ERK to the activation of ADAMTS2 expression, we incubated SK-N-SH cells with SKF 83822 D1 receptor agonist in combination with selective MAPK/ERK (selumetinib- AZD6244) and cAMP/PKA (H89 dihydrochloride) inhi- bitors. Both inhibitors prevented D1 receptor induced expression of ADAMTS2 (Fig. 3d), although only a cAMP/ PKA inhibitor abrogated CREB-dependent transcription and phosphorylation (Fig. 3e, f). Transcriptional mechanisms controlling ADAMTS2 gene expression downstream of dopamine D1 receptors As D1 receptors are known to signal through Gαs, we sought to conﬁrm its role at controlling ADAMTS2 expression in our system, alongside Gαi and Gαq. For this purpose, we incubated SK-N-SH cells with CTX (Gαs activator), PTX (Gαi inhibitor) and YM-254890 (a speciﬁc Gαq inhibitor)20, before activating D1 receptors with SKF 83822. The activation of Gαs (Supplementary Fig. S4A) and the inhibition of Gαi (Supplementary Fig. S4B) per se signiﬁcantly upregulated ADAMTS2 expression. More- over, the inhibition of Gαi potentiated ADAMTS2 expression after D1 receptor activation (Supplementary Fig. S4B). Finally, the inhibition of Gαq did not modify the basal and the SKF 83822-mediated increase of ADAMTS2 expression (Supplementary Fig. S4C). p p 1 p Our previous data suggest that there is a rapid activa- tion of the signalling mechanisms controlling CREB activation after D1 receptor activation that can be modulated pharmacologically. We decided to challenge the contribution of cAMP/CREB signalling to the tran- scriptional activation of ADAMTS2 (Fig. 4a). We initially evaluated the activation of ADAMTS2 gene expression together with that of C-FOS, an ‘early response gene’ known to be a direct transcriptional target of CREB that was used as control. Our data showed that SKF 83822 activated mRNA expression of both ADAMTS2 and C- FOS. Interestingly, incubation with forskolin alone (a selective adenyl cyclase activator) was sufﬁcient to trigger the transcription of both genes, whereas TPA (a PKC/ MAPK activator) uniquely activated C-FOS transcription. To explore the implication of CREB in D1 receptor- mediated ADAMTS2 expression, we generated stable SK- N-SH cells with inducible expression of control or CREB1 shRNAs upon addition of doxycycline to the culture media. Using this approach, we reduced CREB1 mRNA and protein expression, as shown in Fig. 4b. Under these settings, SKF 83822 and forskolin failed to induce the expression of ADAMTS2 (Fig. 4c). Interestingly, SKF Incubation of neuronal-like cells with the selective D1 receptor agonist (SKF 83822) provoked rapid phosphor- ylation of CREB and ERK1/2 (15 min), and no changes in p38 (Supplementary Fig. S4D-F). In addition, rapid phosphorylation of PKA substrates was detected in response to SKF 83822 (Supplementary Fig. S4G). To evaluate CREB-dependent transcriptional activity in neuronal-like cells, we took advantage of a CRE-luciferase reporter. We analysed if APDs could counteract the intracellular signalling activation mediated by selective SKF 83822 D1-class receptor agonist. Our results (Fig. Transcriptional mechanisms controlling ADAMTS2 gene expression downstream of dopamine D1 receptors 3c) To explore the implication of CREB in D1 receptor- mediated ADAMTS2 expression, we generated stable SK- N-SH cells with inducible expression of control or CREB1 shRNAs upon addition of doxycycline to the culture media. Using this approach, we reduced CREB1 mRNA and protein expression, as shown in Fig. 4b. Under these settings, SKF 83822 and forskolin failed to induce the expression of ADAMTS2 (Fig. 4c). Interestingly, SKF Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 8 of 12 Page 8 of 12 83822 did not signiﬁcantly activate C-FOS expression in CREB1 knocked down cells when compared with control cells; in contrast, TPA did (Fig. 4d). A schematic repre- sentation of the signalling pathways and the molecules used in this work is illustrated in Fig. 5 for explanatory purposes. The above-mentioned results were further conﬁrmed using SK-N-SH cells stably expressing control or CREB1 shRNAs (Supplementary Fig. S5). Mechan- istically, reduced CREB expression did not impair ERK phosphorylation elicited downstream of D1 receptor activation by SKF 83822 (Fig. 4e). In this context, we also used forskolin and TPA as controls for selective activation of CREB and ERK, respectively (Fig. 4e and Supplemen- tary Fig. S6). Thus, activation of CREB seems to play a key role in the control of ADAMTS2 gene expression down- stream of D1 receptors. 83822 did not signiﬁcantly activate C-FOS expression in CREB1 knocked down cells when compared with control cells; in contrast, TPA did (Fig. 4d). A schematic repre- sentation of the signalling pathways and the molecules used in this work is illustrated in Fig. 5 for explanatory purposes. The above-mentioned results were further conﬁrmed using SK-N-SH cells stably expressing control or CREB1 shRNAs (Supplementary Fig. S5). Mechan- istically, reduced CREB expression did not impair ERK phosphorylation elicited downstream of D1 receptor activation by SKF 83822 (Fig. 4e). In this context, we also used forskolin and TPA as controls for selective activation of CREB and ERK, respectively (Fig. 4e and Supplemen- tary Fig. S6). Thus, activation of CREB seems to play a key role in the control of ADAMTS2 gene expression down- stream of D1 receptors. investigations. In this respect, clozapine has shown superior efﬁcacy for treatment-resistant and suicidality, as well as its apparent ability to decrease substance use in SCZ24. Transcriptional mechanisms controlling ADAMTS2 gene expression downstream of dopamine D1 receptors Thus, ADAMTS2 might play a key, and yet to be deﬁned, mechanistic role in both the illness onset and clinical responses, regardless of the type of APD used. clinical responses, regardless of the type of APD used. There is scarce information regarding speciﬁc ADAMTS2 expression and activity in the CNS. Therefore, we analysed the expression proﬁles of ADAMTS2 mRNA and protein in mice. Interestingly, these were speciﬁcally localized in brain regions that are part of the mesolimbic and mesocortical dopamine systems (i.e., the dentate gyrus in the hippocampus and the ventral tegmental area21,22,25. At a prenatal stage (E18.5), ADAMTS2 protein was mapped in the neuropiles of anterior brain structures, anterior cingulate cortex, superﬁcial striatum and lateral septum, where neuronal expression of dopamine D1 and D2 receptors was detected by in situ hybridization (http://developingmouse.brain-map.org/). The hypothesis that dopamine and dopaminergic mechanisms are essential to psychosis, and particularly to SCZ, has been one of the most enduring ideas about this disorder26. Elevated presynaptic striatal dopamine correlates most closely with the symptom dimension of psychosis and blockade of this heightened transmission leads to a resolution of symptoms for most patients27. Nonetheless, the functional association between ADAMTS2 and the dopaminergic system has not been previously established, to our best knowledge. ADAMTS2 is a member of the ADAM Metallopeptidase with Thrombospondin family28,29, with a number of targets such as the N-propeptides of procollagens I–III, ﬁbronectin, decorin and Dkk3 participating in extracellular matrix (ECM) organization, as well as in transforming growth factor (TGF)- β and WNT signalling30,31. Alterations in the ECM as well as TGF-β and WNT signalling have been associated with SCZ32–34. Moreover, deregulated mRNA expression of MMPs (MMP-16, -24 and -25) and ADAMTS (ADAMTS-1, -6 and -8) families of proteases have also been reported in SCZ35,36. Thus, considering ADAMTS2 expression and its associated activities in the CNS, as well as the data presented in this work, it is conceivable to speculate that it could participate in SCZ at different stages of the disease. In this regard, a selective D1-class receptor agonist (SKF 83822) signiﬁcantly activated ADAMTS2 expression, and haloperidol and clozapine blocked this activation in neuronal-derived cells. It will be of interest, to address these appealing ques- tions, within the next future, using SCZ and/or ADAMTS2 transgenic mice models. Discussion Our results revealed that treatment with APDs control ADAMTS2 expression, which is directly associated with dopaminergic signalling, primarily with the D1-class receptors and downstream through cAMP/CREB and MAPK signalling. Interestingly, ADAMTS2 mRNA and protein were speciﬁcally found in mesolimbic and meso- cortical dopaminergic regions in mice. Our data suggest that D1 receptor signalling towards CREB activation and its effects on ADAMTS2 expression may be linked to key biological mechanisms in SCZ and the clinical response to APDs21,22. In the context of the local PAFIP programme, we collected a new cohort of 30 drug-naïve SCZ patients with associated clinical data and follow-up. Using these cases alongside ‘healthy’ controls, we attempted an independent validation of our previous results using a different cohort of cases16,17. In this cohort, we con- ﬁrmed ADAMTS2 as a signiﬁcant gene that was highly overexpressed at baseline in PBMCs from drug-naïve SCZ individuals and which returned to ‘normal’ levels in clinical responders treated with APDs (clinical rate scales in Table 1). Although, there is not always a direct correlation between the biomarkers obtained in per- ipheral blood cells and the central nervous system (CNS) samples, a previous study in a rodent model of SCZ have shown a certain parallelism between the expression in human PBMCs16,17 and mouse frontal cortex samples23. Supporting the previously mentioned data, ADAMTS2 mRNA expression was quickly down- regulated by all APDs in neuronal-like cells. Moreover, whereas the activities of paliperidone, risperidone, and aripiprazole downregulating ADAMTS2 mRNA were transitory in such cells, those of clozapine and halo- peridol were sustained to up to 24 h. The molecular effects exerted by clozapine and haloperidol as com- pared with other APDs might deserve further D1 receptors are the most abundant dopaminergic receptor in CNS and their functional crosstalk with D2 receptor is well documented11,37,38. D1 receptor activate adenylyl cyclase (AC), which in turn regulate intracellular cAMP levels leading to PKA activation and CREB phos- phorylation39–41. Our data have shown that selective D1 receptor activation upregulated ADAMTS2 expression Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 9 of 12 Fig. 4 Transcriptional mechanisms that control ADAMTS2 gene expression downstream of dopamine D1-class receptors. Discussion a RT-qPCR showing ADAMTS2 and C-FOS mRNA expression level in SK-N-SH cells incubated for 1 h with SKF 83822 (1 µM; blue bars) (N = 6 and N = 4, respectively), forskolin (10 µM; red bars) (N = 3 and N = 4, respectively) and TPA (10 ng/ml; green bars) (N = 3). b CREB1 knockdown SK-N-SH cells by lentiviral inducible shRNA and GFP reporter construct (yellow bars), incubated with doxycycline (1 µg/ml) for 72 h, CREB1 mRNA level (up) and CREB protein total expression (down) in non-targeted control (NTC) or shCREB1 cells (N = 3). Inducible CREB1 Knockdown SK-N-SH cells were incubated for 1 h with SKF 83822 (1 µM, blue bars), forskolin (10 µM, red bars) and TPA (10 ng/ml, green bars): RT-qPCR showing ADAMTS2 (c) and C-FOS (d) mRNA d bl h d l ( ) ll b d h d l ( l) f h ( ) h Fig. 4 Transcriptional mechanisms that control ADAMTS2 gene expression downstream of dopamine D1-class receptors. a RT-qPCR showing ADAMTS2 and C-FOS mRNA expression level in SK-N-SH cells incubated for 1 h with SKF 83822 (1 µM; blue bars) (N = 6 and N = 4, respectively), forskolin (10 µM; red bars) (N = 3 and N = 4, respectively) and TPA (10 ng/ml; green bars) (N = 3). b CREB1 knockdown SK-N-SH cells by lentiviral inducible shRNA and GFP reporter construct (yellow bars), incubated with doxycycline (1 µg/ml) for 72 h, CREB1 mRNA level (up) and CREB protein total expression (down) in non-targeted control (NTC) or shCREB1 cells (N = 3). Inducible CREB1 Knockdown SK-N-SH cells were incubated for 1 h with SKF 83822 (1 µM, blue bars), forskolin (10 µM, red bars) and TPA (10 ng/ml, green bars): RT-qPCR showing ADAMTS2 (c) and C-FOS (d) mRNA expression in inducible shCREB1 or non-targeted control (NTC) cells incubated with doxycycline (1 µg/ml) for 72 h (N = 3-4). e Shows western blottings using anti-phospho-CREB and anti-phospho-ERK, as well as anti-CREB and anti-ERK antibodies: inducible shCREB1 or non-targeted control (NTC) SK-N-SH cells incubated with doxycycline (1 µg/ml) for 72 h and then incubated for 15 min with SKF 83822 (1 µM), forskolin (10 µM) and TPA (10 ng/ml) (N = 3). Inducible knockdown cells were selected with puromycin (1 µg/ml) at least 7 days. Discussion Two main pathways seem to be involved: (1) Gαs/AC/cAMP/ PKA signalling and (2) MEK/ERK1/2 signalling. Downstream of D1 both PKA and ERK can phosphorylate CREB at Ser133 and activate transcription of ADAMTS2. Speciﬁc activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Speciﬁc inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D1 (dopamine D1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction. Fig. 5 Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D1 receptors by Fig. 5 Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D1 receptors by SKF 83822 (selective D1 receptor agonist) triggers the expression of ADAMTS2. Two main pathways seem to be involved: (1) Gαs/AC/cAMP/ PKA signalling and (2) MEK/ERK1/2 signalling. Downstream of D1 both PKA and ERK can phosphorylate CREB at Ser133 and activate transcription of ADAMTS2. Speciﬁc activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Speciﬁc inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D1 (dopamine D1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction. transcription of ADAMTS2. Speciﬁc activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Speciﬁc inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D1 (dopamine D1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction. alongside a rapid phosphorylation of CREB and ERK pro- teins, which resulted in CREB-mediated transcriptional activity as detected by using speciﬁc reporter assays (Fig. 3). In support of this and in our system, ADAMTS2 tran- scription was speciﬁcally triggered by activation of D1 receptors (by SKF 83822), Gαs (using CTX) and AC (for- skolin). Thus, it is possible that, as part of the dopaminergic activity, ADAMTS2 could act as a major cAMP/CREB effector in SCZ. Interestingly, D1 receptor-mediated CREB activation was abrogated by clozapine (Fig. 3c), providing evidence that cAMP/CREB signalling, and therefore ADAMTS2 expression, can be modulated by APDs. We are highly indebted to the participants and their families for their cooperation in this study. We also thank IDIVAL biobank (Inés Santiuste and Jana Arozamena) for clinical samples and data as well as the PAFIP members (Marga Corredera) for the data collection. This work was supported by: SAF2016-76046-R and SAF2013-46292-R (MINECO and FEDER) to B.C.F., PI16/ 00156 (isciii and FEDER) to J.P.V., LUCHAMOS POR LA VIDA project to F.R.J. and J.P.V., SAF2017-83702-R (MINECO and FEDER), Red TERCEL RD12/0019/0024 (ISCIII) and GVA-PROMETEO 2018/041 (Generalitat Valenciana) to S.M. J.P.V. is Discussion Blots images are representative of independent experiments Data are mean ± SEM; Student’s t test: p < 0 05 p < 0 01 p < 0 001 vs vehicle or doxycycline ( ); and #p < 0 05 ###p < 0 001 vs Fig. 4 Transcriptional mechanisms that control ADAMTS2 gene expression downstream of dopamine D1-class receptors. a RT-qPCR showing ADAMTS2 and C-FOS mRNA expression level in SK-N-SH cells incubated for 1 h with SKF 83822 (1 µM; blue bars) (N = 6 and N = 4, respectively), forskolin (10 µM; red bars) (N = 3 and N = 4, respectively) and TPA (10 ng/ml; green bars) (N = 3). b CREB1 knockdown SK-N-SH cells by lentiviral inducible shRNA and GFP reporter construct (yellow bars), incubated with doxycycline (1 µg/ml) for 72 h, CREB1 mRNA level (up) and CREB protein total expression (down) in non-targeted control (NTC) or shCREB1 cells (N = 3). Inducible CREB1 Knockdown SK-N-SH cells were incubated for 1 h with SKF 83822 (1 µM, blue bars), forskolin (10 µM, red bars) and TPA (10 ng/ml, green bars): RT-qPCR showing ADAMTS2 (c) and C-FOS (d) mRNA expression in inducible shCREB1 or non-targeted control (NTC) cells incubated with doxycycline (1 µg/ml) for 72 h (N = 3-4). e Shows western blottings using anti-phospho-CREB and anti-phospho-ERK, as well as anti-CREB and anti-ERK antibodies: inducible shCREB1 or non-targeted control (NTC) SK-N-SH cells incubated with doxycycline (1 µg/ml) for 72 h and then incubated for 15 min with SKF 83822 (1 µM), forskolin (10 µM) and TPA (10 ng/ml) (N = 3). Inducible knockdown cells were selected with puromycin (1 µg/ml) at least 7 days. Blots images are representative of independent experiments. Data are mean ± SEM; Student’s t-test: p < 0.05, p < 0.01, *p < 0.001 vs. vehicle or doxycycline (−); and #p < 0.05, ###p < 0.001 vs. each condition in NTC cells. Ruso-Julve et al. Translational Psychiatry (2019) 9:306 Page 10 of 12 Page 10 of 12 Fig. 5 Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D1 receptors by SKF 83822 (selective D1 receptor agonist) triggers the expression of ADAMTS2. Two main pathways seem to be involved: (1) Gαs/AC/cAMP/ PKA signalling and (2) MEK/ERK1/2 signalling. Downstream of D1 both PKA and ERK can phosphorylate CREB at Ser133 and activate transcription of ADAMTS2. Discussion Our ﬁndings reveal the contribution of D1 receptors over ADAMTS2 transcription, and also indicate the participation of D2 and 5-HT2A receptors in the effect of clozapine in the control of ADAMTS2 expression. In this regard, it has been reported that clozapine can act as a biased agonist on 5-HT2A receptors42,43 and/or affect the hetero-dimer D2/ 5-HT2A 44. Moreover, disrupting cAMP/CREB and ERK signalling using selective PKA and MEK inhibitors also impaired ADAMTS2 expression and CREB activity (in this case, only when it was PKA dependent) in neuronal-like alongside a rapid phosphorylation of CREB and ERK pro- teins, which resulted in CREB-mediated transcriptional activity as detected by using speciﬁc reporter assays (Fig. 3). In support of this and in our system, ADAMTS2 tran- scription was speciﬁcally triggered by activation of D1 receptors (by SKF 83822), Gαs (using CTX) and AC (for- skolin). Thus, it is possible that, as part of the dopaminergic activity, ADAMTS2 could act as a major cAMP/CREB effector in SCZ. Interestingly, D1 receptor-mediated CREB activation was abrogated by clozapine (Fig. 3c), providing evidence that cAMP/CREB signalling, and therefore ADAMTS2 expression, can be modulated by APDs. Our ﬁndings reveal the contribution of D1 receptors over ADAMTS2 transcription, and also indicate the participation of D2 and 5-HT2A receptors in the effect of clozapine in the control of ADAMTS2 expression. In this regard, it has been reported that clozapine can act as a biased agonist on 5-HT2A receptors42,43 and/or affect the hetero-dimer D2/ 5-HT2A 44. Moreover, disrupting cAMP/CREB and ERK signalling using selective PKA and MEK inhibitors also impaired ADAMTS2 expression and CREB activity (in this case, only when it was PKA dependent) in neuronal-like In conclusion, we have conﬁrmed the association between ADAMTS2 expression and SCZ disease includ- ing its potential role in the clinical efﬁcacy of APDs. Transcription of ADAMTS2 is primarily controlled by the activity of D1-class receptors through cAMP/CREB and MAPK signalling. The unbiased investigation of the molecular mechanisms triggered by APDs, may provide a landscape of novel targets potentially associated with improved therapeutic responses. Discussion Speciﬁc activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Speciﬁc inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D1 (dopamine D1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction. cells. Supporting these observations, recent data pointed at cAMP/CREB signalling as an important mechanism linking dopaminergic signalling with the pathophysiology of SCZ45. Moreover, incubating SK-N-SH cells with forskolin was sufﬁcient to trigger ADAMTS2 expression that occurred alongside PKA and CREB activation. Forskolin directly activates AC in mammals, thereby promoting a rapid phosphorylation of CREB via PKA46,47. In addition, in CREB-knockdown cells, D1 receptor activation failed to increase ADAMTS2 gene expression, reinforcing the idea that cAMP/CREB is an essential mechanism to control ADAMTS2 transcriptional activation. Finally, a number of potential limitations could be considered when interpreting our data and the scope of our ﬁndings: (1) sample size in this study is rather small (N = 30). Noteworthy, we selected a group of patients with a ﬁrst episode of non-affective psychosis, who had not previously taken APDs (not even a single dose) at the time of baseline blood test to avoid any interference with the mRNA levels. Only those individuals who gave written consent and had mRNA samples at baseline and at 3 months were eligible for this study. Therefore, all these stringent inclusion criteria limited the number of patients in this cohort. Further investigations to replicate our ﬁndings using larger and more heterogeneous groups are warranted. (2) Transcriptomic data obtained from blood samples might not resemble that of the brain. (3) ADAMTS2 protein and mRNA expression show modest but detectable levels in ‘healthy’ human and mouse brain samples (https://www.proteinatlas.org). Thus, it is con- ceivable to detect high fold increases when analysing diseased specimens, like in this case ADAMTS2 in SCZ. (4) Failure of some APDs to inhibit CREB activity and/or ADAMTS2 mRNA expression in a D1 receptor-context. Our data herein do not discard that APDs might regulate ADAMTS2 by indirect mechanisms like for example those involving β-arrestin, ERK or AKT signalling42,48. Fig. 5 Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D1 receptors by SKF 83822 (selective D1 receptor agonist) triggers the expression of ADAMTS2. 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https://openalex.org/W4285601896	https://link.springer.com/content/pdf/10.1007/JHEP07(2022)094.pdf	English	null	ALPINIST: Axion-Like Particles In Numerous Interactions Simulated and Tabulated	The Journal of high energy physics/The journal of high energy physics	2,022	cc-by	18,386	Published for SISSA by Springer Received: January 28, 2022 Accepted: June 7, 2022 Published: July 15, 2022 Received: January 28, 2022 Accepted: June 7, 2022 Published: July 15, 2022 ALPINIST: Axion-Like Particles In Numerous Interactions Simulated and Tabulated JHEP07(2022)094 Jan Jerhot,a Babette Döbrich,b Fatih Ertas,c Felix Kahlhoeferc and Tommaso Spadarod Jan Jerhot,a Babette Döbrich,b Fatih Ertas,c Felix Kahlhoeferc and Tommaso Spadarod aCentre for Cosmology, Particle Physics and Phenomenology (CP3), Université catholique de Louvain, Chemin du Cyclotron 2, B-1348 Louvain-la-Neuve, Belgium bCERN, Esplanade des Particules 1, 1211 Geneva 23, Switzerland cInstitute for Theoretical Particle Physics and Cosmology (TTK), RWTH Aachen University, D-52056 Aachen, Germany dLaboratori Nazionali di Frascati INFN, Via E. Fermi 40, 00044 Frascati, Italy E-mail: jan.jerhot@cern.ch, babette.dobrich@cern.ch, ertas@physik.rwth-aachen.de, kahlhoefer@physik.rwth-aachen.de, tommaso.spadaro@cern.ch Jan Jerhot,a Babette Döbrich,b Fatih Ertas,c Felix Kahlhoeferc and Tommaso Spadarod aCentre for Cosmology, Particle Physics and Phenomenology (CP3), Université catholique de Louvain, Chemin du Cyclotron 2, B-1348 Louvain-la-Neuve, Belgium bCERN, Esplanade des Particules 1, 1211 Geneva 23, Switzerland cInstitute for Theoretical Particle Physics and Cosmology (TTK), RWTH Aachen University, D-52056 Aachen, Germany dLaboratori Nazionali di Frascati INFN, Via E. Fermi 40, 00044 Frascati, Italy E-mail: jan.jerhot@cern.ch, babette.dobrich@cern.ch, ertas@physik.rwth-aachen.de, kahlhoefer@physik.rwth-aachen.de, tommaso.spadaro@cern.ch aCentre for Cosmology, Particle Physics and Phenomenology (CP3), Université catholique de Louvain, Chemin du Cyclotron 2, B-1348 Louvain-la-Neuve, Belgium bCERN, Esplanade des Particules 1, 1211 Geneva 23, Switzerland cInstitute for Theoretical Particle Physics and Cosmology (TTK), RWTH Aachen University, D-52056 Aachen, Germany E-mail: jan.jerhot@cern.ch, babette.dobrich@cern.ch, ertas@physik.rwth-aachen.de, kahlhoefer@physik.rwth-aachen.de, tommaso.spadaro@cern.ch Abstract: Proton beam dump experiments are among the most promising strategies to search for light and feebly interacting states such as axion-like particles (ALPs). The interpretation of these experiments is however complicated by the wide range of ALP models and the multitude of diﬀerent production and decay channels that can induce observable signals. Here we propose a new approach to this problem by separating the calculation of constraints and projected sensitivities into model-independent and model-dependent parts. The former rely on extensive Monte Carlo simulations of ALP production and decays, as well as estimates of the detection eﬃciencies based on simpliﬁed detector geometries. Once these simulations have been performed and tabulated, the latter parts only require simple analytical rescalings that can be performed using the public code ALPINIST released together with this work. We illustrate this approach by considering several ALP models with couplings to Standard Model gauge bosons. For the case of ALPs coupled to gluons we show that the sensitivity of proton beam dump experiments can be extended signiﬁcantly by considering hadronic ALP decays into three-body ﬁnal states. Keywords: Axions and ALPs, New Light Particles Keywords: Axions and ALPs, New Light Particles ArXiv ePrint: 2201.05170 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Jan Jerhot,a Babette Döbrich,b Fatih Ertas,c Felix Kahlhoeferc and Tommaso Spadarod Article funded by SCOAP3. https://doi.org/10.1007/JHEP07(2022)094 JHEP07(2022)094 1 2 5 5 6 7 9 10 11 12 13 13 13 14 14 15 15 16 16 17 18 18 19 21 22 24 24 25 26 27 28 3.1 Photo-production JHEP07(2022)094 4.3 Propagation of decay products 1 Introduction In recent years the search for physics beyond the Standard Model (SM) has begun to expand its focus from particles too heavy to be produced in laboratory experiments to light particles with extremely weak interactions [1]. As grows the range of models that predict such light degrees of freedom, so does the number of proposed and ongoing experiments that can probe the corresponding parameter spaces. A central challenge arising from this development is to identify those experiments that promise the greatest improvement in sensitivity to a wide variety of models. A popular way to address this challenge is to deﬁne a number of benchmark scenarios, for which exclusion bounds and sensitivity projections are to be calculated [2–4]. As the ﬁeld matures, however, more ﬂexible and model-independent methods are needed that allow for the reinterpretation of experimental bounds and sensitivities for a wide range of diﬀerent scenarios. JHEP07(2022)094 This need is particularly great in the context of axion-like particles (ALPs), which are hypothetical elementary pseudoscalar particles arising as the pseudo-Nambu-Goldstone modes of an approximate global symmetry that is broken both spontaneously at some large energy scale Λ and explicitly by the non-zero ALP mass ma ≪Λ. ALPs emerge naturally in many theories extending the SM symmetry group, which may feature new accidental global symmetries that are not experimentally observed and therefore assumed to be broken spontaneously [5]. A plethora of unobserved global symmetries also appears in theories with extra dimensions when these are compactiﬁed to four dimensions [6]. From a phenomenological point of view, the relation ma ≪Λ furthermore implies that ALPs can be viable dark matter (DM) candidates or mediate the interactions between SM and DM particles [7–10]. Since the details of the spontaneous symmetry breaking and the corresponding new physics at the energy scale Λ are typically unknown, ALPs can manifest themselves in a wide range of diﬀerent ways at low energies. To approach this problem in a general way, one can express the interactions between ALPs and SM particles in terms of an eﬀective ﬁeld theory valid at energies E ≪Λ [11–13]. This approach makes it possible to experimentally search for ALPs in a model-independent way, i.e. without referring to a speciﬁc symmetry breaking mechanism. 5 Experimental framework p 5.1 Results from past experiments 5.1.1 CHARM 5.1.2 NuCal 5.2 Acceptance of current experiments 5.2.1 NA62 5.3 Acceptance of proposed experiments 5.3.1 SHiP 5.3.2 DarkQuest 5.3.3 DUNE ND 5.3.4 SHADOWS 5.1 Results from past experiments 5.1.1 CHARM 5.1.2 NuCal 5.2 Acceptance of current experiments 5.2.1 NA62 5.3 Acceptance of proposed experiments 5.3.1 SHiP 5.3.2 DarkQuest 5.3.3 DUNE ND 5.3.4 SHADOWS 5.1 Results from past experiments 5.3.3 DUNE ND 5.3.4 SHADOWS 6 Results 18 6.1 Considering only di-photon decays 19 6.2 Including hadronic decays 21 7 Conclusions 22 A Model-dependent calculations 24 A.1 FCNC transitions 24 A.2 ALP-meson mixing 25 B Calculation of decay widths 26 C Brief description of the ALPINIST code 27 D Treatment of kinematics for the mixing production 28 – i – 1 Introduction For a given combination of eﬀective ALP couplings, these tables can then be evaluated and combined in a straight-forward way. In addition to this conceptual innovation, our work also expands on the range of eﬀects included in our calculations in order to extend existing approaches (using for example the automated simulations of beam-dump experiments in Maddump [25]) and to allow for a wide range of applications. On the ALP production side, we consider Primakoﬀ production (both from on-shell [26] and oﬀ-shell [27] photons), ALP-meson mixing [20] and rare decays (both from B mesons [9] and D mesons [28]). On the ALP decay side, we include for the ﬁrst time detailed experimental sensitivities for leptonic ﬁnal states and three-body hadronic ﬁnal states like π+π−π0. For the latter we have implemented an automatic re-weighting of the Dalitz plot density based on detailed analytic calculations of hadronic decay modes [18, 29]. All these calculations are applied to a wide range of past, present and proposed experiments, for which we have implemented a modelling of detector geometries and eﬃciencies. To illustrate the ﬂexibility of our approach, we apply it to several diﬀerent ALP models featuring various eﬀective couplings to SM gauge bosons. Together with this work we release a public code named ALPINIST [30], which provides the relevant tables for ALP production and detection together with helpful routines for model-dependent calculations and a convenient user interface. The remainder of this work is structured as follows. In section 2 we introduce the general framework used to obtain model-independent constraints on ALP models. The various ALP production and decay modes are discussed in sections 3 and 4, respectively. A detailed discussion of the experiments considered in this work is given in section 5. To illustrate our approach, we present experimental sensitivities for a range of ALP scenarios in section 6. The code ALPINIST is described in more detail in appendix C. The calculation of the various eﬀective couplings and the resulting decay widths are discussed in the appendices A and B. 1 Introduction While traditionally much energy has been focused on the eﬀective ALP-photon coupling [14], recent studies have broadened the picture and considered also eﬀective couplings to the other SM gauge bosons [15–19] as well as to SM Higgs bosons [20] and SM fermions [21, 22]. Ideally, one would like to study all of these eﬀective couplings simultaneously, which requires the production of on-shell ALPs and the observation of their subsequent decays back into SM particles. For ma in the MeV–GeV range, the most suitable laboratory for this purpose are particle accelerators. While colliders like the LHC have the advantage of larger ALP production cross sections [23], ﬁxed-target experiments beneﬁt from higher statistics and lower backgrounds and are therefore more sensitive to tiny couplings and small ALP masses [24]. To improve our understanding of ALPs and ultimately achieve their discovery, it is therefore essential to understand the signatures of ALPs in ﬁxed-target experiments for general eﬀective interactions. – 1 – In this work we propose a new approach for evaluating experimental sensitivities, which is based on the observation that the calculation of the predicted number of ALP events in a given experiment can be split into a number of separate steps, each of which can be performed in a largely model-independent way. Speciﬁcally, we • write the diﬀerential ALP production cross section as the sum of several diﬀerent production modes, each of which depends in a well-deﬁned way on the eﬀective ALP couplings; • write the diﬀerential ALP production cross section as the sum of several diﬀerent production modes, each of which depends in a well-deﬁned way on the eﬀective ALP couplings; • make use of the fact that the detection probability depends trivially on the various • make use of the fact that the detection probability depends trivially on the various ALP branching ratios, with only the total ALP lifetime having a more complicated eﬀect. JHEP07(2022)094 ALP branching ratios, with only the total ALP lifetime having a more complicated eﬀect. This makes it possible to perform all relevant calculations in advance and tabulate them in terms of a small number of model parameters. For a given combination of eﬀective ALP couplings, these tables can then be evaluated and combined in a straight-forward way. This makes it possible to perform all relevant calculations in advance and tabulate them in terms of a small number of model parameters. 2 General framework In this section we outline our general approach for simplifying the calculation of exclusion limits and sensitivity projections for ALPs in a model-independent way. For this purpose – 2 – we consider a pseudoscalar particle a with mass ma that couples to the various particles of the SM via eﬀective operators. Limiting ourselves to the case of dimension-5 operators, the strength of each interaction is parametrised by an eﬀective coupling that can be expressed in terms of the new-physics scale Λ and the Wilson coeﬃcients C of the various eﬀective operators (see appendix A for details). For example, the coupling of the ALP to a pair of photons is given by C Laγγ = e2 Cγγ Λ aF µν ˜Fµν , (2.1) (2.1) where F µν denotes the electromagnetic ﬁeld strength tensor and ˜F µν its dual. We denote the set of all couplings that deﬁne a speciﬁc parameter point of a given ALP model by C. More details will be provided in section 6 and appendix A. JHEP07(2022)094 The focus of the present work is on proton beam ﬁxed-target experiments, which aim to produce ALPs using a beam of highly energetic protons and detect the subsequent decays of ALPs into SM particles in a downstream detector. At such experiments ALPs can be produced via various mechanisms (production channels), for example through scattering of beam protons or secondary particles on the target material or through the decay of secondary particles. At ﬁrst sight, the number Ndet of expected ALP events in the detector depends in a complicated non-linear way on the couplings C, which determine the production cross section, the kinematic distributions, the lifetime τa = 1/Γa and the branching ratios into the various ﬁnal states. Upon closer inspection, it however becomes clear that this quantity can be factorized as follows: Ndet = Z dθadEa d2N (ma, C) dθadEa pdet (ma, Γa, θa, Ea, C) . (2.2) (2.2) Here d2N/dθadEa denotes the spectrum of ALPs produced in the target as a function of the angle (relative to the incident beam) and the ALP energy in the laboratory frame, and pdet denotes the probability that an ALP with given angle and energy will lead to an observable signal in the detector, which depends on the position of the ALP decay as well as on the kinematic distribution of the decay products. 2 General framework Here d2N/dθadEa denotes the spectrum of ALPs produced in the target as a function of the angle (relative to the incident beam) and the ALP energy in the laboratory frame, and pdet denotes the probability that an ALP with given angle and energy will lead to an observable signal in the detector, which depends on the position of the ALP decay as well as on the kinematic distribution of the decay products. ALP production in the target can proceed via a number of diﬀerent processes, which will be discussed in detail in section 3. The ALP spectrum is therefore simply a sum over all production channels: d2N (ma, C) dθadEa = X i d2Ni (ma, C) dθadEa . (2.3) (2.3) Now the crucial observation is that for each individual production channel, the spectrum depends in a trivial way on the ALP coupling structure, in the sense that changing the couplings only aﬀects the normalisation but not the shape of the spectrum. In other words, it is possible to calculate the spectrum for a given reference coupling Cref and then perform an appropriate rescaling: Now the crucial observation is that for each individual production channel, the spectrum depends in a trivial way on the ALP coupling structure, in the sense that changing the couplings only aﬀects the normalisation but not the shape of the spectrum. In other words, it is possible to calculate the spectrum for a given reference coupling Cref and then perform an appropriate rescaling: d2Ni (ma, C) dθadEa = fi(C, Cref)d2Ni (ma, Cref) dθadEa . (2.4) (2.4) – 3 – – 3 – For example, if ALPs are produced via their coupling to photons, the production cross section is proportional to C2 γγ and hence For example, if ALPs are produced via their coupling to photons, the production cross section is proportional to C2 γγ and hence fγγ(C, Cref) = C2 γγ C2 γγ,ref . (2.5) (2.5) This simple formula holds even if the ALP eﬀective photon coupling receives contributions from several diﬀerent eﬀective operators, see appendix A for details and ref. [20] for a comprehensive overview of such contributions. We emphasize that the ALP mass, on the contrary, aﬀects the ALP spectrum in a non-trivial way. It is therefore necessary to calculate the spectra d2Ni/dθadEa separately for each ALP mass under consideration. 2 General framework For perturbative production processes such as photon fusion the ALP spectra can be derived from ﬁrst principles. In many cases, however, production proceeds via non-perturbative processes and the spectra can be only obtained via simulations. These simulations however only need to be performed once (for the chosen values of Cref) and can then be applied to any ALP model. JHEP07(2022)094 Similarly, the detection probability can be split into diﬀerent ﬁnal states f: pdet (ma, θa, Ea, C) = X f BRa→f(ma, C)pdet,f(ma, Γa, θa, Ea) , (2.6) (2.6) where BRa→f = Γa→f/Γa denotes the branching ratio into the ﬁnal state f. While these branching ratios may depend in a non-trivial way on the couplings C, the detection probability pdet,f(ma, Γa, θa, Ea) for a given ﬁnal state depends on the couplings only through the ALP lifetime, which determines the probability of the ALP to decay in a given part of the detector. Again, this makes it possible to calculate the detection probabilities pdet,f in advance and then apply an appropriate rescaling through the branching ratios BRa→f. Putting everything together, we can therefore write Ndet = X i,f fi(C, Cref)BRa→f(ma, C) × Z dθadEa d2Ni (ma, Cref) dθadEa pdet,f(ma, Γa, θa, Ea) = X i,f Mif(ma, C) × Edet,if(ma, Γa) , (2.7) (2.7) where Mif denotes the part that depends on the ALP model (i.e. on the couplings C), while Edet,if encapsulates all the experimental details. Note that in the ﬁnal line we have omitted the dependence on Cref, which will be kept implicit in the following. As will become clear in the subsequent sections, the calculation of Edet,if for a given experiment can be quite challenging, typically requiring Monte Carlo (MC) simulations of both the production and the decay. However, these simulations only have to be performed once (for given ma and Γa and a choice of Cref) and the (tabulated) functions Edet,if can easily be made publicly available. This makes it possible to perform fast analyses of any ALP model for which the model-dependent functions Mif have been calculated. Moreover, simulation upgrades including new production mechanisms or decay channels or more realistic experiment modelling can be quickly included in the framework, allowing straightforward estimates of the related phenomenological consequences. – 4 – 3 ALP production 3 In this work we consider three types of ALP production channels, which are described in detail in the remainder of this section: production via the eﬀective coupling of ALPs to photons (section 3.1), ALP mixing with neutral pseudoscalar mesons (section 3.2) and ALPs produced in heavy meson decays (section 3.3). Each of these production channels is eﬃcient for production of ALPs in a diﬀerent mass range and results in a diﬀerent Ea-θa distribution, thus favouring a diﬀerent experimental setup. For example, ALPs produced via photon-fusion tend to be forward-emitted and soft; those produced in heavy meson decays can be emitted at large angles and with higher energies. JHEP07(2022)094 As discussed in the previous section, the diﬀerential ALP yield for each of the production channels can be split into a model-dependent and a model-independent part. For production via the ALP-photon coupling, the model-dependent part is given by eq. (2.5). For production via meson mixing, the corresponding expression is fP (C, Cref) = θaP θaP,ref 2 , (3.1) (3.1) where P = π0, η, η′ and θaP denotes the corresponding ALP-meson mixing angle (see below). Finally, for the production via B meson decay we have where P = π0, η, η′ and θaP denotes the corresponding ALP-meson mixing angle (see below). Finally, for the production via B meson decay we have fBK(∗)(C, Cref) = BR(B →K(∗) + a) BR(B →K(∗) + a)ref , (3.2) (3.2) where where here BR(B →X) = ΓB→X ΓB , (3 BR(B →X) = ΓB→X ΓB , (3.3) (3.3) denotes the B meson branching ratios, and analogous expressions for D meson decays. 3.1 Photo-production The ALP photo-production, also called Primakoﬀprocess, can be realized in two ways at a beam-dump setup: the electro-magnetic (EM) scattering of a beam proton with a target nucleus and the interaction of on-shell photons from decays of secondary neutral pseudoscalar mesons with the nuclear EM ﬁeld. The former process can be in turn either realized through the scattering of the whole proton on the target nuclei (elastic scattering) or through the scattering of their constituents. The elastic scattering, which dominates at beam energies of current beam dump facilities, has been studied in details in ref. [27]. In ref. [26] it has been shown that ALP photo-production via on-shell photons from meson decays constitutes an even a more important source of ALPs. In this work, we include both elastic scattering and Primakoﬀproduction from on-shell photons. g p p In both cases the ALP diﬀerential yield can be expressed as d2N dθadEa = C2 γγ Λ2 8e4 σN sin θa πEbeam Z dp2 t dφa dσγN d cos θa γ(Ea, p2 t ) , (3.4) (3.4) where the diﬀerential cross section for the photon-nucleus interaction is taken from eq. (3.16) of ref. [27] and scales with Z2 target. This cross section has to be normalized to the total cross – 5 – section for the incident particle. In the case of elastic proton scattering, the total cross section for the incident particle is σN ≃53 mb × A0.77 target. For the absorption of an on-shell photon, we use values from ref. [31]. The photon distribution γ can be calculated using the Weizsäcker-Williams approxima- tion [32] in the case of the proton elastic scattering.1 In order to obtain the γ distribution for on-shell photons from π0, η, η′ di-photon decays, we have to simulate the production of these secondary mesons. In this work the meson yields from p-p collisions at experimentally relevant energy values2 were simulated using PYTHIA 8.2 [34] with SoftQCD:all ﬂag and a pomeron ﬂux parametrization SigmaDiffractive:PomFlux(5), a setup which has been validated in ref. [26]. The corresponding photon distribution is then obtained from interpo- lation of the dataset resulting from the ﬂat phase-space decay of these mesons into photon pairs. The decay products are boosted to the laboratory frame and the events are weighted with the π0, η, η′ di-photon branching fractions. JHEP07(2022)094 2For 400, 120 and 70 GeV proton beams the respective p-p c.m. energies are approximately 27.4, 15 and 11.5 GeV. 1It has been pointed out in ref. [33] that this approximation becomes inaccurate for small ALP masses as well as for low beam energies. Given that elastic scattering only gives a sub-dominant contribution compared to ALP production from photons produced in meson decays, we ﬁnd that this approximation is nevertheless fully suﬃcient for our purposes. 3.2 ALP production from meson mixing If the ALP ﬁeld is coupled to the SM quark and/or gluon ﬁelds, it naturally enters the kinetic and mass matrices of the eﬀective Lagrangian of pseudoscalar mesons. Upon diagonalization of these matrices in order to obtain the mass eigenstates, the mixing angle between ALPs and mesons P = π0, η, η′ emerges in the following form: θaP = fπ fa KaP m2 a + m2 aP m2a −m2 P , (3.5) (3.5) where the ALP decay constant fa depends on the underlying ALP couplings. We emphasize that this expression is valid for values ma such that \|θaP \|2 ≪1. For the explicit form of fa, the kinetic mixing terms KaP and the mass mixing terms maP we refer to appendix A and ref. [18]. In the presence of such a mixing it is possible to produce an ALP instead of the meson P via SM processes in a small fraction of cases proportional to \|θaP \|2. The corresponding diﬀerential yield can be written as d2N dθadEa = \|FVMD(ma)\|2 X P ˆα2 s(ma) ˆα2s(mP ) d2NP dθP dEP EP →Ea θP →θa \|θaP \|2 , (3.6) (3.6) where d2NP /dθP dEP denotes the diﬀerential yield of the meson P. To consider ALP masses up to 3 GeV and capture the suppression of ALP production for ma ≫mP , we 1It has been pointed out in ref. [33] that this approximation becomes inaccurate for small ALP masses as well as for low beam energies. Given that elastic scattering only gives a sub-dominant contribution compared to ALP production from photons produced in meson decays, we ﬁnd that this approximation is nevertheless fully suﬃcient for our purposes. 2For 400, 120 and 70 GeV proton beams the respective p-p c.m. energies are approximately 27.4, 15 and 11 5 GeV – 6 – follow ref. 3.2 ALP production from meson mixing [18] and include the vector meson dominance (VMD) form factor FVMD(m) =                  1, for m ≤1.4 GeV 3P i=0 aimi, for 1.4 GeV < m ≤2 GeV (1.4 GeV/m)4, for 2 GeV < m ≤3 GeV 0, for m > 3 GeV, (3.7) (3.7) and the running strong coupling ˆαs(m) in the form and the running strong coupling ˆαs(m) in the form and the running strong coupling ˆαs(m) in the form ˆαs(m) =            1, for m ≤1 GeV 3P i=0 bimi, for 1 GeV < m ≤1.5 GeV 4π/7 log(m/0.34 GeV)2, for m > 1.5 GeV, (3.8) JHEP07(2022)094 (3.8) where parameters ai and bi are determined by requiring continuity of the resulting function and its ﬁrst derivative. Note that compared to ref. [4] we assume a stronger suppression of the ALP yield for large ALP masses to ensure that our estimates are conservative. For obtaining the distribution of d2NP /dθP dEP , we can conveniently re-use the vali- dated datasets from the simulation described in section 3.1. There is however an important subtlety: since the pseudoscalar meson masses are generally diﬀerent from the ALP mass, it is not possible to simply replace a pseudoscalar by an ALP with the same energy and momentum. Even if the replacement is made in such a way that the three-momentum is conserved in a speciﬁc frame, it may not be conserved in a diﬀerent frame. This introduces some degree of arbitrariness in the precise prescription used for the replacement. Here we require that the three-momentum of the ALP be equal to the three-momentum of the pseudoscalar meson in the p-p cms frame, calculate the corresponding ALP energy and then boost the result into the laboratory frame. For further details we refer to appendix D. y pp In ﬁgure 1 we show the predicted distribution of ALPs in the Ea-θa plane for the photon-from-meson production channel (left panel) and for production via ALP-η mixing (right panel). In both cases we ﬁnd an anti-correlation between the two parameters in the sense that higher ALP energies imply smaller production angles and vice versa. 3.2 ALP production from meson mixing For the meson mixing contribution, the diﬀerential yield peaks at larger ALP angles than for Primakoﬀproduction, but still gives a sizeable contribution in typical on-axis beam-dump experiments with an angular acceptance of a few mrad. 3.3 ALP production from rare decays Flavour-changing ALP couplings arise not only from interactions of ALPs with quarks [10] and electroweak gauge bosons [15] but also in higher-order processes from ALP-gluon interactions [35]. In all of these cases ALPs can be produced in ﬂavour-changing neutral current (FCNC) processes and can therefore be probed through rare meson decays in ﬁxed-target experiments [19, 24]. However, in order for the produced ALPs to be focused in the forward direction it is essential that the parent meson decays before interacting with the target material. B and D mesons can thus be of particular importance since when – 7 – 10-16 10-15 10-14 10-13 10-12 d2N a dEadθa [GeV-1] 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] 10-12 10-11 10-10 10-9 d2N a dEadθa [GeV-1] Figure 1. Production diﬀerential yields for an ma = 0.5 GeV ALP produced at a copper target with a 400 GeV proton beam. For a photon-from-meson induced production (left) with a ﬁxed Cγγ/Λ = 10−4 GeV−1 and for an a-η mixing (right) assuming a ﬁxed mixing angle θaη = 10−4. 10-16 10-15 10-14 10-13 10-12 d2N a dEadθa [GeV-1] 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] d 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] 10-12 10-11 10-10 10-9 d2N a dEadθa [GeV-1] JHEP07(2022)094 Ea [GeV] Figure 1. Production diﬀerential yields for an ma = 0.5 GeV ALP produced at a copper target with a 400 GeV proton beam. For a photon-from-meson induced production (left) with a ﬁxed Cγγ/Λ = 10−4 GeV−1 and for an a-η mixing (right) assuming a ﬁxed mixing angle θaη = 10−4. produced at the beam energies of O(100) GeV their decay length is signiﬁcantly smaller than the interaction length of the target material.3 Let us ﬁrst consider the case of B mesons. In this work we include the ALP production from B →K(⋆) + a decays (both charged and neutral). Since the decay is isotropic in the rest frame of the B meson, the diﬀerential ALP yield depends only on the distribution of B mesons and the branching ratios for the decays involving ALPs (see eq. (3.3)). The B meson distributions are again obtained using simulations of p-p collisions with PYTHIA 8.2. We follow the setup of ref. [24], i.e. 3The nuclear collision length for most of the target materials is ≈10 cm [36]. The decay length for 100 GeV momentum B meson is < 1 cm and ≈1 cm for a D meson, while for K it is > 500 cm. 3.3 ALP production from rare decays we allow only the bottom quark production hard-QCD processes in order to have suﬃcient statistics and then reweight the ﬁnal distribution according to the bottom production cross section at the given beam energies. The value reported by PYTHIA is σpp = 39.85 × 109 pb and σbb = 1.866 × 103 pb for 400 GeV beam, which has already been validated in ref. [24] using the values reported in ref. [37]. For 120 (and 70 GeV) the values are σpp = 38.54×109 pb and σbb = 3.8 pb (σpp = 38.38×109 pb and σbb = 1.15×10−7 pb respectively), for which no measurement has been found in the existing literature for validation. In any case, for experiments operating with such small beam energies the ALP production via B meson decays is found to be negligible (see section 6). A separate simulation involving the two-body decay kinematics of B →K(⋆) + a is performed to obtain the resulting ALP distribution as a function of Ea and θa. In an analogous way, we simulate the production of D mesons with σcc =          3.601 × 106 pb, for Ebeam = 400 GeV 0.518 × 106 pb, for Ebeam = 120 GeV 1.551 × 105 pb, for Ebeam = 70 GeV (3.9) (3.9) and a subsequent decay D →π + a. Despite a larger production cross section of D mesons, for the ALP models considered in this work the branching ratio BRD→π+a is many orders of and a subsequent decay D →π + a. Despite a larger production cross section of D mesons, for the ALP models considered in this work the branching ratio BRD→π+a is many orders of 3The nuclear collision length for most of the target materials is ≈10 cm [36]. The decay length for a 100 GeV momentum B meson is < 1 cm and ≈1 cm for a D meson, while for K it is > 500 cm. – 8 – 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] 10-27 10-26 10-25 10-24 10-23 10-22 10-21 10-20 10-19 10-18 d2N a dEadθa [GeV-1] 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θa [rad] 10-18 10-17 10-16 10-15 10-14 d2N a dEadθa [GeV-1] Figure 2. 4For example we do not include veto detectors in the simulation and we do not account for eﬃciencies of individual detectors and possible scattering or absorption of ﬁnal state particles in the material of the experiment before they are detected. 3.3 ALP production from rare decays Production diﬀerential yield of an ma = 0.5 GeV ALP produced in decays B±,0 →K±,0+a (left) and D±,0 →π±,0 + a (right) of B and D produced at a copper target with 400 GeV proton beam assuming a referential value BRB→K+a = BRD→π+a = 10−10. 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θa [rad] 10-18 10-17 10-16 10-15 10-14 d2N a dEadθa [GeV-1] 100 101 102 10-4 10-3 10-2 10-1 Ea [GeV] θ a [rad] 10-27 10-26 10-25 10-24 10-23 10-22 10-21 10-20 10-19 10-18 d2N a dEadθa [GeV-1] JHEP07(2022)094 Figure 2. Production diﬀerential yield of an ma = 0.5 GeV ALP produced in decays B±,0 →K±,0+a (left) and D±,0 →π±,0 + a (right) of B and D produced at a copper target with 400 GeV proton beam assuming a referential value BRB→K+a = BRD→π+a = 10−10. magnitude smaller than BRB→K(⋆)+a as discussed in appendix A. Nevertheless, we enclose the D mesons data sets to allow also studies of speciﬁc ALP models enhancing the up-type quark transition (see e.g. ref. [28]). The ALP distribution resulting from B →K + a and D →π + a decays are plotted in ﬁgure 2. Compared to the distributions shown in ﬁgure 1 we observe that the ALP distribution from rare meson decays peaks at even larger angles and energies. In particular ALPs produced from B mesons have very high energies, making this production mode particularly promising for oﬀ-axis experiments searching for relatively heavy ALPs with short lifetimes. 4 ALP decay and detection of ﬁnal states To determine the probability that an ALP induces an observable signal, we have modelled the various ﬁxed-target experiments under consideration in a simpliﬁed4 and generic MC simulation of ALP propagation and decays. The layout consists of a decay volume, a spectrometer, analyzing magnets and a forward calorimeter (see schematic drawing in ﬁgure 3). Our set-up is described in detail in section 4.1. The outcome of the simulation is the function Edet,if(ma, Γa) deﬁned in eq. (2.7). We provide tabulated data sets of Edet,if based on a simulation with N = 106 trials on a grid of Γa and ma values for each of the production and decay channels i,f, and for each of the experiments described in section 5. We note that the function Edet,if includes the eﬃciency ϵdet,f of the experiment for the detection of a given ﬁnal state f. For past experiments we use the eﬃciencies as determined by the experiment (see section 5), while for future experiments we simply set ϵdet,f = 1. However, it is straight-forward in our approach to – 9 – dump decay volume magnetic spectrometer B calorimeter p beam ALP + Figure 3. Schematic view of the experimental setup for the case that the ALP decays via a →π+π−γ. magnetic spectrometer dump decay volume p beam Figure 3. Schematic view of the experimental setup for the case that the ALP decays via a →π+π−γ. JHEP07(2022)094 redo our analysis for diﬀerent values of ϵdet,f making our framework a powerful tool to assess the impact of the experimental design on sensitivity projections. 4.1 General principles of the ALP Monte Carlo simulation The probability pdet,f that an ALP with given ma, Γa, θa and Ea decaying to channel f will be detected at the experiment is a potentially complicated function of the distance l that the particle travels before decaying and the momenta of the ﬁnal-state particles, which can be characterised by a set of kinematical variables a1, . . . an. The conditions imposed on the decay products by experimental cuts can be summarized by a function Θdet,f(ma, θa, Ea, l, a1, . . . an), which is equal to 1 if all experimental conditions are met and 0 otherwise. Since it is typically impossible to perform the integration over all of these variables, it is useful to deﬁne pdet,f instead as a sum over randomly generated decay processes pdet,f(ma, Γa, θa, Ea) = N X k=1 ϵdet,f Θdet,f ma, Γa, θa, Ea; {l, a1, . . . an}k ran N , (4.1) (4.1) where {l, a1, . . . an}k ran denotes the k-th simulated event of the ALP decay. To carry out these simulations we ﬁrst calculate the ALP decay length in the labo- ratory frame la = γaβa/Γa with γa = Ea/ma and βa = pa/ma and use the distribution p(l) = l−1 a exp(−l/la) to choose a random value of l. For the decay itself we generate the four-momenta of the second generation particles, as well as extra sets for the third gener- ation particles if the former include unstable particles (the speciﬁc distributions used to simulate the ALP decays will be discussed in section 4.2). Given the magnetic ﬁelds of the experiment, these four-momenta enable us to calculate the trajectories of the ﬁnal-state particles and check the experimental conditions in order to evaluate Θdet,f. In practice, in order to obtain the distribution Edet,if(ma, Γa) for given values of ma and Γa we ﬁrst read the distribution ∆2Ni/∆θa∆Ea for the production mode i from the previous section into a θa-Ea histogram using ROOT [38]. At each iteration we sample a random bin {θa, Ea}k ran according to the histogram and simulate the ALP decay for these values. By summing up a suﬃciently large sample N, we then obtain a reliable estimate of Edet,if(ma, Γa) in the form Edet,if(ma, Γa) = N X k=1 ϵdet,f Θdet,f ma; {θa, Ea, l, a1, . . . an}k ran N . 5In all of the decays that we consider, there are at least two particles with the same mass. These are taken to calculate m12. 4.1 General principles of the ALP Monte Carlo simulation (4.2) (4.2) N – 10 – – 10 – neutral charged 2-body 2γ e+e− µ+µ− 3-body 3π0 π+π−γ 2π0η π+π−π0 π+π−η Table 1. Allowed decay channels for ALPs with masses up to about 1 GeV. Table 1. Allowed decay channels for ALPs with masses up to about 1 GeV. JHEP07(2022)094 Note that the right-hand side depends implicitly on Γa through the distribution used to obtain l. Although the ALP detection probability depends sensitively on the ALP energy Ea and angle θa, the left-hand side of the equation above is independent of these quantities and only depends on the chosen production mechanism (represented by the index i). In other words, Edet,if can be thought of as the expectation value of the detection probability for the distribution of ALP energies and angles predicted by the chosen production mechanism. 4.2 Decay channels Since beam dump experiments can probe ALPs of masses up to O(1) GeV, there are a number of kinematically allowed decay channels, listed in table 1. We restrict ourselves to two- or three-body decays, therefore accounting for the dominant contribution to the total decay width for the ALP masses and coupling scenarios under consideration (see section 6). For the purposes of the MC simulation, we consider charged particles to be stable and π0 and η to decay instantaneously into a γ-γ pair with the appropriate branching fraction. While two body decays of ALPs are treated with a ﬂat phase space, for three-body decays a more careful treatment is necessary. In a three-body decay, the invariant masses of ﬁrst and second and second and third particles, m12 and m23, can be used to deﬁne a Dalitz plot.5 If the transition amplitude square \|M\|2 of the process depends on the momenta of outgoing particles, the resulting Dalitz plot density is generally non-uniform. Such a momentum dependence arises in a wide range of models, for example if the decay proceeds via a resonance, as in the case of a →ππγ, which receives a contribution from virtual ρ meson exchange. In this work, for the simulation of hadronic decays, we employ the framework derived in ref. [18] using chiral perturbation theory. The results from ref. [18] are derived under the assumption that ALPs are coupled only to gluons, but they remain valid also for ALPs with ﬂavour-universal couplings to light quarks. In such a case, the decay width Γa→hadrons changes but the ratio \|M\|2 a→hadrons/Γa→hadrons, which determines the distribution of the ﬁnal-state hadrons, remains invariant. However, for models with non-universal couplings to light quarks, as considered for example in ref. [29], there may be signiﬁcant diﬀerences in both the individual branching ratios and the corresponding distributions of the decay products. 4.2 Decay channels – 11 – 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 0.8 1.0 1.2 1.4 1.6 m12 2 [GeV2] m23 2 [GeV2] ma = 1.43 GeV 0.2 0.4 0.6 0.8 1.0 0.8 1.2 1.6 2.0 m12 2 [GeV2] ma = 1.61 GeV 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.8 1.2 1.6 2.0 2.4 m12 2 [GeV2] ma = 1.80 GeV 102 103 104 105 \|ℳ\|2 Γ [GeV-1] 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 m12 2 [GeV2] m23 2 [GeV2] ma = 2.02 GeV 0.5 1.0 1.5 2.0 2.5 1.0 2.0 3.0 4.0 m12 2 [GeV2] ma = 2.26 GeV 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1.0 2.0 3.0 4.0 5.0 m12 2 [GeV2] ma = 2.54 GeV 102 103 104 105 \|ℳ\|2 Γ [GeV-1] Figure 4. Dalitz plot density for six values of ALP masses used for the a →π+π−η decay. 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 0.8 1.0 1.2 1.4 1.6 m12 2 [GeV2] m23 2 [GeV2] ma = 1.43 GeV 0.2 0.4 0.6 0.8 1.0 0.8 1.2 1.6 2.0 m12 2 [GeV2] ma = 1.61 GeV 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.8 1.2 1.6 2.0 2.4 m12 2 [GeV2] ma = 1.80 GeV 102 103 104 105 \|ℳ\|2 Γ [GeV-1] JHEP07(2022)094 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 m12 2 [GeV2] m23 2 [GeV2] ma = 2.02 GeV 0.5 1.0 1.5 2.0 2.5 1.0 2.0 3.0 4.0 m12 2 [GeV2] ma = 2.26 GeV 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1.0 2.0 3.0 4.0 5.0 m12 2 [GeV2] ma = 2.54 GeV 102 103 104 105 \|ℳ\|2 Γ [GeV-1] Figure 4. Dalitz plot density for six values of ALP masses used for the a →π+π−η decay. Figure 4. Dalitz plot density for six values of ALP masses used for the a →π+π−η decay. For a given ALP mass ma, we use \|M\|2 a→hadrons/Γa→hadrons from ref. [18] to weight a ﬂatly distributed Dalitz-plot density. The procedure is repeated for each ma bin. As an example, the density for the decay a →π+π−η is shown in ﬁgure 4. 4.3 Propagation of decay products The photons, either emitted as daughter particles of the ALP or as tertiary decay products, are propagated in straight line up to the upstream face of the forward calorimeter. When extrapolating any charged particle, we account for the bending induced by the spectrometer analyzing magnets, if present: we treat the ﬁeld as that of an ideal solenoid and do not model eﬀects due to detailed ﬁeld maps, stray ﬁelds, etc. Photons and e± are assumed to release all their energy in the calorimeter. No resolution eﬀects are simulated and any experimental condition is directly applied on the photon or e± energy. A minimum separation between photon or e± showers is required for “cluster counting”: a pair of energy releases is merged into a single “cluster” if the relative distance of the calorimeter impact points is below an experiment-dependent radius. In this case, the cluster energy is the energy sum of the two merged particles. For cluster counting, the particle impact point is required to be within the calorimeter sensitive region and an experiment-dependent minimum energy is required. Muons are extrapolated forward up to the front face of a muon detector, if present. For muon detection, the muon impact point at the front face of the muon detector is required to be in the sensitive region. The presence – 12 – Experiment Status Ebeam NPOT Target ln,DV lc,DV zDV θoﬀ Ωcov [GeV] [1018] [m] [m] [m] [mrad] [µsr] CHARM completed 400 2.4 Cu 35 35 480 10 34 NuCal completed 70 1.7 Fe 23 23 64 0 700 NA62 running 400 10 Cu 139 81 82 0 84 DUNE ND6 proposed 120 1100 C 10 10 575 0 36 DarkQuest proposed 120 1.44 Fe 13.5 1 5 0 12000 SHADOWS proposed 400 10 Cu 26 23 10 75 9200 SHiP proposed 400 200 Mo 50 28 45 0 4500 Table 2. Overview of the basic parameters for the experimental setups considered in this work. See text for details and deﬁnitions. JHEP07(2022)094 Table 2. Overview of the basic parameters for the experimental setups considered in this work. See text for details and deﬁnitions. of a passive shield in front of a muon detector is only accounted for by applying a loose minimum-energy threshold. of a passive shield in front of a muon detector is only accounted for by applying a loose minimum-energy threshold. 6Since the operational time is not yet decided, we evaluate the data sets for one year of data-taking at DUNE. Our data sets can be easily re-scaled and the resulting contours in 6 correspond to ten years of operation. In ref. [4], a ten year operation is considered with a larger number of POT integrated per year than that considered here. We assume the DUNE near detector to be operated on-axis. Changing the detector vertical position in our simulation is straightforward and can be performed on request. 5.1 Results from past experiments To explore future opportunities, ﬁrst of all we need to recast the results of the past CHARM and NuCal experiments in terms of their sensitivity for ALPs. 4.3 Propagation of decay products It should be emphasized that we consider ALP decays into photons not only in the decay volume but also in the spectrometer area, eﬀectively enlarging the decay volume up to the calorimeter location. 5 Experimental framework In this section we brieﬂy discuss how we have modelled the experimental setups considered in this work and the related acceptance conditions. Table 2 provides an overview of the basic parameters used for each experiment. A beam of protons with energy Ebeam is made to interact with an absorber based on the element listed in the column “Target”. The total number of protons on target (POT) is denoted by NPOT. The experiment should be sensitive to ALPs produced in the target and reaching a decay volume (DV) located zDV meters downstream. The longitudinal axis of the DV has an angle θoﬀwith respect to the beam axis, and Ωcov denotes the solid angle covered by the calorimeter. Neutral (charged) ALP-daughter particles can be detected if the ALP decays within ln(c),DV meters from the DV entrance. Details are given in the following subsections. 5.1.1 CHARM For CHARM (using a copper target), an oﬀ-axis search for two photons in the ﬁnal state was presented in ref. [39], with an average eﬃciency of 0.51. The search required at least – 13 – one photon detected at the calorimeter. The sensitive area of the calorimeter has transverse dimensions of 3 m × 3 m. In the analysis quoted, the shower energy was required to be between 5 and 50 GeV and this condition has been implemented in our model. The same reference includes results for the detection of ﬁnal states with two daughter muons.7 The detection eﬃciency is reported to be 0.85. In our simulation, the muons are required to have at least 1 GeV at the ﬁnal detector plane in order to be seen as minimum ionizing particles throughout the detector material. Regarding hadronic ﬁnal states, we are only aware of on-axis searches, which go hand in hand with a larger background rate. Ref. [40] reports around 80 muon-less events based on a statistics of 7 × 1017 POT [41], after requiring a shower-like energy release of more than 20 GeV. According to the quoted analysis, 4 ± 21 events can be attributed to the interaction of other neutrinos or neutrino-like particles. Due to the limitation in statistics and due to the uncertainty induced by the subtraction of the background, the strongest exclusion by CHARM on hadronic decay modes is again given by a re-interpretation of the search for decays to ﬁnal states with emitted photons (ref. [39]): ﬁnal states such as π+π−γ, π+π−π0, and π+π−η are considered and at least one and at most two photons are required to be detected in the calorimeter, while all of the charged hadrons are required to escape the volume. JHEP07(2022)094 5.1.2 NuCal The sensitivity of NuCal to ﬁnal states with photons was evaluated in ref. [42], requiring the detection of one electromagnetic shower. While NuCal operated with a 70 GeV proton beam, it beneﬁted from a relatively small distance between target and detector (64 m) and from a comparably large decay volume length (23 m). After rejecting hadronic-like showers, NuCal observed 1 event compared to a background expectation of 0.3 events. ALP signals corresponding to a prediction of more than 3.6 events are excluded with a conﬁdence level of 90%. For the detection of a two-track ﬁnal state, we follow the procedure from ref. [43] and require both tracks to reach a circle of 2.6 m diameter at the end of the decay volume with a combined energy of at least 10 GeV. As in ref. [43], we assume an overall detection eﬃciency of 0.7 for the photonic ﬁnal state, and 0.8 for the di-muon ﬁnal state. By contrast, we are not aware of corresponding results for hadronic ﬁnal states in NuCal. Thus, we proceed similarly as for CHARM: we reinterpret the search for ﬁnal states with photons as appropriate (i.e. if the ﬁnal state contains at least one γ, π0, or η) and require that the charged hadrons escape detection. 7This is in contrast to the simulation performed in ref. [24], where it was mistakenly assumed that CHARM would be sensitive also to ﬁnal states with just one muon. 5.2.1 NA62 The primary goal of NA62 is the precise measurement of the rare decay of K+ →π+ν¯ν, but the experiment is also sensitive to a variety of exotics scenarios. To search for neutral ﬁnal states, NA62 has to be run in beam-dump mode, otherwise the residual background can limit the sensitivity. For a dump-mode run, the standard T10 target of NA62 must be removed, and the movable TAX collimators located 22 m downstream of T10 must be closed and used to dump the proton beam. This operation has been validated during the 2016–2018 data-taking and about 1016 POT have been collected in beam-dump mode. Triggers sensitive to decay channels with neutral and charged daughter particles have been deployed. During the current 2021–2023 run ∼1018 POT will be collected in beam dump mode. Around 1017 POT have been collected in 2021 [45]. Here we discuss the implications of collecting data in beam dump mode in the data-taking period between the so-called long-shutdown 3 (LS3) and LS4 to obtain ∼1019 POT.8 We assume no background limitation, in line with the present knowledge based on 2021 data [45]. JHEP07(2022)094 We model NA62 in a toy MC as follows: the beam-deﬁning collimator for ‘regular’ data- taking is used to dump the beam and its distance to the start of the ﬁducial volume is 82 m. A decay region in vacuum contains a spectrometer with a ﬁrst station 81 m downstream of the ﬁducal volume entrance. The spectrometer hosts a 0.7 T solenoidal magnet 95 m downstream of the entrance of the decay volume. The Liquid Krypton Calorimeter (LKr) is located about 139 m downstream of the entrance of the decay volume and it is modelled as an octagon, see e.g. ref. [47]. For the neutral ﬁnal states, we require the following acceptance conditions: both photons from the ALP decay must be detected at a minimum mutual distance of 10 cm at the LKr plane. In addition, the energy of each photon must be above 1 GeV and each photon impact point at the LKr must be at least 15 cm away from the LKr central hole through which the beam-pipe passes. Finally, the energy sum of the two photons must exceed 3 GeV. The NA62 target material (i.e. the material of the upstream section of the ﬁnal collimator) is copper. 5.2.1 NA62 For the di-muon decay, we require both muons to be in the acceptance of the NA62’s ﬁrst and last tracking stations. The acceptance loss due to the central hole allowing the passage of the beam pipe is modelled, cf. also ref. [24]. Each of the muons should have at least 5 GeV in order to be tracked eﬃciently. Finally, for hadronic ﬁnal states that include photons, not only the π+ and π−must be tracked, but also the additional requirement is made that all photons reach the calorimeter and deposit at least 3 GeV of combined energy. 8Note that there are several projects in consideration for NA62 in the post-LS4 period which would allow collection of even larger statistics [46]. These are not considered in this work. 5.2 Acceptance of current experiments The only operational proton beam experiment that we will consider in the following is NA62. Some ﬁrst data taken in beam dump also exist from the KOTO experiment [44]. The modelling of KOTO might be easily added to our code. Doing so would however require us to re-run and validate the PYTHIA simulation for a 30 GeV beam energy, which is non-trivial because of limited available data. – 14 – 10While this work was undergoing ﬁnalization, an independent study of the DarkQuest sensitivity to ALPs has appeared [54], using 7-8 m as ﬁducial volume (following [53], whereas our ﬁducial volume deﬁnition follows [52]). Apart from this major diﬀerence in the acceptance simulation, there are slight diﬀerences regarding the overall required energy and the photon separation. 5.3.1 SHiP Since the envisaged geometry for SHiP has changed since the publication of the SHiP proposal [48], we follow the layout of ref. [49] for our estimates. The prospects for detection of photons from ALP decays in the SHiP calorimeter is modelled as follows. The ﬁducial region is taken to be 45 m downstream of the production point of the ALPs and contains a spectrometer with the ﬁrst spectrometer station located 50.76 m after the entrance of the decay volume and a 0.15 T magnet. The calorimeter is situated after the forth spectrometer station,9 which is 60.16 m downstream of the entrance of the decay volume. JHEP07(2022)094 We ask both photons to be in an acceptance area of 5 m × 10 m. The energy of each photon must be above 1 GeV. The sum of the photon energies should be above 3 GeV. The photon impact points at the calorimeter should be at least 10 cm apart. The target material is molybdenum and the POT are 1020. Note that the proposed SHiP calorimeter has the potential of reconstructing the photon direction, which allows for an ALP mass reconstruction. To model the detection of charged particles in SHiP we mimic the steps provided in ref. [50]. The decay vertex must lay in the decay volume and the two tracks must hit all spectrometer chambers in their sensitive volume, with a minimum distance of 5 cm away from the decay vessel walls. Each particle must have a minimum energy of 5 GeV. Finally, for hadronic ﬁnal states that include also photons, all photons should reach the calorimeter and deposit at least 3 GeV of combined energy. 5.3 Acceptance of proposed experiments To evaluate the sensitivity of future experiments, we base our analysis on the available pro- posals. Some of the endeavours we considered are more deﬁned, while others might be subject – 15 – even to signiﬁcant change in the future. Moreover, some of the proposed experiments may face non-negligible backgrounds, which we do not attempt to estimate in the present work. 9The exact distance between the calorimeter and the last spectrometer station is not stated in the used literature, so we use an approximate value of 50 cm inferred from the schematics. 9The exact distance between the calorimeter and the last spectrometer station is not stated in the used literature, so we use an approximate value of 50 cm inferred from the schematics. 10While this work was undergoing ﬁnalization, an independent study of the DarkQuest sensitivity to ALPs has appeared [54] using 7-8 m as ﬁducial volume (following [53] whereas our ﬁducial volume deﬁnition 12The DUNE ND will also have the opportunity to take data up to 60 mrad oﬀ-axis as so-called DUNE- PRISM. This option is not considered in this work since the amount of time allocated to oﬀ-axis operation is unknown. We would like to stress, however, that since in certain scenarios ALPs in beam dumps are dominantly produced oﬀ-axis, DUNE-PRISM can have a large potential for this type of hidden sector physics searches even if operated for a short period of time. 11Note that this requirement goes slightly beyond that made in ref. [52], where no minimum energy requirement is made. There, instead, only the transverse pT kick induced by KMAG and the third tracking station are considered. 5.3.2 DarkQuest To make projections for DarkQuest, we consider the proposed extension of the SeaQuest experiment [51], particularly the proposed phase-I parameters. Details of the DarkQuest setup can be found in ref. [52]. The target material is iron. Following ref. [53], we assume for the di-photon ﬁnal state the need to detect 10 signal events to claim a signal beyond the background ﬂuctuations. It is assumed that the experiment is upgraded with a calorimeter placed between tracking stations 3 and 4 (at ∼18.5 m downstream of the target). The ﬁducial volume entrance is 5 meters downstream of the target.10 The ﬁrst spectrometer station is located about 6 m from the target and a geometric acceptance of 2 m × 2 m is assumed in the transverse direction. We assume using the “phase-I statistics” of 1.44 × 1018 POT. As with the other setups, we require a minimum energy of 1 GeV for each detected photon and a total energy of at least 3 GeV and — given the photon shower Moliere radius – 16 – — a minimum mutual distance of 10 cm at the calorimeter plane to avoid shower overlap. For the di-muon ﬁnal state, we model tracking in the simulation using the KMAG magnet and impose that the particles be in the acceptance of the ﬁrst and third tracking stations. In addition we require 1 GeV of minimum energy for each muon so that it is not stopped in the iron absorber.11 Following ref. [51], muon track pairs should be detected with a ∼60% eﬃciency for di-muon masses above 4.2 GeV. Here, we do not consider the possibility to also detect hadronic ﬁnal states, as the KL background expected for the phase-I set-up is expected to be a limiting factor [52]. 5.3.3 DUNE ND JHEP07(2022)094 Motivated by the recent results of ref. [4] we also include the DUNE near detector [55] (ND) in our study. DUNE ND uses a neutrino beam produced at the Long Baseline Neutrino Facility (LBNF) at Fermilab by a 120 GeV proton beam impacting on a graphite target with integrated intensity of 1.1 × 1021 POT per year [56]. We note that the nominal intensity can increase with further updates and the target material can be changed to beryllium [56]. Since DUNE ND is located underground separated by approximately 0.5 km of earth from the target, it can also eﬀectively serve as an on-axis12 proton beam dump experiment. The essential parts of DUNE ND used in our analysis are the Liquid Argon detector (ND-LAr) and a multipurpose detector based on gaseous argon (ND-GAr). The ND-LAr is a 5 m long time projection chamber with height of 3 m and width of 7 m located 574 m downstream of the target. The ND-GAr located right downstream of the ND-LAr is composed of a 5 m long cylindrical high pressure gaseous time projection chamber of 5.2 m diameter surrounded by an electromagnetic calorimeter, in a 0.5 T magnetic ﬁeld and a muon system. In this analysis we treat the ND-LAr and ND-GAr as two independent decay volumes. Following the reconstruction in ref. [55], we allow the ALP decays to happen in 6 m × 3 m × 2 m volume of ND-LAr, excluding 50 cm from the sides and upstream and 150 cm downstream corresponding to roughly 5 cm × 9 cm Moliere radius to allow a full reconstruction of the showers. For ND-GAr we assume a 4 m long, 2 m high and 4.8 m wide block located 580 m from the target and exclude the outer layers following the exclusion in the active volume calculation of ref. [55]. The photons in the 2γ decays in the ND-GAr must have a minimum 0.8 deg (14 mrad) separation angle and a minimum 20 MeV energy each. Charged hadrons from hadronic ALP decays must have a minimum kinetic energy of 5 MeV, based on the measured resolution [55]. For decays taking place in the volume of ND-LAr we do not put any conditions on the minimum energy until the expected performance of the detector based on the ProtoDUNE [57] results is known. 5.3.4 SHADOWS SHADOWS is a proposed oﬀ-axis experiment in the NA62 experimental cavern, which can take data concurrently with NA62 operated in beam dump mode. It would use a 400 GeV proton beam dumped in the copper TAXes and can take up to 1019 POT in the data-taking period between LS3 and LS4. The layout of the detector we have employed is based on the 2021 PBC proposal [58] and can be subject to changes in the near future. The decay volume starts 10 m downstream of the target and accommodates a 2.5 m long spectrometer with four tracking stations. The spectrometer is located 33 m downstream of the target. The distance among the ﬁrst two stations is 50 cm. A 1.5 m gap between the second and the third station is foreseen to accommodate a 1 T dipole magnet. The fourth station is 50 cm downstream of the third. A calorimeter is located about 50 cm behind the fourth spectrometer station. The calorimeter sensitive area has a rectangular shape of 2.5 m × 2.5 m and in our implementation the detector is oﬀaxis by θoﬀ= 75 mrad, so that the calorimeter center is shifted by 2.25 m from the beam axis. The calorimeter is likely to be succeeded by a muon detector13 since muons generated in the TAXes present the main background component based on the preliminary studies in ref. [58]. In this analysis we optimistically assume negligible background, which may overestimate the sensitivity. The request on muons is similar to that of NA62 and SHiP: two spectrometer chambers should be hit as well as a minimum energy of 5 GeV for each particle. JHEP07(2022)094 5.3.3 DUNE ND – 17 – For muons in DUNE, following ﬁgure 2.30 of [55], we assume muons with energies below 1 GeV are reconstructed with full eﬃciency. Above those energies, the angle w.r.t. to the beam axis must be smaller than 40 deg (700 mrad). 13We place the muon detector plane 1 m behind the calorimeter plane. 14This conclusion might change when considering direct couplings of the ALPs to leptons. We provide all necessary data sets as well as the implementation in the code to consider such a scenario. Note however that we do not currently consider ALP decays via the Bethe-Heitler process [59], which may be relevant in the case of ALPs coupled dominantly to leptons. 6 Results [26]), except that it includes additional interactions between ALPs and Z bosons, which are however of no relevance for ﬁxed-target experiments. The second scenario additionally predicts FCNC processes involving ALPs, eﬀectively enhancing the ALP production. The third scenario features ALP-meson mixing and decays of ALPs into hadronic ﬁnal states. Finally, the fourth scenario, ﬁrst proposed in ref. [19] and explored further in ref. [4] investigates the potential interplay between the diﬀerent couplings. JHEP07(2022)094 In order to predict ALP signals in beam dump experiments, one ﬁrst needs to obtain explicit expressions for the functions fi(C, Cref), which describe how the contributions from the diﬀerent production channels depend on the fundamental interactions in eq. (6.1). These functions are readily available in the literature and reproduced in appendix A. The second step is to calculate the ALP decay length and branching ratios as a function of the couplings and the ALP mass. In most cases the dominant decay mode will be the one into a pair of photons, with decays into leptons only appearing at the one-loop level and therefore giving a negligible contribution.14 For scenarios with Cgg ̸= 0 and ma > 3mπ there will also be a relevant contribution from three-body decays into hadronic ﬁnal states. Additional details are provided in appendix B. Once the functions Mif have been obtained, it is straight-forward to calculate the predicted number of ALP events Ndet for each experiment as a function of the fundamental couplings and the ALP mass. We provide these numbers in tabulated form together with this work for convenience. In the remainder of this section we show the exclusion limits (projected sensitivities) for past (future) experiments at 90% conﬁdence level. We ﬁrst consider the case that only the di-photon ﬁnal state can be detected and subsequently explore the improvement in reach that can be achieved when also hadronic three-body ﬁnal states are considered. 6 Results Having obtained the model-independent functions Eif by combining the diﬀerential ALP yields from section 3 with the various ALP decay models discussed in section 4 and the experimental set-ups considered in section 5, we are now in the position to calculate the predicted number of ALPs in a given experiment for any ALP model for which the model- dependent functions Mif = fi(C, Cref)BRa→f(ma, C) are known. The necessary formalism has been worked out in great detail for a broad class of ALP eﬀective theories that include interactions of ALPs with SM quarks and leptons, as well as gauge and Higgs bosons. In the present work we will illustrate this procedure for the case that the ALP interacts dominantly with SM gauge bosons: La,int = g′2 CBB Λ aBµν ˜Bµν + g2 CWW Λ aW µν ˜Wµν + g2 s Cgg Λ aGµν ˜Gµν. (6.1) (6.1) Here g′, g and gs denote the hypercharge, weak and strong gauge couplings respectively, Xµ = Bµ, W µ, Gµ denote the corresponding gauge ﬁelds (with the group generator index suppressed), Xµν denotes the ﬁeld strength tensor and ˜Xµν = 1 2ϵµνρσXρσ denotes its dual. – 18 – Rather than varying all three couplings independently, we will consider four diﬀerent benchmark scenarios: Rather than varying all three couplings independently, we will consider four diﬀerent benchmark scenarios: (i) B dominance: CBB ̸= 0; CWW = Cgg = 0 (ii) W dominance: CWW ̸= 0; CBB = Cgg = 0 (iii) Gluon dominance: Cgg ̸= 0; CBB = CWW = 0 (iv) Co-dominance: CBB = CWW = Cgg ̸= 0 The ﬁrst three scenarios are chosen to highlight the diﬀerent production channels. In particular, the ﬁrst scenario is similar to the frequently studied case of photon dominance (see e.g. ref. [26]), except that it includes additional interactions between ALPs and Z bosons, which are however of no relevance for ﬁxed-target experiments. The second scenario additionally predicts FCNC processes involving ALPs, eﬀectively enhancing the ALP production. The third scenario features ALP-meson mixing and decays of ALPs into hadronic ﬁnal states. Finally, the fourth scenario, ﬁrst proposed in ref. [19] and explored further in ref. [4] investigates the potential interplay between the diﬀerent couplings. The ﬁrst three scenarios are chosen to highlight the diﬀerent production channels. In particular, the ﬁrst scenario is similar to the frequently studied case of photon dominance (see e.g. ref. 6.1 Considering only di-photon decays For both scenarios (i) and (ii) one ﬁnds BR(a →γγ) ≈1 since the one-loop processes a →ℓℓare suppressed by many orders of magnitude. Moreover, since we assume Cgg = 0 in both cases, the production via meson mixing vanishes (fP = 0), while fγγ ̸= 0 thanks to the contribution from CBB and CWW to the eﬀective ALP-photon coupling (see appendix A). – 19 – 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] CBB/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CWW = Cgg = 0 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] CWW/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CBB = Cgg = 0 Figure 5. 90% CL exclusions limits for scenarios (i) (left) and (ii) (right). The exclusion from past experiments which accounts for signal and background studies is indicated by a full contour while the future experiments, assuming a zero background hypothesis, are indicated by a corresponding line. 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] CWW/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CBB = Cgg = 0 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] CBB/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CWW = Cgg = 0 JHEP07(2022)094 Figure 5. 90% CL exclusions limits for scenarios (i) (left) and (ii) (right). The exclusion from past experiments which accounts for signal and background studies is indicated by a full contour while the future experiments, assuming a zero background hypothesis, are indicated by a corresponding line. The only diﬀerence between the two scenarios is that for (i) there are no FCNCs and hence fBK(⋆) = 0, whereas they give an important contribution for (ii). Likewise, the contribution of fDπ vanishes for (i) and gives a marginal contribution for (ii) (see appendix A). The only diﬀerence between the two scenarios is that for (i) there are no FCNCs and hence fBK(⋆) = 0, whereas they give an important contribution for (ii). Likewise, the contribution of fDπ vanishes for (i) and gives a marginal contribution for (ii) (see appendix A). 16Note that we include the two-loop contribution to B →K(⋆) + a from ALP-gluon couplings ﬁrst calculated in ref. [35]. See appendix A for details. 15Note that the slight diﬀerences in reach compared to ref. [26] can be mainly attributed to the inclusion of η′ decays and of a wider range of meson momenta. 6.1 Considering only di-photon decays 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP Cgg = CWW = CBB mπ mη mη′ 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CBB = CWW = 0 mπ mη mη′ JHEP07(2022)094 Figure 6. 90% CL exclusions limits for scenarios (iii) (left) and (iv) (right) assuming a di-gamma search only. in the exclusion plots whenever the ALP mass approaches one of the pseudoscalar meson masses. Note that if the two masses become very close, the condition \|θaP \| ≪1 no longer holds. These regions are indicated by a dark shading and masked in our analysis. In scenario (iv) there furthermore occurs a partial cancellation in the eﬀective ALP- photon coupling, see eqs. (A.5) and (A.14), which has two eﬀects: ﬁrst of all, for small ALP masses (ma ≲100 MeV) it shifts all exclusion contours to somewhat larger couplings, which are necessary to achieve comparable ALP production yields. Second, it leads to additional “poles” outside of the masked regions whenever the diﬀerent contributions to Cγγ cancel accidentally. 6.1 Considering only di-photon decays The existing exclusion limits and projected sensitivities for the two scenarios are compared in ﬁgure 5.15 As expected, all experiments under consideration follow a similar pattern, with the shape of the (projected) exclusions dictated by the ALP decay length (which decreases for large couplings and large ALP masses) and the production cross section (which decreases for small couplings and large ALP masses). Another notable feature in these plots is the low-mass cut-oﬀin sensitivity for most future experiments. This cut-oﬀ results from the required separation distance between the two photons in the ﬁnal state, which decreases for smaller ALP masses and correspondingly larger boost factors. One ﬁnds that in order to extend the reach towards smaller couplings, the decisive quantity is the assumed POT, which is largest for DUNE and SHiP. To make improvements towards larger couplings and masses, the beam energy and detector geometry are decisive. In particular, we observe that the contribution from rare B meson decays in (ii) is especially relevant for experiments with Ebeam = 400 GeV and clearly favours oﬀ-axis experiments like SHADOWS as well as on-axis experiments with large angular coverage like SHiP. In scenarios (iii) and (iv) the gluon coupling is non-zero and therefore all production channels considered in this work become relevant.16 Moreover, one ﬁnds BR(a →γγ) ≪1 for ma > 1 GeV where hadronic decays dominate [18]. The eﬀects of ALP-meson mixing in both production and decay is clearly visible in ﬁgure 6 and imprints a “pole structure” – 20 – 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP CBB = CWW = 0 mπ mη mη′ 10-4 10-3 10-2 10-1 100 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP Cgg = CWW = CBB mπ mη mη′ Figure 6. 90% CL exclusions limits for scenarios (iii) (left) and (iv) (right) assuming a di-gamma search only. 6.2 Including hadronic decays For scenarios with ALP-gluon couplings the branching ratio into photons is strongly suppressed for heavy ALP masses and whenever there is a cancellation in the eﬀective ALP-photon coupling. The incorporation of hadronic decays is therefore essential to probe these parameter regions and utilize the full potential of beam dump experiments for ALP searches. This can be seen in ﬁgure 7, which compares the exclusions and sensitivities for the di-photon channel only and the combination of all channels. We ﬁnd that the improvement in sensitivity is most signiﬁcant for SHiP, where the reach in terms of the ALP mass is extended by almost a factor of 2. The reason for this is simply that SHiP is the overall most sensitive experiment and therefore most dependent on the branching ratios of GeV-scale ALPs. Nevertheless, the eﬀect of including hadronic ﬁnal states is clearly visible also for all other experiments that we consider. We emphasize that even experiments vetoing hadronic ﬁnal states can partially cover regions with suppressed decays into photons thanks to hadronic decays with only photons in the ﬁnal state and with charged hadrons escaping detection (provided that a partial reconstruction of the event is allowed). For experiments sensitive to a range of diﬀerent – 21 – 100 10-10 10-9 10-8 10-7 10-6 10-5 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP 2γ only Cgg = CWW = CBB mη mη′ 100 10-10 10-9 10-8 10-7 10-6 10-5 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP 2γ only CWW = CBB = 0 mη mη′ 100 10-10 10-9 10-8 10-7 10-6 10-5 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP 2γ only Cgg = CWW = CBB mη mη′ Figure 7. 90% CL exclusions limits for scenarios (iii) (left) and (iv) (right) given by a di-gamma decay only search (dashed line), compared with the exclusion when all mentioned hadronic channels are also taken into account (full line). 100 10-10 10-9 10-8 10-7 10-6 10-5 ma [GeV] Cgg/Λ [GeV-1] NuCal CHARM NA62 DUNE DarkQuest SHADOWS SHiP 2γ only CWW = CBB = 0 mη mη′ JHEP07(2022)094 ma [GeV] Figure 7. 90% CL exclusions limits for scenarios (iii) (left) and (iv) (right) given by a di-gamma decay only search (dashed line), compared with the exclusion when all mentioned hadronic channels are also taken into account (full line). 6.2 Including hadronic decays ﬁnal states, on the other hand, our analysis highlights the exciting possibility that beam dump experiments may not only discover ALPs, but also infer their dominant branching ratios, giving us crucial clues regarding the underlying model. 7 Conclusions The search for light feebly interacting particles at experiments with low centre-of-mass energy but high intensity is one of the most exciting frontiers of modern particle physics. A particularly attractive target are axion-like particles (ALPs), for which the smallness of the mass and the weakness of the interactions can simultaneously be motivated by their origin as Pseudo-Goldstone bosons of a spontaneously broken global symmetry. However, the wide range of possible ALP models raises the question how to systematically explore the parameter space and how to present experimental results in a model-independent way. In this work we have addressed this challenge by developing a novel framework that al- lows to evaluate constraints and projected sensitivities from proton beam dump experiments for diﬀerent ALP models without the need to re-run expensive Monte Carlo simulations. This is achieved by splitting the calculation into model-independent parts, which are com- putationally hard but can be performed and tabulated in advance, and model-dependent parts, which only require simple analytical rescaling functions. Speciﬁcally, we have shown that ALP production can be written as a sum over a number of diﬀerent production channels, each of which scales in a well-deﬁned way with the eﬀective ALP parameters at low energies. Most of these production channels require Monte Carlo simulations of the hadron shower created by the incident proton in the absorber and the subsequent decays of the resulting mesons. We have performed all necessary simulations using state-of-the-art tools and explicitly validated the resulting cross sections. – 22 – Regarding ALP propagation and decay, there are two main diﬃculties. The ﬁrst one is to accurately simulate ALP three-body decays, which become important for GeV-scale ALPs with hadronic couplings. The second diﬃculty is to estimate the detection probability for a given ﬁnal state in a wide range of experiments. To simulate three-body decays, we have developed a Monte Carlo technique based on the reweighting of Dalitz plots obtained from analytical calculations of hadronic ALP decays in chiral perturbation theory. Detector eﬃciencies are then estimated by propagating the decay particles through simpliﬁed detector geometries, which we have implemented for a range of past, present and future experiments. To illustrate our approach we present exclusion limits and sensitivity projections for ALPs with couplings dominantly to Standard Model gauge bosons. To illustrate the impact of the detailed coupling structure, we consider four diﬀerent scenarios, called B dominance, W dominance, gluon dominance and co-dominance. 7 Conclusions We ﬁnd crucial diﬀerences between these four scenarios regarding the relative importance of diﬀerent production and decay channels. In particular, for scenarios with gluon couplings, we ﬁnd that experimental sensitivities can be signiﬁcantly extended by including hadronic ﬁnal states from three-body decays in the analysis. JHEP07(2022)094 The tabulated simulations and a Python script to perform the necessary rescaling for a user-deﬁned combination of the various ALP interactions with SM particles are publicly available at https://github.com/jjerhot/ALPINIST. This code allows for the calculation of experimental constraints and sensitivities for a wide range of ALP models. Furthermore, it can be easily extended to include additional experiments, production and decay channels. Further details are provided in appendix C. We emphasize that our implementation is still not fully model-independent. In particular the simulation of three-body decays presently assume the Dalitz plot densities predicted for ALPs with couplings to gluons and/or ﬂavour-independent couplings to SM quarks. Diﬀerent distributions are expected in more complicated new-physics scenarios, such as the one discussed in ref. [29]. We therefore aim to make also the code for Monte Carlo simulations publicly available, such that the user can easily modify decay distributions and experimental geometries. At the same time, we hope that independent Monte Carlo simulations will be developed by the experimental collaborations to produce similar tables to the ones used in our analysis based on more reﬁned detector simulations. This will ultimately allow for a complete reinterpretation of experimental results in the general parameter space of ALP models. A Model-dependent calculations Although our analysis in section 6 considers only couplings to B and W bosons and gluons (see eq. (6.1)), it is useful to start with a more general eﬀective Lagrangian of ALP-SM interactions: La ⊃1 2∂µa∂µa −1 2m2 aa2 + X q Cqq 2Λ (∂µa)¯qγµγ5q + X ℓ Cℓℓ 2Λ (∂µa)¯ℓγµγ5ℓ + g2 CWW Λ aW µν ˜Wµν + g′2 CBB Λ aBµν ˜Bµν + g2 s Cgg Λ aGµν ˜Gµν , (A.1) (A.1) JHEP07(2022)094 where Λ denotes the unknown scale of new physics (which may or may not be connected to the symmetry breaking scale [60]) and the parameters C denote the Wilson coeﬃcients of the various eﬀective operators. For the remainder of this discussion we will set Cqq = 0 at tree level. For a more detailed discussion of the eﬀects of ALP-quark couplings, we refer to ref. [22]. At energy scales below EW symmetry breaking the most relevant interactions of ALPs with electroweak gauge bosons will then have the following form: La,EW ⊃e2 Cγγ Λ aF µν ˜Fµν + 2e2 CγZ Λ aF µν ˜Zµν + 4g2 CWW Λ aεµναβ∂µW + ν ∂αW − β , (A.2) (A.2) where e = g sin θW = g′ cos θW and the Wilson coeﬃcients are given by Cγγ = CWW + CBB (A.3) CγZ = CWW cos2 θW −CBB sin2 θW sin θW cos θW . (A.4) (A.3) (A.4) At the one-loop level, Cγγ receives additional contributions from lepton and gauge boson loops, leading to At the one-loop level, Cγγ receives additional contributions from lepton and gauge boson loops, leading to Cγγ = CWW + CBB + X ℓ Cℓℓ 16π2 B1(4mℓ/ma) + 2α π CWW sin2 θW B2(4mW /ma) , (A.5) (A.5) where the functions B1,2(τ) are deﬁned in eq. (14) of ref. [20]. The analogous one-loop contributions to the eﬀective leptonic coupling Cℓℓ,eﬀare given in eq. (26) of ref. [20], which is also implemented in our code. Acknowledgments We thank Yotam Soreq for useful discussions and Gaia Lanfranchi for comments on our implementation of SHiP and SHADOWS. This work is funded by the Deutsche Forschungs- gemeinschaft (DFG) through the Collaborative Research Center TRR 257 “Particle Physics Phenomenology after the Higgs Discovery” under Grant 396021762 — TRR 257 and the Emmy Noether Grant No. KA 4662/1-1. This work is also supported through the European Research Council under grant ERC-2018-StG-802836 (AxScale project). JJ is supported through a FRIA grant by the F.R.S.-FNRS (Fonds de la Recherche Scientiﬁque — FNRS), Belgium. – 23 – A.1 FCNC transitions Flavour-changing interactions are generated from the a-W interaction at the one-loop level [15] and from the a-g interaction at the two-loop level [35]. The corresponding eﬀective interactions take the following form: Lb→sa ⊃−Cbs Λ (∂µa)¯slγµbl + h.c. , Lc→ua ⊃−Ccu Λ (∂µa)¯ulγµcl + h.c. . (A.6) (A.6) – 24 – The eﬀective FCNC couplings can be written as Cqq′ = CWW gqq′,W +Cgggqq′,g. The one-loop contribution from W bosons is given by The eﬀective FCNC couplings can be written as Cqq′ = CWW gqq′,W +Cgggqq′,g. The one-loop contribution from W bosons is given by gbs,W = 3α2 W X q=u,c,t VqbV ⋆ qsF(ξq) , gcu,W = 3α2 W X q=d,s,b VuqV ⋆ cqF(ξq) , (A.7) (A.7) where αW = g2/(4π), ξq = m2 q/m2 W and where αW = g2/(4π), ξq = m2 q/m2 W and F(x) = x [1 + x(ln x −1)] (1 −x)2 . (A.8) (A.8) JHEP07(2022)094 The two-loop contribution gbs,g has been calculated for the ﬁrst time in ref. [35]: gbs,g = α2 sαW 4π  f(µ) + 2ACF g(µ) + 2BCF X q=u,c,t VqbV ⋆ qsξq   (A.9) (A.9) with functions f(µ) and g(µ) given in eqs. (B6) and (B7) of ref. [35]. The analogous expression for gcu,g is obtained by the obvious replacements. In our code, we associate µ with the new-physics scale Λ, which can be speciﬁed by the user as well as the model-dependent parameters A and B. For the results presented in section 6, we set A = B = 0, which was shown in ref. [35] to yield neither overly optimistic nor overly conservative exclusions. We emphasize that both the contribution to the eﬀective FCNC couplings from W bosons and the contribution from gluons are approximately proportional to ξQ, where Q is the heaviest quark in the loop. The resulting decay rates are then proportional to ξ2 Q. This leads to a strong suppression of the c →u + a transition (where Q = b) relative to the b →s + a transition (where Q = t), such that the process B →K(⋆) + a turns out to be experimentally more important than D →π + a in spite of the greater D meson production cross section. A.2 ALP-meson mixing π /( ) The ALP-gluon coupling as well as ALP-meson mixing give rise to further contributions to the eﬀective ALP-photon coupling, which is found to be [61] The ALP-gluon coupling as well as ALP-meson mixing give rise to further contributions to the eﬀective ALP-photon coupling, which is found to be [61] Cγγ,eﬀ= Cγγ −1.92Cgg + ˜αs(m2 a)FVMD X P Λ 16π2fP θaP (A.14) (A.14) with fη ≈93 MeV and fη′ ≈73 MeV. The functions FVMD and ˜αs, deﬁned in eqs. (3.7) and (3.8) respectively, allow us to extrapolate the mixing eﬀects for ma > mη′. with fη ≈93 MeV and fη′ ≈73 MeV. The functions FVMD and ˜αs, deﬁned in eqs. (3.7) and (3.8) respectively, allow us to extrapolate the mixing eﬀects for ma > mη′. A.2 ALP-meson mixing At energies below the QCD conﬁnement scale, the ALP-gluon coupling gives rise to both kinetic and mass mixing of ALPs with the SM neutral pseudoscalars P ∈{π0, η, η′}. Removing this mixing requires a redeﬁnition of the various ﬁelds: P →P + θaP a (A.10) (A.10) with the mixing angle with the mixing angle with the mixing angle θaP = fπ fa KaP m2 a + m2 aP m2a −m2 P , (A.11) (A.11) where fπ = 93 MeV and fa = Λ/32π2 \|Cgg\| denote the pion and ALP decay constants.17 17We note that our deﬁnition of fa and the ALP-meson mixing is not invariant under a chiral rotation of the SM quark ﬁelds, even though such a rotation should not aﬀect physical observables. Rather than providing a more general treatment, we follow the common approach in the literature and assume that the chiral rotation is ﬁxed in such a way that ﬂavour-diagonal couplings to light quarks are absent. – 25 – The kinetic mixing terms are given by The kinetic mixing terms are given by Kaπ0 = −κu−κd 2 Kaη = − (κu+κd) 1 √ 3 cosθηη′− q 2 3 sinθηη′ −κs 2 √ 3 cosθηη′+ q 2 3 sinθηη′ 2 Kaη′ = − (κu+κd) 1 √ 3 sinθηη′+ q 2 3 cosθηη′ −κs 2 √ 3 sinθηη′− q 2 3 cosθηη′ 2 (A.12) (A.12) where θηη′ ≈−13◦is the η-η′ mixing angle and we make the convenient choice κq = m−1 q /Tr[m−1] with m = diag{mu, md, ms} to achieve maπ0 = maη8 = 0 [11]. The remaining mass mixing terms are where θηη′ ≈−13◦is the η-η′ mixing angle and we make the convenient choice κq = m−1 q /Tr[m−1] with m = diag{mu, md, ms} to achieve maπ0 = maη8 = 0 [11]. The remaining mass mixing terms are JHEP07(2022)094 JHEP07(2022)094 m2 aη = − √ 6B0 sin θηη′ Tr[m−1] m2 aη′ = √ 6B0 cos θηη′ Tr[m−1] (A.13) (A.13) where B0 = m2 π0/(mu + md). where B0 = m2 π0/(mu + md). where B0 = m2 π0/(mu + md). B Calculation of decay widths Based on the eﬀective couplings derived in appendix A we can calculate the various decay widths relevant for this study. We begin by noting that in all cases of interest the decaying particle has spin 0 and hence the squared matrix element does not depend on the phase space parameters, leading to an isotropic decay. Hence no information on the decay kinematics is lost when performing the phase space integration and it is suﬃcient to quote the corresponding decay width. The width of the decay a →γγ is given by Γa→γγ = C2 γγ,eﬀ Λ2 e4m3 a 4π (B.1) (B.1) with an eﬀective ALP-photon coupling Cγγ,eﬀ. For the ALP leptonic decays a →ℓℓ(ℓ= e, µ) with decay width 2 Γa→ℓℓ= C2 ℓℓ,eﬀ Λ2 m2 ℓ 8π q m2a −4m2 ℓ (B.2) (B.2) where mℓdenotes the lepton mass and Cℓℓ,eﬀthe eﬀective leptonic coupling. – 26 – – 26 – Other possible two-body decays for ALP masses up to 3 GeV are the hadronic decays a →ρρ, a →ωω, a →K⋆¯K⋆and a →φφ, which (in the absence of direct couplings to quarks) have a decay width proportional to C2 gg. Since these ﬁnal states are not detector- stable for ﬁxed-target experiments, we do not calculate them explicitly but we account for them in the decay widths for three-body decays. The corresponding decay widths used in this work employ amplitudes derived in ref. [18] using chiral perturbation theory and vector meson dominance. Furthermore, we are also interested in the production of ALPs via ﬂavour-changing two-body decays. The decay widths for the rare decays B →K(∗) + a can be calculated in terms of the ﬂavour-changing eﬀective couplings Cbs: JHEP07(2022)094 ΓB→K+a = \|Cbs\|2 Λ2 1 64πm3 B (m2 B −m2 K)2f2 0 (ma)λ1/2(mB, mK, ma) ΓB→K⋆+a = \|Cbs\|2 Λ2 1 64πm3 B A2 0(ma)λ3/2(mB, mK⋆, ma) , (B.3) (B.3) where λ(x, y, z) = x2 −(x + z)2 x2 −(y −z)2. Since we apply this result on a broad range of ALP masses, we use the following parametrization of the form factors [62, 63]: f0(q) = 0.330 1 −q2/37.46 , (B.4) A0(q) = 1.364 1 −q2/m2 B − 0.990 1 −q2/36.78 GeV2 , (B.5) (B.4) (B.5) which depend on the momentum transfer q. which depend on the momentum transfer q. B Calculation of decay widths In complete analogy, we ﬁnd for the decay D →π + a: ΓD→π+a = \|Ccu\|2 Λ2 1 64πm3 D (m2 D −m2 π)2f2 0 (ma)λ1/2(mD, mπ, ma) , (B.6) (B.6) where we use f0(0) = 0.612 [64] (see also ref. [65]). To account for the q-dependence we use an analogous parametrization as above: where we use f0(0) = 0.612 [64] (see also ref. [65]). To account for the q-dependence we use an analogous parametrization as above: f0(q) = f0(0) 1 −q2/m2 ﬁt (B.7) (B.7) For ﬁtted m2 ﬁt = 6.46 GeV2 we reproduce the mean values of ref. [64] with a relative error < 1%, which is suﬃcient for our purpose. For ﬁtted m2 ﬁt = 6.46 GeV2 we reproduce the mean values of ref. [64] with a relative error < 1%, which is suﬃcient for our purpose. C Brief description of the ALPINIST code Since the MC simulations of ALP production and decay are computing power demanding, our goal is to avoid as much as possible re-doing these simulations for each change in the model-dependent parameters. Thus we separate the code into several standalone modules, each of which processes the input tabulated data and passes the output data, again in the form of a table, to the next module in the sequence. This sequence is shown in the form of a diagram in ﬁgure 8, where the code-modules are represented by square boxes and the tabulated data by triangular boxes pointing in the direction of the data ﬂow. – 27 – ALP production ALP decay ALP rescale /tab prod /tab decay Pythia /tab mesons /tab gammas decay mesons decay width calculation /Dalitz /integrated /tab toPlot plots Lagrangian Figure 8. Diagram representing the internal structure of the ALPINIST framework. Pythia Lagrangian /Dalitz /integrated JHEP07(2022)094 Figure 8. Diagram representing the internal structure of the ALPINIST framework. • The ALP production module uses Wolfram Mathematica [66]. It corresponds to section 3, i.e. it generates the ALP distribution table for each production mode and for each experiment for a ﬁxed value of the model-dependent parameters. For the ALP production from mesons it reads directly the output from the PYTHIA simulations. For the production from on-shell photons, it reads the photon distributions generated by a separate module, which simulates 2γ decays of π0, η and η′ mesons. This module uses the ROOT framework [38]. • The ALP decay module also uses the ROOT framework libraries. It contains the main MC simulation described in sections 4 and 5, i.e. it handles the ALP decay kinematics for various decay modes in the conditions of the speciﬁc experiment. For the three- body decays it also requires the Dalitz plot densities, which are generated according to the speciﬁc Lagrangian (eq. (6.1) in this case) by a separate module. • The ALP rescale module requires only Python [67] and the input tables to be run. For the set of model-dependent parameters given by the user it generates the table with the number of signal events in the common ma-Ca plane used for plotting the exclusion limits. The equations used for the calculations can be found in appendices A, B as well as the references therein. C Brief description of the ALPINIST code The ALP rescale module is the most relevant one for most users and thus is provided publicly at https://github.com/jjerhot/ALPINIST together with its input tables. Other modules may be needed for potential updates of experimental conditions and for including new experiments, production or decay modes to the simulation. Access to the full repository can be obtained upon request from the authors. The speciﬁc instructions for using of each module can be found in the respective README.md ﬁles. D Treatment of kinematics for the mixing production The mixing production serves as an approximation to the various processes in which an ALP can be produced. The total yield of produced ALPs can be derived from the yield – 28 – of the neutral pseudoscalar mesons and the respective mixing angles with the ALP. This approach, however, does not say anything about the change of the kinematics when the ALP is produced instead of the meson. The general minimal adjustment, which can be applied irrespective of the speciﬁc process which lead to the ALP production, is the change of the kinematics when the mass of the original pseudoscalar is changed. Since the center-of-mass frame of the processes that lead to the meson production is unknown, we choose a common frame for the mass adjustment to be the p-p collision cm frame. We emphasize that there is a certain level of arbitrariness in this choice, with the obvious alternative being the laboratory frame. However, we expect that the p-p cm frame leads to a more accurate description for highly-energetic ALPs, where only a smaller portion of the available energy is spent on the ALP production and the majority of the energy is carried in the ALP boost. These highly boosted ALPs are particularly important for the sensitivity of beam dump experiment searches. JHEP07(2022)094 The cm momentum of a particle with mass m is then simply found as pcm(plab, m) = γbeamplab −γbeamβbeam q p2 lab + m2 (D.1) (D.1) pcm(plab, m) = γbeamplab −γbeamβbeam q p2 lab + m2 (D.1) and analogous relations can be used for the Ecm and, by inversion, for plab and Elab. By adjusting the particle mass one necessarily violates the momentum or energy conservation (or both) in the original process. In our simulation we ﬁx the momentum of the ALP in the p-p cm frame to be the same as the momentum of the original meson. The energy and angle of the ALP a in the laboratory frame then translates to the energy and angle of the original meson P in the laboratory frame as EP pﬁxed = q plab (pcm (pa, ma) , mP ) + m2 P , θP pﬁxed = arcsin sin(θa) pa plab (pcm (pa, ma) , mP ) . (D.2) (D.2) The plotted distributions for the case of P = η′ can be found in the upper row of ﬁgure 9. D Treatment of kinematics for the mixing production If one chooses to ﬁx the ALP energy to the meson energy in the p-p cm frame, one obtains in complete analogy the following relations: EP Eﬁxed = Elab (Ecm (pa, ma) , mP ) θP Eﬁxed = arcsin  sin(θa) pa q Elab (Ecm (Ea, ma) , mP )2 −m2 P  . (D.3) (D.3) The resulting distributions are plotted in the bottom row of ﬁgure 9. By comparing the two distributions, we can see that even in the borderline case of choosing the p-p collision as a universal cm frame, we can get a signiﬁcantly diﬀerent Ea-θa distribution solely by choosing a diﬀerent method of ﬁxing the kinematics in the cm frame. This choice, however, impacts mostly ALPs with a small boost (large masses and low energies), which are of limited relevance for beam dump experiments. The resulting distributions are plotted in the bottom row of ﬁgure 9. By comparing the two distributions, we can see that even in the borderline case of choosing the p-p collision as a universal cm frame, we can get a signiﬁcantly diﬀerent Ea-θa distribution solely by choosing a diﬀerent method of ﬁxing the kinematics in the cm frame. This choice, however, impacts mostly ALPs with a small boost (large masses and low energies), which are of limited relevance for beam dump experiments. – 29 – 0.0 0.5 1.0 1.5 2.0 2.5 3.0 50 100 150 200 250 300 ma [GeV] Ea [GeV] 0 50 100 150 200 250 300 Eη′ [GeV] 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.02 0.04 0.06 0.08 0.10 ma [GeV] θa [rad] 0 0.02 0.04 0.06 0.08 0.1 θη′ [rad] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 50 100 150 200 250 300 ma [GeV] Ea [GeV] 0 50 100 150 200 250 300 Eη′ [GeV] 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.02 0.04 0.06 0.08 0.10 ma [GeV] θa [rad] 0 0.02 0.04 0.06 0.08 0.1 θη′ [rad] Figure 9. Dependence of the energy (left) and the angle of the ALP (right) on the ALP mass in the laboratory frame for the case of the ﬁxed momentum (upper panel) or ﬁxed energy (lower panel) in the p-p cm frame when the original particle was the η′ meson, produced by a 400 GeV proton beam. The angular distribution is evaluated for the case Ea = 100 GeV. D Treatment of kinematics for the mixing production 0.0 0.5 1.0 1.5 2.0 2.5 3.0 50 100 150 200 250 300 ma [GeV] Ea [GeV] 0 50 100 150 200 250 300 Eη′ [GeV] 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.02 0.04 0.06 0.08 0.10 ma [GeV] θa [rad] 0 0.02 0.04 0.06 0.08 0.1 θη′ [rad] JHEP07(2022)094 ma [GeV] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 50 100 150 200 250 300 ma [GeV] Ea [GeV] 0 50 100 150 200 250 300 Eη′ [GeV] 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.02 0.04 0.06 0.08 0.10 ma [GeV] θa [rad] 0 0.02 0.04 0.06 0.08 0.1 θη′ [rad] Figure 9. Dependence of the energy (left) and the angle of the ALP (right) on the ALP mass in the laboratory frame for the case of the ﬁxed momentum (upper panel) or ﬁxed energy (lower panel) in the p-p cm frame when the original particle was the η′ meson, produced by a 400 GeV proton beam. The angular distribution is evaluated for the case Ea = 100 GeV. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. SCOAP3 supports the goals of the International Year of Basic Sciences for Sustainable Development. [1] P. Agrawal et al., Feebly-interacting particles: FIPs 2020 workshop report, Eur. Phys. J. C 81 (2021) 1015 [arXiv:2102.12143] [INSPIRE]. [1] P. Agrawal et al., Feebly-interacting particles: FIPs 2020 workshop report, Eur. Phys. J. C 81 (2021) 1015 [arXiv:2102.12143] [INSPIRE]. [2] J. 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https://openalex.org/W2346376459	https://europepmc.org/articles/pmc4861599?pdf=render	English	null	Decoding obesity in the brainstem	eLife	2,016	cc-by	2,713	JONATHAN CEDERNAES AND JOSEPH BASS neurons within the brainstem have received less attention. Specifically, we don’t know how the parts of the hypothalamus that respond to leptin communicate with brainstem regions that respond to more short-term signals of satiety. The specific pathways of neurons that carry and process signals from the gut have also remained less characterized. Related research article D’Agostino G, Lyons DJ, Cristiano C, Burke LK, Madara JC, Campbell JN, Garcia AP, Land BB, Lowell BB, Dileone RJ, Heisler LK. 2016. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. eLife 5:e12225. doi: 10.7554/eLife.12225 Image Nerve fibers (red) that originate in the lower brainstem terminate next to a subset of neurons (green) in the hypothalamus Related research article D’Agostino G, Lyons DJ, Cristiano C, Burke LK, Madara JC, Campbell JN, Garcia AP, Land BB, Lowell BB, Dileone RJ, Heisler LK. 2016. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. eLife 5:e12225. doi: 10.7554/eLife.12225 Image Nerve fibers (red) that originate in the lower brainstem terminate next to a subset of neurons (green) in the hypothalamus Now, in eLife, Giuseppe D’Agostino, Lora Heisler and colleagues – who are based at the Universities of Aberdeen and Cambridge, Har- vard Medical School and Yale University School of Medicine – advance this area of study by genetically dissecting a circuit of neurons that originates from the brainstem (Figure 1). D’Agostino et al. report how this circuit contrib- utes to satiety and how it integrates with long- term control of appetite and energy balance via direct connections with neurons in the hypothal- amus (D’Agostino et al., 2016). W W hen an obese person loses weight they will often continue to experience hunger after they stop dieting and merely try to maintain a lower body weight (Sumithran et al., 2011). This situation exempli- fies the complexity of regulating appetite and energy balance. Feelings of hunger or satiety result from integrating a wide range of signals from our environment with a similarly large spec- trum of signals from inside the body. These inter- nal signals might be nerve impulses from the gut, nutrient levels in the blood, or protein-based hor- mones. For example, fat tissue makes and releases a hormone called leptin that inhibits hun- ger to regulate the body’s energy balance. Copyright Cedernaes and Bass. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. INSIGHT NEURAL CIRCUITS Decoding obesity in the brainstem NEURAL CIRCUITS Neurons in the brainstem are the input for a neural circuit that integrates nutrient signals to control feeding behavior. Neurons in the brainstem are the input for a neural circuit that integrates nutrient signals to control feeding behavior. JONATHAN CEDERNAES AND JOSEPH BASS JONATHAN CEDERNAES AND JOSEPH BASS Over 40 years of research has implicated a molecule called cholecystokinin (or CCK) as a key regulator of satiety (Gibbs et al., 1973; Cai et al., 2014). CCK produced by the small intestine acti- vates a part of the brainstem called the nucleus of the solitary tract (or NTS), but only if so-called melanocortin 4 receptors are expressed in this region (Fan et al., 2004). The NTS sends this nutritional information onto parts of the hypo- thalamus and other nearby regions of the brain (Cai et al., 2014). These brain regions then inte- grate signals about the animal’s nutrient state and appetite (Grill and Hayes, 2012) The brainstem is heavily involved in these processes, integrating signals from the gut and relaying this information to a part of the brain called the hypothalamus. However, while many researchers have focused on the neurons within the feeding centers in the hypothalamus, the D’Agostino et al. have now characterized a neural circuit in the NTS that expresses its own CCK instead. First, they showed that these CCKNTS neurons became active in mice that 1 of 3 Cedernaes and Bass. eLife 2016;5:e16393. DOI: 10.7554/eLife.16393 Figure 1. A neural circuit in the brainstem integrates signals from peripheral tissues to control feeding behavior and energy balance. The lower brainstem (light blue) receives input (green arrow) from peripheral tissues such as the gut, and also sends output (red arrow) to these tissues. Now, in experiments on mice, D’Agostino et al. have characterized a neuronal pathway that starts with neurons that express CCK in the nucleus of the solitary tract (NTS), which is part of the lower brainstem. Genetically engineering the CCKNTS neurons so that a chemical called CNO can activate them showed that activating the neurons signals satiety and strongly decreases eating in mice. This leads to significant weight loss in the mice. The CCKNTS neurons send signals to a part of the hypothalamus (green) called the paraventricular nucleus (PVH). Specifically, the ends of the CCKNTS neurons form connections with neurons expressing melanocortin 4 receptors (MC4RPVH) in the PVH; the MC4RPVH neurons are thought to regulate appetite. Using light to activate the endings of the CCKNTS neurons that contact the PVH (via an optogenetics approach) also promoted satiety and a positive emotional state (or positive valence) in behavior tests. This strongly suggests that the connections between the CCKNTS neurons and the PVH are important. JONATHAN CEDERNAES AND JOSEPH BASS Inhibiting the CCK-A receptors (CCK-A-RPVH) with a chemical called devazepide meant that the chemogenetic and optogenetic activation of CCKNTS neurons no longer inhibited food intake. This figure was created with help from Billie Marcheva. Neural circuits Decoding obesity in the brainstem Insight Neural circuits Decoding obesity in the brainstem Figure 1. A neural circuit in the brainstem integrates signals from peripheral tissues to control feeding behavior and energy balance. The lower brainstem (light blue) receives input (green arrow) from peripheral tissues such as the gut, and also sends output (red arrow) to these tissues. Now, in experiments on mice, D’Agostino et al. have characterized a neuronal pathway that starts with neurons that express CCK in the nucleus of the solitary tract (NTS), which is part of the lower brainstem. Genetically engineering the CCKNTS neurons so that a chemical called CNO can activate them showed that activating the neurons signals satiety and strongly decreases eating in mice. This leads to significant weight loss in the mice. The CCKNTS neurons send signals to a part of the hypothalamus (green) called the paraventricular nucleus (PVH). Specifically, the ends of the CCKNTS neurons form connections with neurons expressing melanocortin 4 receptors (MC4RPVH) in the PVH; the MC4RPVH neurons are thought to regulate appetite. Using light to activate the endings of the CCKNTS neurons that contact the PVH (via an optogenetics approach) also promoted satiety and a positive emotional state (or positive valence) in behavior tests. This strongly suggests that the connections between the CCKNTS neurons and the PVH are important. Inhibiting the CCK-A receptors (CCK-A-RPVH) with a chemical called devazepide meant that the chemogenetic and optogenetic activation of CCKNTS neurons no longer inhibited food intake. This figure was created with help from Billie Marcheva. were fed nutrients (e.g. amino acids or sucrose) but not in mice fed only water. This shows that the activity of these neurons depends on the nutrient state of the animal. D’Agostino et al. then engineered these neu- rons so that they respond to a specific chemical. Activating the neurons with this chemical stopped the mice from eating even after fasting overnight. The mice lost a lot of weight if their CCKNTSneurons were activated for pro- longed periods, but rapidly regained weight when the chemical activation was stopped. How- ever, this chemical activator had no effect on the mice’s feeding behavior when D’Agostino et al. References Cai H, Haubensak W, Anthony TE, Anderson DJ. 2014. Central amygdala PKC-d+ neurons mediate the influence of multiple anorexigenic signals. Nature Neuroscience 17:1240–1248. doi: 10.1038/nn.3767 D’Agostino G, Lyons DJ, Cristiano C, Burke LK, Madara JC, Campbell JN, Garcia AP, Land BB, Lowell BB, Dileone RJ, Heisler LK. 2016. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. eLife 5:e012225. doi: 10. 7554/eLife.12225 Cai H, Haubensak W, Anthony TE, Anderson DJ. 2014. Central amygdala PKC-d+ neurons mediate the influence of multiple anorexigenic signals. Nature Neuroscience 17:1240–1248. doi: 10.1038/nn.3767 D’Agostino G, Lyons DJ, Cristiano C, Burke LK, Madara JC, Campbell JN, Garcia AP, Land BB, Lowell BB, Dileone RJ, Heisler LK. 2016. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. eLife 5:e012225. doi: 10. 7554/eLife.12225 At present, it is still not known what signal first activates the CCKNTS neurons. This means that it remains unknown how the CCKNTS neuro- circuit integrates information from the gut. Also, while activating the CCKNTS circuit did not pro- mote satiety if the CCK receptors were blocked, it is not clear whether gut-derived CCK or other gut hormones can activate the CCKNTS neurons to promote satiety. Fan W, Ellacott KL, Halatchev IG, Takahashi K, Yu P, Cone RD. 2004. Cholecystokinin-mediated suppression of feeding involves the brainstem melanocortin system. Nature Neuroscience 7:335–336. doi: 10.1038/ nn1214 Flak JN, Patterson CM, Garfield AS, D’Agostino G, Goforth PB, Sutton AK, Malec PA, Wong JM, Germani M, Jones JC, Rajala M, Satin L, Rhodes CJ, Olson DP, Kennedy RT, Heisler LK, Myers MG. 2014. Leptin- inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance. Nature Neuroscience 17:1744–1750. doi: 10.1038/nn.3861 Gibbs J, Young RC, Smith GP. 1973. Cholecystokinin elicits satiety in rats with open gastric fistulas. Nature 245:323–325. doi: 10.1038/245323a0 Flak JN, Patterson CM, Garfield AS, D’Agostino G, Goforth PB, Sutton AK, Malec PA, Wong JM, Germani The pathway characterized by D’Agostino et al. clearly has a strong effect on regulating body weight. As such, it will be important to ask whether this specific neurocircuit can be exploited to treat obesity. If this is the case, it’s worth noting that such a treatment is unlikely to use CCK itself. This is partly because this mole- cule is rapidly degraded in the body, and partly because people may become tolerant to CCK’s appetite-suppressing effects if it is given for pro- longed periods. JONATHAN CEDERNAES AND JOSEPH BASS prevented CCK from binding to its receptors on its target neurons. This suggests that the CCK receptor is directly responsible for the signa sent from the NTS D’Agostino et al. found that the CCKNTS neu rons form direct connections with the paraven tricular nucleus in the hypothalamus. Stimulatin this connection via an optogenetic techniqu stopped the mice from eating, even after the had been deprived of food. Further analys showed the nerve endings of the CCKNTS neuron at the paraventricular nucleus were also ve close to a subset of paraventricular nucleus cel that express melanocortin 4 receptors. Leptin signals how much fat is stored in th body and it was already known that this ho mone regulates the activity of neurons in th brainstem that respond to CCK (Flak et al receptor is directly responsible for the signals sent from the NTS were fed nutrients (e.g. amino acids or sucrose) but not in mice fed only water. This shows that the activity of these neurons depends on the nutrient state of the animal. D’Agostino et al. found that the CCKNTS neu- rons form direct connections with the paraven- tricular nucleus in the hypothalamus. Stimulating this connection via an optogenetic technique stopped the mice from eating, even after they had been deprived of food. Further analysis showed the nerve endings of the CCKNTS neurons at the paraventricular nucleus were also very close to a subset of paraventricular nucleus cells that express melanocortin 4 receptors. D’Agostino et al. then engineered these neu- rons so that they respond to a specific chemical. Activating the neurons with this chemical stopped the mice from eating even after fasting overnight. The mice lost a lot of weight if their CCKNTSneurons were activated for pro- longed periods, but rapidly regained weight when the chemical activation was stopped. How- ever, this chemical activator had no effect on the mice’s feeding behavior when D’Agostino et al. prevented CCK from binding to its receptors on its target neurons. This suggests that the CCK Leptin signals how much fat is stored in the body and it was already known that this hor- mone regulates the activity of neurons in the brainstem that respond to CCK (Flak et al., Cedernaes and Bass. eLife 2016;5:e16393. DOI: 10.7554/eLife.16393 2 of 3 Insight Neural circuits Decoding obesity in the brainstem Neural circuits Decoding obesity in the brainstem 2014). D’Agostino et al. Published 09 May 2016 Published 09 May 2016 JONATHAN CEDERNAES AND JOSEPH BASS now give new insight into how leptin and CCK signals are integrated in the brainstem and the paraventricular nucleus, and provide further support for the idea that the connection between these signals can act in both directions (Wu et al., 2012; Morton et al., 2005; Hayes et al., 2010). Collectively, D’Agos- tino et al.’s findings broaden our understanding of the neuronal circuitry that processes an ani- mal’s appetite and behavioral responses to its own energy state. The findings also describe the underlying communication, and important “gateways”, between the peripheral tissues, brainstem and hypothalamus. United States j-bass@northwestern.edu http://orcid.org/0000-0002-1602-8601 http://orcid.org/0000-0002-1602-8601 Competing interests: The authors declare that no competing interests exist. Competing interests: The authors declare that no competing interests exist. References Importantly, other promising molecules that can activate CCK receptors have already been investigated in mice as potential weight loss therapies (Irwin et al., 2012). Per- haps these potential drugs could be improved and made more specific such that they only tar- get a subset of CCK-expressing neurons within the gut and the brain. Thus, characterizing the CCKNTS neurons further, and uncovering how they are activated by the gut, may reveal specific molecules that could be targeted by drugs to suppress appetite for long enough to combat obesity. Grill HJ, Hayes MR. 2012. Hindbrain neurons as an essential hub in the neuroanatomically distributed control of energy balance. Cell Metabolism 16:296– 309. doi: 10.1016/j.cmet.2012.06.015 Hayes MR, Skibicka KP, Leichner TM, Guarnieri DJ, DiLeone RJ, Bence KK, Grill HJ. 2010. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation. Cell Metabolism 11:77–83. doi: 10.1016/j. cmet.2009.10.009 Grill HJ, Hayes MR. 2012. Hindbrain neurons as an essential hub in the neuroanatomically distributed control of energy balance. Cell Metabolism 16:296– 309. doi: 10.1016/j.cmet.2012.06.015 Hayes MR, Skibicka KP, Leichner TM, Guarnieri DJ, DiLeone RJ, Bence KK, Grill HJ. 2010. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation. Cell Metabolism 11:77–83. doi: 10.1016/j. cmet.2009.10.009 Irwin N, Frizelle P, Montgomery IA, Moffett RC, O’Harte FP, Flatt PR. 2012. Beneficial effects of the novel cholecystokinin agonist (pGlu-Gln)-CCK-8 in mouse models of obesity/diabetes. Diabetologia 55: 2747–2758. doi: 10.1007/s00125-012-2654-6 Morton GJ, Blevins JE, Williams DL, Niswender KD, Gelling RW, Rhodes CJ, Baskin DG, Schwartz MW. 2005. Leptin action in the forebrain regulates the hindbrain response to satiety signals. Journal of Clinical Investigation 115:703–710. doi: 10.1172/ JCI200522081 Jonathan Cedernaes is in the Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, United States jonathan.cedernaes@northwestern.edu Jonathan Cedernaes is in the Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, United States jonathan.cedernaes@northwestern.edu Clinical Investig JCI200522081 Sumithran P, Prendergast LA, Delbridge E, Purcell K, Shulkes A, Kriketos A, Proietto J. 2011. Long-term persistence of hormonal adaptations to weight loss. New England Journal of Medicine 365:1597–1604. doi: 10.1056/NEJMoa1105816 Sumithran P, Prendergast LA, Delbridge E, Purcell K, Shulkes A, Kriketos A, Proietto J. 2011. Long-term persistence of hormonal adaptations to weight loss. New England Journal of Medicine 365:1597–1604. doi: 10.1056/NEJMoa1105816 Sumithran P, Prendergast LA, Delbridge E, Purcell K, Shulkes A, Kriketos A, Proietto J. 2011. Long-term persistence of hormonal adaptations to weight loss. New England Journal of Medicine 365:1597–1604. doi: 10.1056/NEJMoa1105816 Wu Q, Clark MS, Palmiter RD. 2012. Deciphering a neuronal circuit that mediates appetite. Nature 483: 594–597. doi: 10.1038/nature10899 Wu Q, Clark MS, Palmiter RD. 2012. Deciphering a neuronal circuit that mediates appetite. Nature 483: 594–597. doi: 10.1038/nature10899 Wu Q, Clark MS, Palmiter RD. 2012. Deciphering a neuronal circuit that mediates appetite. Nature 483: 594–597. doi: 10.1038/nature10899 Joseph Bass is in the Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, 3 of 3 Cedernaes and Bass. eLife 2016;5:e16393. DOI: 10.7554/eLife.16393
https://openalex.org/W4308726580	https://www.researchsquare.com/article/rs-2231530/latest.pdf	English	null	Use of inhaled nitric oxide prognosticates poor survival in severe ARDS with venovenous ECMO: a retrospective analysis	Research Square (Research Square)	2,022	cc-by	7,042	Stefan Muenster (  stefan.muenster@ukbonn.de ) Stefan Muenster (  stefan.muenster@ukbonn.de ) Stefan Muenster (  stefan.muenster@ukbonn.de ) Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Jennifer Nadal Institute of Medical Biometry, Informatics and Epidemiology, University Hospital Bonn Jens-Christian Schewe Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Heidi Ehrentraut Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Stefan F. X. Kreyer Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Christian Putensen Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Stefan F. Ehrentraut Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Analysis of patients with severe ARDS on VV ECMO with pulmonary hypertension or right ventricular failure treated with iNO: a retrospective observational study Analysis of patients with severe ARDS on VV with pulmonary hypertension or right ventric failure treated with iNO: a retrospective observational study Stefan Muenster (  stefan.muenster@ukbonn.de ) Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Jennifer Nadal Institute of Medical Biometry, Informatics and Epidemiology, University Hospital Bonn Jens-Christian Schewe Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Heidi Ehrentraut Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Stefan F. X. Kreyer Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Christian Putensen Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Stefan F. Ehrentraut Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn Research Article Keywords: Inhaled nitric oxide, ARDS, VV ECMO, right heart failure, pulmonary hypertensio responder Posted Date: April 25th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2231530/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 Internation Read Full License Research Article Posted Date: April 25th, 2023 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. d ll Page 1/21 Abstract Background: In a retrospective observational study, we investigated patients with severe acute respiratory distress syndrome (ARDS) undergoing veno-venous (VV) extracorporeal membrane oxygenation (ECMO) support with known pulmonary arterial hypertension (PAH) and/or right ventricular (RV) failure treated with iNO. Objective: We analysed clinical characteristics such as time on mechanical ventilation, weaning from VV ECMO, organ dysfunction, the iNO therapy in terms of indication, dosing and duration, the ability of iNO to lower PAH in responder and non-responder patients, the survival rate, in-hospital mortality and long-term survival. Results: Of the 657 patients identified with ECMO support, 292 under VV ECMO support were evaluated and n=55 patients with iNO were included. We found that in-hospital mortality and long-term survival were poor in patients with severe ARDS under VV ECMO support with persistent PAH and/or RV failure. A rescue therapy with the pulmonary vasodilator iNO showed that only one-third of the cohort responded to the therapy with a sufficient decrease of the mean PAP. Survival between iNO responder and non- responder showed a trend albeit data between the groups were not statistically significant. We did not find a higher rate of continuous renal replacement therapy as marker for acute renal failure when iNO concentrations were ≤ 20 ppm and when the treatment duration stayed ≤ 3 days. Conclusions: This retrospective observational study suggests that persistent PAH and/or RV failure is associated with worse clinical outcome in patients with severe ARDS and VV-ECMO support. Concomitant treatment with the pulmonary vasodilator iNO reduced PAH and/or reversed RV dysfunction in only one-third of the patient population (iNO responder patients) but did not alter the rate of acute kidney failure. Conclusions: This retrospective observational study suggests that persistent PAH and/or RV failure is associated with worse clinical outcome in patients with severe ARDS and VV-ECMO support. Background Inhaled nitric oxide (iNO) has been widely used as a selective pulmonary vasodilator to treat pulmonary arterial hypertension (PAH) and/or right ventricular failure (RV failure).1 iNO is only distributed to ventilated lung regions and exerts the unique ability of selectively inducing smooth muscle relaxation in the pulmonary vasculature in the said regions.1,2 Thus, iNO improves arterial blood oxygenation, decreases intrapulmonary shunting, and enhances blood flow distribution towards ventilated alveolar regions of the lungs.2,3 The high affinity of nitric oxide (NO) to oxyhaemoglobin in red blood cells leads to the rapid formation of nitrate and methaemoglobin; this mechanism limits the vasodilation effects to the pulmonary vasculature and thereby avoids systemic arterial hypotension.4,5 Inhaled NO concentrations of up to 80 parts per million (ppm) have been administered safely without unfavourable systemic side effects.6–8 Page 2/21 Patients suffering from acute respiratory distress syndrome (ARDS) frequently present with PAH whereas increased intrapulmonary shunting caused by perfusion of non-aerated alveoli contributes to severe arterial hypoxemia.9,10 Moreover, elevated pulmonary artery pressure raises transcapillary pressure and thereby increases the risk of alveolar oedema, which may aggravate ARDS.11,12 In addition, PAH may lead or contribute to RV failure and is an independent risk factor for mortality in patients with ARDS.13,14 Given its pharmacological properties, iNO may lower pulmonary arterial pressure, thereby reducing the risk of RV failure and intrapulmonary shunting.15–17 Although randomised controlled trials (RCTs) on iNO use have shown both improved oxygenation and hemodynamics in the acute phase of ARDS in adults, all studies thus far have failed to demonstrate any clinically significant benefit of iNO on survival or ventilator-free days.18,19 Patients suffering from acute respiratory distress syndrome (ARDS) frequently present with PAH whereas increased intrapulmonary shunting caused by perfusion of non-aerated alveoli contributes to severe arterial hypoxemia.9,10 Moreover, elevated pulmonary artery pressure raises transcapillary pressure and thereby increases the risk of alveolar oedema, which may aggravate ARDS.11,12 In addition, PAH may lead Venovenous extracorporeal membrane oxygenation (VV ECMO), widely considered a life-saving procedure, may restore oxygenation and eliminate carbon dioxide accumulation when conventional mechanical ventilation fails to ensure a sufficient gas exchange in severe ARDS.20–22 Patients with severe ARDS and VV ECMO support with concomitant iNO administration represent a very specific and limited cohort. Data that elucidate the use of iNO in this specific cohort are lacking and little is known about survival and mortality in this population. Study design This retrospective observational study was conducted following the Strengthening the Reporting of Observational Studies in Epidemiology statement. Background To gain more insight into this specific and limited group of critically ill patients, we performed a retrospective observational study of patients with severe ARDS and VV ECMO support with PAH and/or RV failure treated with iNO as rescue therapy. Exclusion criteria Veno-arterial ECMO support. Veno-arterial ECMO support. Objectives The objectives were: to describe iNO treatment in terms of indication, dosing, and duration of application. to describe iNO treatment in terms of indication, dosing, and duration of application. to measure the iNO ability to lower mean pulmonary arterial pressure (PAP) in patients under VV ECMO support who are responders and non-responders to iNO. to measure the iNO ability to lower mean pulmonary arterial pressure (PAP) in patients under VV ECMO support who are responders and non-responders to iNO. to describe the clinical characteristics, such as time on mechanical ventilation, weaning from VV ECMO, and organ dysfunction, as defined using the sequential organ failure assessment (SOFA) score,23 to describe the clinical characteristics, such as time on mechanical ventilation, weaning from VV ECMO, and organ dysfunction, as defined using the sequential organ failure assessment (SOFA) score,23 Page 3/21 Page 3/21 Page 3/21 to measure outcome parameters, such as survival on ICU, in-hospital mortality, and long-term survival; to measure outcome parameters, such as survival on ICU, in-hospital mortality, and long-term survival; Study population Data on all ECMO patients treated on the intensive care unit of the Department of Anaesthesiology and Intensive Care Medicine at the quaternary level of the University Hospital, Bonn, Germany, between May 2015 and May 2021 were collected. Inclusion criteria Age >18 years old; Electronic medical records available, including VV ECMO run parameters, vital parameters, and laboratory measurements (both point-of-care and laboratory diagnostics); Electronic medical records available, including VV ECMO run parameters, vital parameters, and laboratory measurements (both point-of-care and laboratory diagnostics); VV ECMO support; VV ECMO support; ARDS following the Berlin definition,20 and iNO administration. iNO administration. Indication for ECMO Indications for VV ECMO support complied with the Extracorporeal Life Support Organization General Guidelines.24 Indications included treatment of severe hypoxaemia and hypercapnia and prevention of possible harmful mechanical ventilation (i.e., prolonged use of exceedingly high peak inspiratory pressures or driving pressure > 15 cmH2O) to ensure sufficient gas exchange according to ARDS network definitions.25 All decisions for initiating VV ECMO support were based on the consensus between at least two experienced senior critical care physicians of the ARDS/ECMO team of the current study. Indication for iNO delivery and definition of PAH and RV failure Page 4/21 Page 4/21 iNO (NO-A nitric oxide delivery system, EKU Elektronik GmbH, Germany) was administered at the treating physician’s discretion after bedside evaluation of pulmonary hemodynamics and right heart function, as indicated by invasive pulmonary hemodynamics (assessed using a Swan–Ganz catheter) and/or transoesophageal echocardiography. A mean PAP (mPAP) ≥ 25 mmHg was used as a cut-off value for iNO application.26 A positive response to iNO was determined as an mPAP reduction of ≥ 6 mmHg following administration of iNO within 30min. Other indicators, such as pulmonary vascular resistance or cardiac index, were not used due to insufficient data regarding their validity during extracorporeal life support. In the present study, RV failure is defined by transoesophageal echocardiography, as suggested by Vieillard-Baron et al.27 In severe ARDS, acute cor pulmonale or severe RV dilatation accurately reflects RV failure, particularly when right atrial pressure is increased. Ethical approval/informed consent Ethical approval for the current study was provided by the ethics committee (no. 492/20) of the University Hospital of Bonn, Germany, and the need for informed consent was waived. Statistical analyses All data are presented as the median and interquartile range (IQR) for non-normally distributed variables or mean ± standard deviation for normally distributed continuous variables, as appropriate, and frequency distributions with percentages for categorical variables. The paired t-test was used to analyse group differences in normally distributed variables, whereas the Wilcoxon test was used for non-normally distributed variables. Nominal variables were assessed using McNemar’s test for 2 × 2 tables. The Kaplan–Meier method and the stratified log-rank test were performed to analyse survival.28 All analyses were performed on R version 4.1.2.29 All tests were two-sided, and p < 0.05 was determined as the cut-off for significance. No adjustments were made for multiple tests, and P values should be interpreted as exploratory only. Ability of iNO to lower mean PAP in responder and nonresponder patients As mentioned in the Methods section, a positive response to iNO was defined as a mPAP decrease ≥ 6 mmHg. In n=37 patients, a Swan-Ganz catheter was inserted to continuously monitor pulmonary hemodynamics, allowing us to distinguish between iNO responders and nonresponders in these cases (in contrast to patients who were monitored discontinuously and solely by echocardiography). Responder patients with ARDS under VV ECMO showed a significant decrease in mPAP when iNO was administered (mPAP before iNO [40.1 ± 5.6 mmHg] vs. mPAP during iNO [30.5 ± 5.0 mmHg]; p < 0.0001; Figure 2C). In contrast, nonresponder patients with ARDS did not show a significant decrease in mPAP during iNO treatment (mPAP before iNO [36.9 ± 8.1 mmHg] vs. mPAP during iNO [35.9 ± 8.2 mmHg]; p = 0.1873; Figure 2D). Identification and characteristics of the eligible study cohort To identify the eligible study cohort, 657 patients under ECMO were screened during the study period. Patients with complete electronic medical records were analysed (Figure 1). In addition, 292 patients who underwent VV ECMO support were identified as the eligible study cohort, which included 55 individuals with known PAH and/or RV failure administered with iNO as rescue therapy. This study cohort will be Page 5/21 Page 5/21 further analysed and discussed and represents the group of interest for the retrospective observational analysis. Both the baseline and clinical characteristics are provided in Tables 1 and 2. Indication, dosing, and duration of iNO treatment in patients under VV ECMO To evaluate whether patients under VV ECMO support may benefit from iNO administration, transoesophageal echocardiography, and/or invasive monitoring using a pulmonary artery catheter were performed before the start of iNO. Subsequently, three groups of indications were identified for the use of iNO in patients undergoing VV ECMO (Table 1): PAH, RV failure, or a combination of both pathologies. In addition, the iNO doses administered in the VV ECMO patient cohort were investigated (Figure 2A). iNO was administered at an average dose of 14.5 ± 5.5 ppm (ranging from a minimum dose of 6.9 ppm to a maximum of 20 ppm). The duration of treatment was 3 days (IQR, 1.76–4.41; Figure 2B). Standard ventilation parameters and blood gas analyses both on days 1, 3, and 7 during VV ECMO To avoid the onset of PAH and/or RV failure, optimizing respiratory conditions in patients with ARDS is strongly recommended because a driving pressure ≥ 18 cmH2O, arterial partial pressure of carbon dioxide (PaCO2) ≥ 48 mmHg, and PaO2/FIO2 < 150 mmHg have been reported as risk factors.31 Ventilation parameters and arterial blood gas analyses were both recorded on days 1, 3, and 7 during VV ECMO with iNO treatment (Table 3). These findings showed no differences in minute ventilation, positive end-expiratory pressure (PEEP), or peak inspiratory pressure between the different days. Ventilation parameters, as well as arterial blood gas analyses, showed results that comply with the respective ARDS guidelines on mechanical ventilation.32 Outcomes Table 4 presents the data on the length of ICU and hospital stay, in-hospital mortality rate, and median survival time of the study cohort. The length of ICU stay was 23 days and total hospital stay 27 days in iNO treated patients. The in-hospital mortality rate was n=38 (69.1%). Median survival was 22 days [95% CI, 14,37] in patients with iNO breathing. To evaluate whether survival may depend on the ability of iNO to lower elevated mPAP, a subgroup analysis of the survival rates was performed in the iNO responder and non-responder groups. Figure 3B shows that the survival in iNO treated patients differed between iNO responders (36.4%) and non- responders (23.1%), albeit not statistically significant (p = 0.0623, Figure 3). The Kaplan–Meier curve in Figure 4 shows the long-term survival rate (90 days) of patients with severe ARDS on VV ECMO support with PAH and/or RV failure treated with iNO. Organ failure, ECMO circuit weaning, and rate of tracheostomy Weaning failure from VV ECMO support was observed in n = 37 [67.3%] iNO-treated patients. The total time on mechanical ventilation was 24 days. Also, the time on mechanical ventilation before VV ECMO, duration of VV ECMO, and rate of tracheostomy in iNO-treated patients have been reported in Table 2. Adjunctive therapies, i.e., continuous kidney replacement therapy and prone positioning before or during VV ECMO support, were also reported in Table 2. Apart from the SOFA score on ICU admission, the severity of further organ failure was assessed using the cardiopulmonary resuscitation rate before ECMO and the rate of sepsis, as defined by SEPSIS-3.30 Values obtained can be found in Table 2. Page 6/21 Discussion We investigated in a retrospective observational study patients with severe ARDS undergoing VV ECMO support with concomitant iNO administration to gain more insight into this specific population. We analysed clinical characteristics such as time on mechanical ventilation, weaning from VV ECMO, organ dysfunction, the iNO treatment in terms of indication, dosing and duration, the ability of iNO to lower PAP in responder and non-responders patients, the survival rate, in-hospital-mortality and long-term survival. We found that in-hospital mortality and long-term survival were generally poor in patients with severe ARDS under VV ECMO support with persistent PAH and/or RV failure. A rescue therapy with the pulmonary vasodilator iNO showed that only one-third of the cohort responded to the therapy with a sufficient decrease of the mean PAP. Survival between iNO responder and non-responder showed a trend albeit data between the groups were not statistically significant. We did not find an increased rate of Page 7/21 Page 7/21 continuous kidney replacement therapy as marker for acute renal failure when iNO concentrations were ≤ 20 ppm and when the treatment duration stayed ≤ 3 days. continuous kidney replacement therapy as marker for acute renal failure when iNO concentrations were ≤ 20 ppm and when the treatment duration stayed ≤ 3 days. Pulmonary vascular dysfunction is one of the pathophysiological hallmarks of ARDS and ultimately leads to a certain degree of PAH.33 Recent data suggest that PAH and subsequent RV failure are medical burdens that occur in every second patient with moderate to severe ARDS and are independently associated with the risk of mortality.33,34 In ARDS, multiple pathophysiological mechanisms that directly cause injury to pulmonary circulation include endothelial dysfunction, distal pulmonary vascular occlusion at the capillary level, pulmonary vasoconstriction, extrinsic vessel occlusion by alveoli distention, and, ultimately, vascular remodeling.35 These mechanisms lead to increased pulmonary vascular resistance, precapillary pulmonary hypertension, and increased RV afterload.35 The uncoupling between pulmonary circulation and the right heart ultimately leads to the breakdown of oxygen delivery. Discussion Various strategies, including limiting volume loading and correcting blood pressure by infusing norepinephrine, have been suggested to decrease RV wall stress and RV end-diastolic pressure, thereby improving RV stroke volume.36 The patients of the current study received a restrictive fluid regimen and norepinephrine to avoid hypotension, as described previously.32 Both hypoxia and hypercapnia strongly increase pulmonary vasoconstriction and contribute to PAH.37 Severe ARDS per se is associated with profound hypoxia, which may be accompanied by hypercapnia.38 Hypercapnia is the consequence of protective ventilatory strategies designed to reduce ventilator-induced lung injury. It also reflects increased dead space due to alveolar overdistension and ARDS severity.39 Higher PEEP levels are frequently required in severe ARDS to avoid life-threatening hypoxia. However, transpulmonary pressure, despite low tidal ventilation when lung compliance decreases due to alveolar collapse, may be associated with increased end-inspiratory airway pressure.40 Consequently, pulmonary capillaries become stretched and their caliber is reduced, resulting in increased pulmonary vasoconstriction.41,42 By controlling arterial oxygenation and decarboxylation, even during ultraprotective ventilation,38 VV ECMO suppresses the major factors that increase pulmonary vascular resistance and cause PAH in severe ARDS, thereby sufficiently unloading the RV.43 In this study cohort, VV ECMO was indicated to correct hypoxaemia and hypercapnia, as well as allow ultraprotective ventilation, to prevent peak inspiratory (>27 cmH2O) and/or driving (>15 cmH2O) pressure (Table 3). Although VV ECMO initiation in the current study resulted in adequate arterial oxygenation and normocapnia at a peak inspiratory and driving pressures < 25 cmH2O and < 10 cmH2O, respectively, PAH with or without RV failure persisted in these patients with severe ARDS. Although iNO in ARDS has been widely abandoned by intensivists because RCTs and meta-analyses have demonstrated no benefits for survival despite temporal improvement in oxygenation,18,19 it may be an Page 8/21 Page 8/21 option for decreasing RV afterload by lowering PAH.33 Intriguingly, a positive response to iNO was observed in only 30% of the patients of the current study. Similar findings were reported by Manktelow44 on severe ARDS with septic shock. These studies were conducted before the widespread availability of VV ECMO and ultraprotective ventilator strategy. The data of the current study confirms the validity of this observation despite the use of current protective ventilation treatment regimens. Discussion iNO use in ARDS has been widely studied over the last decades, and no evidence of direct NO toxicity has been observed at clinically relevant doses below 20 ppm.45 However, conflicting evidence has been reported on whether iNO contributes to increased acute kidney injury.46–48 CKRT in this study did not indicate an increased rate of acute kidney injury during VV ECMO (Table 2). Thus, the potentially detrimental effects of iNO on kidney function cannot be confirmed in our study. Indeed, the rate of CKRT during VV ECMO and concomitant iNO treatment reflects similar reported rates in other cohort studies on severe ARDS and VV ECMO without iNO therapy.49 iNO use in ARDS has been widely studied over the last decades, and no evidence of direct NO toxicity has been observed at clinically relevant doses below 20 ppm.45 However, conflicting evidence has been reported on whether iNO contributes to increased acute kidney injury.46–48 CKRT in this study did not indicate an increased rate of acute kidney injury during VV ECMO (Table 2). Thus, the potentially detrimental effects of iNO on kidney function cannot be confirmed in our study. Indeed, the rate of CKRT during VV ECMO and concomitant iNO treatment reflects similar reported rates in other cohort studies on severe ARDS and VV ECMO without iNO therapy.49 The optimal dose and time of iNO treatment in ARDS remains controversial. A European expert recommendation on the use of iNO in adults with ARDS suggested that toxic side effects (e.g., methemoglobinaemia and the formation of relevant nitrogen dioxide levels) are less likely when inhaled NO doses stay <20 ppm.45 Initiating iNO treatment as early as 24–72 h after the onset of ARDS has been suggested because iNO is mainly effective during the early onset of ARDS. In this study, an average iNO dose of 14.5 ppm was administered, and iNO delivery was performed for a median duration of 3 days. iNO was initiated within 24 h after the diagnosis of either PAH and/or RV failure. In most of the patients, iNO was initiated during VV ECMO, as opposed to before or after VV ECMO, as administered in a small cohort. In terms of survival, persistent PAH and/or RV failure are known to contribute to worse outcome such as in-hospital death, length of ICU and hospital stay. Discussion In this study, we found an in-hospital mortality rates comparable to those reported in a recently published small single-centre retrospective trial.50 In a subgroup analysis, we found that the survival in iNO treated patients might differ between iNO responders and non-responders but results were not statistically significant; likely because the subgroups were too small. Of note, this cohort suffers from multiple comorbidities, all of which will worsen the clinical course of the patient regardless of iNO administration (please refer to Table S1 which reports the Charlson Comorbidity index for the study cohort). A limitation of this study is the retrospective and monocentric nature of the analyses. The availability of RCTs involving patients with severe ARDS is limited after initial RCTs failed to demonstrate any beneficial effects of iNO on survival and mortality. Also, retrospective analyses are difficult to control for an unbiased selection process of patients.51 However, in this study all included patients were analysed depending on treatment and thus were not subjected to selection bias for iNO treatment. Lastly, a major limitation of this study is the small number of participants because patients with VV EMO with persistent PAH and/or RV failure treated with iNO represents a very limited patient cohort. Page 9/21 In conclusion, this retrospective observational study suggests that persistent PAH and/or RV failure is associated with poor clinical outcome in patients with severe ARDS and VV-ECMO support. Surprisingly, only one-third of the patient population responded to iNO with a significant decrease of PAH and/or RV failure. In other words, elevated pulmonary arterial pressure or onset of RV failure in ECMO-supported ARDS patients appears to be very difficult to treat with the pulmonary vasodilator iNO. Further research will be needed to elucidate the underlying potential mechanisms. We did not find an increased rate of continuous renal replacement therapy as marker for acute renal failure when iNO concentrations stayed below 20 ppm and when the duration of treatment was less then 3 days. References 1. Yu B, Ichinose F, Bloch DB, et al. Inhaled nitric oxide. British Journal of Pharmacology 2019; 176:246–255. 1. Yu B, Ichinose F, Bloch DB, et al. Inhaled nitric oxide. British Journal of Pharmacology 2019; 176:246–255. 2. Pison U, López FA, Heidelmeyer CF, et al. Inhaled nitric oxide reverses hypoxic pulmonary vasoconstriction without impairing gas exchange. Journal of Applied Physiology (Bethesda, Md.: 1985) 1993; 74:1287–1292. 2. Pison U, López FA, Heidelmeyer CF, et al. Inhaled nitric oxide reverses hypoxic pulmonary vasoconstriction without impairing gas exchange. Journal of Applied Physiology (Bethesda, Md.: 1985) 1993; 74:1287–1292. 2. Pison U, López FA, Heidelmeyer CF, et al. Inhaled nitric oxide reverses hypoxic pulmonary vasoconstriction without impairing gas exchange. Journal of Applied Physiology (Bethesda, Md.: 1985) 1993; 74:1287–1292. 3. Kinsella JP, Neish SR, Shaffer E, et al. Low-dose inhalation nitric oxide in persistent pulmonary hypertension of the newborn. Lancet (London, England) 1992; 340:819–820. 3. Kinsella JP, Neish SR, Shaffer E, et al. Low-dose inhalation nitric oxide in persistent pulmonary hypertension of the newborn. Lancet (London, England) 1992; 340:819–820. 4. Berra L, Pinciroli R, Stowell CP, et al. Autologous transfusion of stored red blood cells increases pulmonary artery pressure. American Journal of Respiratory and Critical Care Medicine 2014; 190:800– 807. 5. Muenster S, Beloiartsev A, Yu B, et al. Exposure of Stored Packed Erythrocytes to Nitric Oxide Prevents Transfusion-associated Pulmonary Hypertension. Anesthesiology 2016; 125:952–963. 6. The Neonatal Inhaled Nitric Oxide Study Group (NINOS). Inhaled Nitric Oxide and Hypoxic Respiratory Failure in Infants With Congenital Diaphragmatic Hernia. Pediatrics 1997; 99:838–845. 7. Roberts JD, Fineman JR, Morin FC, et al. Inhaled Nitric Oxide and Persistent Pulmonary Hypertension of the Newborn. New England Journal of Medicine 1997; 336:605–610. 8. Clark RH, Kueser TJ, Walker MW, et al. Low-Dose Nitric Oxide Therapy for Persistent Pulmonary Hypertension of the Newborn. New England Journal of Medicine 2000; 342:469–474. 9. Ryan D, Frohlich S, McLoughlin P. Pulmonary vascular dysfunction in ARDS. Annals of Intensive Care 2014; 4:28. 10. Ware LB, Matthay MA. The acute respiratory distress syndrome. The New England Journal of Medicine 2000; 342:1334–1349. Page 10/21 11. Chetham PM, Babál P, Bridges JP, et al. Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry. The American Journal of Physiology 1999; 276:L41-50. 12. Ochoa CD, Stevens T. Studies on the cell biology of interendothelial cell gaps. 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The New England Journal of Medicine 2004; 351:327–336. 41. Whittenberger JL, McGREGOR M, Berglund E, et al. Influence of state of inflation of the lung on pulmonary vascular resistance. Journal of Applied Physiology 1960; 15:878–882. 42. West JB, Dollery CT, Naimark A. Distribution of blood flow in isolated lung; relation to vascular and alveolar pressures. Journal of Applied Physiology 1964; 19:713–724. 43. Reis Miranda D, van Thiel R, Brodie D, et al. Right ventricular unloading after initiation of venovenous extracorporeal membrane oxygenation. American Journal of Respiratory and Critical Care Medicine 2015; 191:346–348. 44. Manktelow C, Bigatello LM, Hess D, et al. Physiologic Determinants of the Response to Inhaled Nitric Oxide in Patients with Acute Respiratory Distress Syndrome. Anesthesiology 1997; 87:297–307. 45. Germann P, Braschi A, Della Rocca G, et al. Inhaled nitric oxide therapy in adults: European expert recommendations. Intensive Care Medicine 2005; 31:1029–1041. 46. Adhikari NKJ, Burns KEA, Friedrich JO, et al. Effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and meta-analysis. BMJ 2007; 334:779. 47. Lei C, Berra L, Rezoagli E, et al. Nitric Oxide Decreases Acute Kidney Injury and Stage 3 Chronic Kidney Disease after Cardiac Surgery. American Journal of Respiratory and Critical Care Medicine 2018; 198:1279–1287. 48. Gebistorf F, Karam O, Wetterslev J, et al. Inhaled nitric oxide for acute respiratory distress syndrome (ARDS) in children and adults. The Cochrane Database of Systematic Reviews 2016; 2016:CD002787. 49. Haneya A, Diez C, Philipp A, et al. References Impact of Acute Kidney Injury on Outcome in Patients With Severe Acute Respiratory Failure Receiving Extracorporeal Membrane Oxygenation. Critical Care Medicine 2015; 43:1898–1906. Page 13/21 Page 13/21 50. Ortiz F, Brunsvold ME, Bartos JA. Right Ventricular Dysfunction and Mortality After Cannulation for Venovenous Extracorporeal Membrane Oxygenation. Critical Care Explorations 2020; 2:e0268. 51. Pannucci CJ, Wilkins EG. Identifying and avoiding bias in research. Plastic and Reconstructive Surgery 2010; 126:619–625. 51. Pannucci CJ, Wilkins EG. Identifying and avoiding bias in research. Plastic and Reconstructive Surgery 2010; 126:619–625. Tables Table 1. baseline characteristics. characteristic severe ARDS patients on VV ECMO with PAH and/or RV failure - concomitant iNO rescue therapy (n=55) demographics age – (mean ± SD) [years] 54.6 ± 11.6 male sex – n (%) [male] 40 (72.7) weight – (mean ± SD) [kg] 107.9 ± 40.2 height – (mean ± SD) [cm] 175.76 ± 8.5 BMI – (mean ± SD) 34.8 ± 12.6 cause of ARDS viral pneumonia – n (%) 22 (40) bacterial pneumonia – n (%) 8 (14.5) others – n (%) 25 (45.5) indication for iNO administration pulmonary arterial hypertension (PAH)– no. [%] 26 (47) RV failure – no. [%] 20 (36) PAH and RV failure – no. [%] 9 (16) 50. Ortiz F, Brunsvold ME, Bartos JA. Right Ventricular Dysfunction and Mortality After Cannulation for Venovenous Extracorporeal Membrane Oxygenation. Critical Care Explorations 2020; 2:e0268. 51. Pannucci CJ, Wilkins EG. Identifying and avoiding bias in research. Plastic and Reconstructive Surgery 2010; 126:619–625. Tables characteristic severe ARDS patients under VV ECMO with PAH and/or RV failure - concomitant iNO rescue therapy (n=55) day 1 day 3 day 7 ventilation parameters minute ventilation – (median (IQR)) [l/min] 2.04 (1.6-3.2) 2.21 (1.8-4.1) 3.82 (2.9-4.8) PEEP – (median (IQR)) [mbar] 20 (18-20) 20 (17-20) 18 (16-20) peak inspiratory pressure – (median (IQR)) [mbar] 26 (25-28) 26 (25-28) 26 (26-28) blood gas analysis PaO2 – (median (IQR)) [mmHg] 103 (100-107) 102 (77-117) 101 (90-113) PaCO2 – (median (IQR)) [mmHg] 39 (35-43) 39 (36-43) 41 (35-46) pH – (median (IQR)) 7.30 (7.3-7.4) 7.34 (7.3-7.4) 7.32 (7.3-7.4) PaO2/FIO2 – (median (IQR)) 84 (35-103) 92 (75-111) 95 (77-130) minimal FIO2 of a 24 hrs period 80 (70-90) 70 (60-85) 70 (50-85) iNO, inhaled nitric oxide; IQR, interquartile range; PEEP, positive end-expiratory pressure; PaO2, arterial partial pressure of oxygen; PaCO2, arterial partial pressure of carbon dioxide; FIO2, fraction of inspired oxygen. Table 4. survival analysis. characteristic severe ARDS patients under VV ECMO with PAH and/or RV failure - concomitant iNO rescue therapy (n=55) outcome length of ICU stay – (median (IQR)) [days] 23.3 (13.55 – 39.1) length of hospital stay – (median (IQR)) [days] 27.1 (13.75 – 40) in-hospital death – n(%) 38 (69.1) median survival time – [95% CI] [days] 22 [14; 37] iNO, inhaled nitric oxide; ICU, intensive care unit; IQR, interquartile range; CI, confidence interval. Tables cs. severe ARDS patients on VV ECMO with PAH and/or RV failure - concomitant iNO rescue therapy (n=55) iNO, inhaled nitric oxide; SD, standard deviation; BMI, body mass index; ARDS, acute respiratory distress syndrome; PAH, pulmonary arterial hypertension; RV failure, right ventricular failure. Page 14/21 Table 2. clinical characteristics. characteristic severe ARDS patients under VV ECMO with PAH and/or RV failure - concomitant iNO rescue therapy (n=55) invasive mechanical ventilation total time on mechanical ventilation – (median (IQR)) [days] 24 (14 – 41.5) time on mechanical ventilation prior to VV ECMO support – (median (IQR)) [days] 2 (0.79 – 8) tracheostomy during stay on ICU – n (%) 21 (38.2) VV ECMO duration of VV ECMO support – (median (IQR)) [days] 13.6 (9 – 19.9) weaning failure from VV ECMO support – n (%) 37 (67.3) adjunctive therapies CKRT during VV ECMO – n (%) 29 (52.7) proning prior to VV ECMO 21 (38.2) proning during VV ECMO 30 (54.5) organ dysfunction SOFA-score on ICU admission– (mean ± SD) 8 ± 3 RESP-score – (mean ± SD) -1 ± 4 CPR prior to ECMO – n (%) 0 (0.0) sepsis – n (%) 7 (14) Table 2. clinical characteristics. characteristic Table 2. clinical characteristics. characteristic iNO, inhaled nitric oxide; SD, standard deviation; IQR, interquartile range; VV, venovenous; ECMO, extracorporeal membrane oxygenation; ICU, intensive care unit; CKRT, continuous kidney replacement therapy; SOFA, sequential organ failure assessment; RESP, Respiratory ECMO Survival Prediction; CPR, cardiopulmonary resuscitation. Page 15/21 Page 15/21 Table 3. ventilation parameters and blood gas analysis. Declarations Author Contributions: All authors meet the ICMJE recommendations regarding authorship. S.M., J.N., J- C.S., H.E., S.K., C.P., S.F.E made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; AND Page 16/21 Page 16/21 S.M., J.N., J-C.S., H.E., S.K., C.P., S.F.E drafted the work or revised it critically for important intellectual content; AND All authors gave final approval of the version to be published; AND All authors gave final approval of the version to be published; AND All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding: The Data Integration Centre of the University Hospital Bonn was funded by the Federal Ministry of Education and Research of the Federal Republic of Germany (grant number: 01ZZ1803Q). Funding: The Data Integration Centre of the University Hospital Bonn was funded by the Federal Ministry of Education and Research of the Federal Republic of Germany (grant number: 01ZZ1803Q). Institutional Review Board Statement: Ethical approval for the current study was provided by the ethics committee (no. 492/20) of the University Hospital of Bonn, Germany, and the need for informed consent was waived. Informed Consent Statement: Patient consent was waived due to the retrospective nature of the study. Data Availability Statement: The clinical datasets generated and/or analysed during the current study are not publicly available due to the local data protection law but are available from the corresponding author on reasonable request. Conflicts of Interest: Stefan Muenster, M.D. is currently a member of the scientific advisory board that advises Air Liquide in the use of inhaled nitric oxide in adult patients undergoing cardiac surgery. All the other authors have no conflicts of interest to declare. Figures Page 17/21 Page 17/21 Figure 1 Inclusion process for the selected patients of the retrospective observational study. ECMO extracorpore membrane oxygenation. VV venovenous. VA venoarterial. iNO inhaled nitric oxide. PAH pulmonary arte hypertension. RV right ventricular failure. The observation period is from 2015 to 2021. Figure 1 Figure 1 Inclusion process for the selected patients of the retrospective observational study. ECMO extracorporeal membrane oxygenation. VV venovenous. VA venoarterial. iNO inhaled nitric oxide. PAH pulmonary arterial hypertension. RV right ventricular failure. The observation period is from 2015 to 2021. Page 18/21 Page 18/21 NO treatment. C Mean PAP before and during iN ned as iNO responders (decrease in mPAP durin Figure 2 A Treatment summary of iNO therapy. B Duratio Figure 2 A Treatment summary of iNO therapy. B Duration of iNO treatment. C Mean PAP befo treatment in patients with ARDS under VV ECMO defined as iNO responders (decreas treatment ≥6 mmHg) D Mean PAP before and during iNO treatment in patients with Figure 2 A Treatment summary of iNO therapy. B Duration of iNO treatment. C Mean PAP before and during iNO treatment in patients with ARDS under VV ECMO defined as iNO responders (decrease in mPAP during treatment ≥ 6 mmHg). D Mean PAP before and during iNO treatment in patients with ARDS under VV ECMO defined as iNO nonresponders (decrease in mPAP during treatment < 6 mmHg). Differences in mPAP were compared using the paired t-test with statistical significance at p< 0.01. ppm parts per milli iNO inhaled nitric oxide, mPAPmean pulmonary artery pressure. Figure 2 A Treatment summary of iNO therapy. B Duration of iNO treatment. C Mean PAP before and during iNO treatment in patients with ARDS under VV ECMO defined as iNO responders (decrease in mPAP during treatment ≥ 6 mmHg). D Mean PAP before and during iNO treatment in patients with ARDS under VV ECMO defined as iNO nonresponders (decrease in mPAP during treatment < 6 mmHg). Differences in mPAP were compared using the paired t-test with statistical significance at p< 0.01. ppm parts per million, iNO inhaled nitric oxide, mPAPmean pulmonary artery pressure. Page 19/21 Figure 3 A Subgroup analysis of the survival rates of inhaled nitric oxide responders and nonresponders. Figure 3 Figure 3 A Subgroup analysis of the survival rates of inhaled nitric oxide responders and nonresponders. A Subgroup analysis of the survival rates of inhaled nitric oxide responders and nonresponders. Page 20/21 A Subgroup analysis of the survival rates of inhaled nitric oxide responders and nonresponders. Figure 4 Figure 4 Page 20/21 Figure 4 Figure 4 Figure 4 Figure 4 Page 20/21 Page 20/21 Kaplan–Meier curve reporting the long-term survival rate (90 days) of patients with severe ARDS on VV ECMO support with PAH and/or RV failure treated with iNO. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. graphabstract.png SupplementaryTableS1.docx Page 21/21
https://openalex.org/W2617167973	http://juser.fz-juelich.de/record/829628/files/A%CE%B242%20pentamers%20hexamers%20are%20the%20smallest%20detectable%20oligomers%20in%20solution.pdf	English	null	Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution	Scientific reports	2,017	cc-by	12,933	Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution Received: 25 October 2016 Accepted: 11 April 2017 Published: xx xx xxxx Amyloid β (Aβ) oligomers may play a decisive role in Alzheimer’s disease related neurodegeneration, but their structural properties are poorly understood. In this report, sedimentation velocity centrifugation, small angle neutron scattering (SANS) and molecular modelling were used to identify the small oligomeric species formed by the 42 amino acid residue long isoform of Aβ (Aβ42) in solution, characterized by a sedimentation coefficient of 2.56 S, and a radius of gyration between 2 and 4 nm. The measured sedimentation coefficient is in close agreement with the sedimentation coefficient calculated for Aβ42 hexamers using MD simulations at µM concentration. To the best of our knowledge this is the first report detailing the Aβ42 oligomeric species by SANS measurements. Our results demonstrate that the smallest detectable species in solution are penta- to hexamers. No evidences for the presence of dimers, trimers or tetramers were found, although the existence of those Aβ42 oligomers at measurable quantities had been reported frequently. An increasing number of human diseases are characterized by the accumulation of specific protein aggregates found in proteinaceous depositions with a common structural motif, which is characterized by a cross-β-sheet architecture called the amyloid fold1. While these amyloid fibril folds are already clarified for a number of proteins (reviewed in ref. 2), little is known about the first stages of the amyloid formation process, where dynamic, heter- ogeneous and often toxic intermediates are most likely populated. Such oligomeric species appear to be currently elusive to high-resolution structural methods such as solution based NMR or X-ray crystallography. Aside from the size limit of solution based NMR, the rather low fraction of the quested oligomer, its transient nature and heterogeneity renders NMR measurements at least extremely difficult3. Only with stabilized oligomeric prepa- rations such studies have been feasible thus far4. Remarkably, Kotler et al. recently succeeded in the structural characterization of an Aβ40 oligomer of 5 to 15 nm size contributing to only 7% of the sample by magic angle spinning recoupling 1H-1H NMR experiments5. The above mentioned solution heterogeneity also interferes with the formation of diffracting crystals as required for X-ray crystallography. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 25 October 2016 Accepted: 11 April 2017 Published: xx xx xxxx Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution f g y q y y g p y In the case of Alzheimer’s disease (AD), which is the most common form of dementia worldwide, the accu- mulation of aggregated amyloid β protein (Aβ) in brain tissue is one of the disease hallmarks6. Aβ is generated as a proteolytic fragment from the transmembrane amyloid β-protein precursor (APP). Among the various isoforms that arise from variation of the location of the termini or by post-translational modification, the 42 amino acid long isoform (Aβ42) is the main constituent of extracellular senile plaques found post mortem in AD brains7.i Soluble Aβ oligomers, rather than the deposited fibrillar forms, are considered by many to be responsible for neurodegeneration in AD patients8, 9. The smallest Aβ assemblies reported in the literature are dimers, which exist in body fluids and brain tissues of AD patients10, 11. It should be noted that the finding of brain derived neurotoxic dimers by Shankar et al.10 had been refuted by later work12. Several studies of in vitro and in vivo experiments indicate the existence of a monomer-dimer equilibrium for Aβ13, 14. In photo-induced cross-linking 1Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225, Düsseldorf, Germany. 2Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425, Jülich, Germany. 3Jülich Centre for Neutron Science & Institute of Complex Systems, Neutron Scattering (JCNS-1&ICS-1), Forschungszentrum Jülich, 52425, Jülich, Germany. 4Jülich Centre for Neutron Science, Outstation at MLZ (JCNS-MLZ), Forschungszentrum Jülich, 85747, Garching, Germany. 5Present address: Physikalische Biochemie, University Potsdam, 14476, Golm, Germany. M. Wolff and B. Zhang-Haagen contributed equally to this work. Correspondence and requests for materials should be addressed to L.N. (email: l.nagel-steger@fz-juelich.de) Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 1 www.nature.com/scientificreports/ of unmodified proteins (PICUP) experiments using Aβ, covalently linked dimers and other larger oligomeric species12 were detected for Aβ42 and for Aβ40, the shorter isoform missing the last two c-terminal residues15, 16. While for Aβ40 trimers and tetramers were the abundant species, for Aβ42 penta-/hexamers and also dode- camers and octadecamers could be detected16. Based on the PICUP technique the involvement of a penta- to hexameric oligomers in the Aβ42 assembly process had been reported in several studies17–19. Additionally, non-cross-linked Aβ oligomers have been identified in polyacrylamide gel electrophoresis as sodium dodecyl sulphate (SDS)-resistant bands and interpreted as dimers to tetramers20. Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution Cultured neuronal cells were found to release small Aβ oligomers, among which the dimer represents a major species, whose size was determined by SDS-PAGE combined with western blotting21–23. Furthermore, dimers were found along with monomers and trimers as stable components, which could be purified by size exclusion chromatography under denaturing con- ditions, in neuritic plaques obtained from AD brain material24. Other studies have identified Aβ paranuclei as penta- or hexamers, which were detected by mass spectrometry based techniques25, 26. Recently, characterization of an enriched Aβ42 oligomer by NMR combined with AFM was achieved27. Further information on Aβ oli- gomers can be found in recently published reviews28–34.f g y p Considering the plethora of different experimental approaches, even those techniques which produced the most consistent results on the early assembly states of Aβ42, i.e., PICUP and ISM-MS, possess some shortcomings which should be addressed for the sake of gain in knowledge. Crosslinked oligomers are only well reported by PICUP if all subunits become stably connected to withstand SDS-denaturation prior to SDS-PAGE. Detergence below or above their critical micellar concentration might stabilize assemblies by shielding hydrophobic regions, which would not be stable in hydrous solutions. Ionization coupled with transfer to the gas phase as required for mass spectrometry, on the other hand, might be a selective as well as destructive process. Recently, a critical evalu- ation of PICUP followed by SDS-PAGE analysis performed by IMS-MS challenged the penta- to hexameric build- ing block in Aβ42 aggregation35. A clear understanding of the early steps in the self-association of Aβ is critical in order to understand the formation of toxic oligomeric species and the initiation of fibrillisation in Alzheimer’s disease. We therefore chose an approach with high detection sensitivity, which is applicable to proteins in solu- tion, for the characterization of the smallest oligomers of Aβ. g β Recently, sedimentation velocity (SV) analysis, which is an application of analytical ultracentrifugation (AUC), was shown to be a highly sensitive detection method for trace amounts of aggregates in protein samples. Sedimentation velocity centrifugation allows distinguishing multiple sedimenting species in free solution while maintaining reversibly formed complexes in a bath of their components at all times. This permits the study of self-association as well as heterogeneous protein interactions36. Using SV, <1% aggregates in a sample can be reli- ably detected, provided instrument and equipment are handled with sufficient care37. Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution SV analysis can determine the size- and shape distributions for self-assembling proteins38–41. Due to the fractionating property of the sedi- mentation process, with large particles sedimenting before small particles, the method is resistant to the presence of large particles such as unresolved protein aggregates or dust particles. The method is characterized by an excel- lent signal-to-noise ratio. The evolution of the shape of the formed sedimentation boundary over time reveals not only information about the sedimentation velocity but also about the diffusion properties of the analytes42, 43. Importantly, the method requires no interactions of Aβ with surfaces that might induce aggregation. SANS is a well-established, non-destructive method to examine structure on length scales of 1 to 1000 nm. While only one report on a SANS study of full length Aβ was found in PubMed44, which is about the characterization of Aβ40 at acidic solvent conditions, some more reports exist on small angle X-ray scattering (SAXS) studies of Aβ4245, 46. X-ray scattering has the disadvantage of being destructive to proteins and the generation of radicals might impair the aggregation process. In a previous study, we demonstrated the applicability of SANS using monomeric Aβ47. While in SV the hydrodynamic properties of macromolecules are evaluated, yielding the hydrodynamic radius, in SANS the properties of macromolecules as neutron scatterers are evaluated yielding also the radius of gyration, aside from information about size, shape and interactions. Because the two solution-based methods are exploiting different physical properties they are ideally suited to complement each other. f p y p p y y p By SV analysis, we previously demonstrated the existence of discrete oligomeric species in the s-value range from 4 to 15 S during the lag phase of fibril formation48. Analysis reproducibly yielded two Aβ42 assembly species at low salt conditions and physiological pH, independent of the total Aβ42 concentration; one corresponding to a 12-mer and the other to an 18-mer of Aβ42. These sizes suggest the involvement of a trimeric or hexameric build- ing block. A pentameric to hexameric building block could be evaluated by PICUP for Aβ42 and has been defined by Bitan et al. as paranucleus15, 16, 44. We interpreted the recently observed gap in the determined s-value distri- butions between 0.69 S, corresponding to the Aβ42 monomer and 4 S as the range for the nucleus size48. Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution In this s-value region corresponding to molecular weights between 5 and 55 kDa, we hypothesize further on-pathway to fibril reaction intermediates. Here, we aim to close this gap by combining two techniques for the structural characterization of macromolecules in solution, namely SV analysis and small angle neutron scattering (SANS). Because the two solution-based methods are exploiting different physical properties they are ideally suited to cor- roborate each other in order to provide important information about the first steps of the nucleation process. In addition, we further complement our experimental results by including results from molecular dynamics (MD) simulations of Aβ42 assemblies, which bridge our SV and SANS results. This combined approach reveals clear experimental evidence for a penta- or hexameric assembly of Aβ42 in solution. Resultsh This model can then be tested by F-statistics for the significance of the high molecular weight species. This was achieved by calculating a critical relative root mean square devi- ation (rmsd) value based on the model including the oligomeric species. A species is considered as a significant part of a sample if the rmsd of a fit excluding this species exceeds the critical relative rmsd. Thus the significance of the oligomeric species was shown for four data sets by observing an increase of the rmsd above the critical rmsd upon removal of the larger species (Fig. 1a,b).h p g p g The total Aβ42 concentration was varied from 0.1 μM to 160 μM. All SV measurements were performed with Aβ42 incubation times of ~2 h before SV centrifugation. The s-value for the monomeric AF488-Aβ42 was deter- mined as 0.80 ± 0.01 S, which is slightly higher than the s-value 0.6 ± 0.02 S found for the unlabelled monomer (Table 1). The monomer accounts for 61% of the total fluorescence signal. The s-values of the smallest detectable oligomers in this experiment were determined as 2.32 ± 0.14 S for AF488-Aβ42 and 2.56 ± 0.32 S for Aβ42. The smaller s-value for AF488-Aβ42 oligomer in comparison to the unlabelled Aβ42 oligomer could be explained by an increase of the frictional coefficient f or shape factor f/f0 for AF488-Aβ42 due to the fluorophores protruding away from the oligomer. For the monomer, which is unstructured in aqueous solution46, the increase in mass seems to be the dominating effect leading to an increase of the s-value for AF488-Aβ42. In Fig. 1b the c(s) distri- butions for four independent samples of 0.1 μM AF488-Aβ42 dissolved in 10 mM phosphate buffer are shown. At this low concentration aside from the monomer and the residual free dye only a small fraction of small oligo- meric species were detected. These oligomers are dominated by a species between 2 S and 3 S, which represents the smallest s-value of the oligomer distribution. The fraction of this species accounts for about 1.4% of the total signal. After subtracting the contribution of the free dye the value increases to 2% of total AF488-Aβ42. In order to evaluate whether by changing the temperature the equilibrium could be shifted from monomers to penta-/ hexamers, we performed SV experiments at different temperatures between 10 and 30 °C. Resultsh The goal of this study is to identify metastable intermediates during the aggregation of Aβ42 in solution and thus to elucidate the initial steps of Aβ42 assembly. Sedimentation velocity centrifugation allows sensitive detection of Aβ oligomers in solution The first step in searching for small Aβ assemblies was to identify the smallest species, i.e., the dimers, and prove Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 2 www.nature.com/scientificreports/ that SV analysis can discriminate them from monomers. As a model, a stable synthetic Aβ40 dimer was gener- ated from a monomer with a cysteine residue at position 0, which formed an intermolecular disulphide bridge under oxidizing conditions. SV analysis revealed that the Aβ monomer and the covalently linked dimer are suffi- ciently different, with s-values of 0.65 and 0.9 S, respectively (Fig. S2). None of the SV experiments performed for non-linked Aβ42 provided evidence of an Aβ42 dimer in solution. If Aβ42 dimers or trimers were in rapid equi- librium with the monomers, one would expect that at higher Aβ42 concentrations the equilibrium were shifted from monomers towards the dimers and trimers. For such a sample, the peak in c(s) would not represent a single s-value species, but a so-called reaction boundary. A diagnostic for the identification of such a reaction boundary would be a shift towards higher s-values at higher Aβ42 concentrations or at least peak broadening. The super- position of the c(s) peaks for the monomer from different data sets revealed no Aβ42 concentration dependent shift in s-value nor peak broadening of the c(s) signal at 0.62 S. We thus conclude that the dimer fraction is below the detection limit. The smallest detectable assembly state in the SV experiments is a penta- to hex- amer. Experimental evidence for oligomeric species between trimers and 18-mers were found mainly from SDS-PAGE analysis after PICUP15, 16 and mass spectrometry studies. For example, a trimer is postulated as the building block for the Aβ56 aggregate found in in-vivo studies by Lesné and colleagues49, 50, and trimers along with tetramers were also detected by Bernstein et al.17, 51. We decreased the total Aβ42 concentrations to the low micro- to nanomolar concentration range in our SV experiments, expecting that at Aβ42 concentrations below the critical concentration for the formation of higher-order structures, the assembly of larger assemblies from small oligomers is suppressed. Resultsh The necessary increase in detection sensitivity was gained by using AUC cou- pled with a fluorescence detection system. Thus, a lower limit of about 100 nM fluorophore labelled Aβ42 was achieved. For the position of the fluorescence label we chose the N-terminus of Aβ, because the N-terminus is not primarily involved in intermolecular contacts required for aggregate formation52 and similar conjugates had been successfully used before53. Additionally, those regions of Aβ42, which had been identified as critical for paranucleus formation, i.e. the central hydrophobic core as well as the C-terminal residues15, are distantly located from the fluorophore. A linker further separates the fluorophore from the Aβ42 peptide and introduces flexibility. Moreover, charged fluorophores also possess a smaller propensity to stack on each other than uncharged fluoro- phores. From our AFM measurements (Fig. S3) we concluded that the fluorescence label neither prohibits fibril formation nor leads to extensive amorphous aggregate formation. Nevertheless, it cannot be fully excluded that the presence of a fluorescence label at the N-terminus favours certain paranuclei conformations, which would be less or not at all populated by the wild-type Aβ42 peptide. Thus, we conclude that the application of the fluorophore-labelled conjugate allow us to expand our measurements into the submicromolar Aβ42 concentra- tion range and to draw conclusions that can be transferred with the above mentioned restrictions to wild-type Aβ42. SV analysis is a technique, which recently gained special relevance in the determination of aggregate con- centration in samples of pharmaceutically relevant proteins54–57. According to theoretical considerations at high signal-to-noise ratios of absorbance and fluorescence detection, a superior sensitivity for traces well below 1% of total signal is achievable in sedimentation velocity experiments57. For example; this would correspond to 0.01 absorbance units of an oligomer in the presence of 0.99 absorbance units of the monomer at a suitable wave- length. The limit of detection (0.2% of total signal) and the limit of quantification (about 2.8% of total signal) have been determined from practical studies on a dimeric antibody37. For confirmation of the detected high molecular weight species, the calculation of the c(s) distribution was extended by applying a Bayesian approach followed by F statistics, as reported recently by Wafer et al.54. After repeating the c(s) calculation by alternating between the simplex and Marquardt-Levenberg algorithms until no further change in rmsd was observed, the c(s) model is exchanged by a model of non-interacting species. Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 Resultsh However, variation of the experimental temperature in this range did not lead to measurable changes in the oligomeric fractions. To exclude the possibility of contamination or that we observe a covalently bonded aggregate species, we treated the Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 3 www.nature.com/scientificreports/ Figure 1. SV analysis of 0.1 µM AF488-Aβ42 with fluorescence detection reveals the existence of small oligomers in addition to the monomer. (a) Raw data of SV experiment with overlaid lines for the fitted data, showing a signal intensity of about 2500 RFU. Only every third scan, as well as every third data point is shown for clarity. In the box below the corresponding residuals within the fitted data range are shown. Rmsd for this sample was 11.86. (b) Area normalized c(s) results for four independent samples of 0.1 µM AF488-Aβ42 have been superimposed in the graph. The 0.8 S species is assigned to the monomer, the peak at 0.3 S is the result of either incomplete removal of unincorporated dye or the dark count rate or a combination of both. The inset shows the magnification of c(s) distributions. Sedimentation was performed at 60,000 rpm, 20 °C. Figure 1. SV analysis of 0.1 µM AF488-Aβ42 with fluorescence detection reveals the existence of small i Figure 1. SV analysis of 0.1 µM AF488-Aβ42 with fluorescence detection reveals the existence of small oligomers in addition to the monomer. (a) Raw data of SV experiment with overlaid lines for the fitted data, showing a signal intensity of about 2500 RFU. Only every third scan, as well as every third data point is shown for clarity. In the box below the corresponding residuals within the fitted data range are shown. Rmsd for this sample was 11.86. (b) Area normalized c(s) results for four independent samples of 0.1 µM AF488-Aβ42 have been superimposed in the graph. The 0.8 S species is assigned to the monomer, the peak at 0.3 S is the result of either incomplete removal of unincorporated dye or the dark count rate or a combination of both. The inset shows the magnification of c(s) distributions. Sedimentation was performed at 60,000 rpm, 20 °C. oligomers in addition to the monomer. (a) Raw data of SV experiment with overlaid lines for the fitted data, showing a signal intensity of about 2500 RFU. Only every third scan, as well as every third data point is shown for clarity. Resultsh In the box below the corresponding residuals within the fitted data range are shown. Rmsd for this sample was 11.86. (b) Area normalized c(s) results for four independent samples of 0.1 µM AF488-Aβ42 have been superimposed in the graph. The 0.8 S species is assigned to the monomer, the peak at 0.3 S is the result of either incomplete removal of unincorporated dye or the dark count rate or a combination of both. The inset shows the magnification of c(s) distributions. Sedimentation was performed at 60,000 rpm, 20 °C. Monomer (S) Small Oligomer (S)* Aβ42 0.62 ± 0.02 2.56 ± 0.32 AF488-Aβ42 0.80 ± 0.01 2.32 ± 0.14 Table 1. Experimentally determined sedimentation coefficients for Aβ42 monomer and small oligomer. Sedimentation coefficients for the monomeric and small oligomeric species of Aβ42 were determined from c(s) distributions. Weight averaged s20,w-values and standard deviations were calculated from at least 4 independent sample preparations. Table 1. Experimentally determined sedimentation coefficients for Aβ42 monomer and small oligomer. Sedimentation coefficients for the monomeric and small oligomeric species of Aβ42 were determined from c(s) distributions. Weight averaged s20,w-values and standard deviations were calculated from at least 4 independent sample preparations. sample with 6 M guanidine hydrochloride. Under these denaturing conditions this oligomer species disappeared (data not shown). Figure 2 shows the c(s) distributions obtained for 5 (Fig. 2a,c) and 10 μM Aβ42 (Fig. 2b,c). At these medium Aβ42 concentrations, larger aggregates become detectable in addition to the small oligomers. Figure 3 shows three c(s) distributions obtained for high Aβ42 concentrations, which demonstrate that the small oligomer is still detectable under these conditions, where larger aggregates have already formed. In Table 1, the mean s20,w-values for the monomer and for the small oligomers are summarized for Aβ42 and AF488-Aβ42. g In summary, SV measurements covering a concentration range between 0.1 μM and 160 μM consistently reveal the presence of a small oligomer at 2.56 S or 2.32 S for Aβ42 and AF488-Aβ42, respectively. According to MD simulations described below, these values correspond to Aβ42 pentamers or hexamers. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d Small angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- to hexamer species. In contrast to SV with fluorescence detection, SANS measurements do not require dye labelling; although, an equivalent D2O buffer is used to reduce buffer scattering. For the relative low concentra- tions used in the aggregation studies, a long counting time was required to obtain reasonable statistical accuracy because the detected signal at larger wave vectors is two orders of magnitude below the buffer scattering. To slow down the aggregation process and reduce measurement times to ~8 h for several detector distances without sig- nificant change in the sample, the measurements have been performed at 7 °C. SANS measurements have been performed for Aβ42 with incubation times between 0.5 and 316 h (Fig. 4) for concentrations of 221 μM (1 mg/ml), 55 μM 0.25 mg/ml) and 22 μM (0.1 mg/ml). SANS measurements show that even at low temperature and relative low concentrations aggregates of all sizes are formed after the addition of buffer to the dry Aβ. This in in contrast to SV experiments, where at the speed applied to study the small oligomers larger oligomers, protofibrils or fibrils are not detectable due to their fast sedimentation. The scattering intensity of the samples did not change measurably within 10 h as monitored by dynamic light scattering measurements (data not shown). The measured intensities after buffer subtraction were analysed by a model adding contributions of larger aggregates and complexes, midsize aggregates, small oli- gomers and monomers as shown in Fig. 4a. Except for the monomer, the populations of the different assembly states were determined in accompanying measurements by dynamic light scattering (Fig. S4). Large aggregates or complexes, such as assemblies of fibrils with radii of gyration (Rg) larger than 50 nm contribute at low Q by a power law − ~Q d with d between 5/3 and 3 as expected for networks/gels58. Midsize aggregates, such as protofibrils Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 4 www.nature.com/scientificreports/ Figure 2. The SV analysis at low micromolar Aβ42 concentrations yields the existence of the small oligomer between 2 S and 3 S and a fraction of larger assemblies with s-values between 3 and 12 S. The raw data at 5 µM (a) and 10 µM Aβ42 (b) together with overlaid fit results and residuals are shown. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d The corresponding c(s) results for 5 µM (c) and 10 µM Aβ42 (d) are shown below. Sedimentation was performed at 50,000 rpm, 20 °C and monitored with absorbance detection. Detection wavelength for 5 µM was 208 and 220 nm for 10 µM Aβ42. The c(s) distribution was normalized to a maximum c(s) value. The insets show the distributions with an expanded y scale. igure 2. The SV analysis at low micromolar Aβ42 concentrations yields the existence of the small oligomer h Figure 2. The SV analysis at low micromolar Aβ42 concentrations yields the existence of the small oligomer between 2 S and 3 S and a fraction of larger assemblies with s-values between 3 and 12 S. The raw data at 5 µM (a) and 10 µM Aβ42 (b) together with overlaid fit results and residuals are shown. The corresponding c(s) results for 5 µM (c) and 10 µM Aβ42 (d) are shown below. Sedimentation was performed at 50,000 rpm, 20 °C and monitored with absorbance detection. Detection wavelength for 5 µM was 208 and 220 nm for 10 µM Aβ42. The c(s) distribution was normalized to a maximum c(s) value. The insets show the distributions with an expanded y scale. Figure 2. The SV analysis at low micromolar Aβ42 concentrations yields the existence of the small oligomer between 2 S and 3 S and a fraction of larger assemblies with s-values between 3 and 12 S. The raw data at 5 µM (a) and 10 µM Aβ42 (b) together with overlaid fit results and residuals are shown. The corresponding c(s) results for 5 µM (c) and 10 µM Aβ42 (d) are shown below. Sedimentation was performed at 50,000 rpm, 20 °C and monitored with absorbance detection. Detection wavelength for 5 µM was 208 and 220 nm for 10 µM Aβ42. The c(s) distribution was normalized to a maximum c(s) value. The insets show the distributions with an expanded y scale. or fibrils were modelled by an ellipsoid of revolution with Rg between 50 and 10 nm1. Small oligomers were mod- elled by a Beaucage model F R d ( ( , )) g providing the Rg of the small aggregates with fixed dimensionality59. Monomers contribute with a fixed Rg = 1 nm as a background contribution and cannot be resolved. Small oli- gomers with Rg between 1 and 5 nm were observed at different concentrations throughout the experiments (Fig. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d 4b). h ld b h d h b l d f d ff h h ll d g It should be emphasized that we observe oligomers and aggregates of different sizes, which are well separated in the SANS signal. If objects of all sizes were present in the sample, we would observe an undefined, decay- ing scattering intensity without a shoulder. Importantly, the small oligomers (identified as hexa-/pentamers, see below) can only be observed if species between the small oligomers and monomers are significantly missing and do not contribute intensity above the shoulder at ~0.6 nm−1. If smaller oligomers than pentamers were present, the scattered intensity could not be described by a Beaucauge function separated from the monomer background. Nevertheless, the presence of smaller species cannot be completely ruled out, but is limited to amounts which contribute to the scattering signal at the scale of the noise of the measurements. MD simulations provide size and shape information about early oligomeric species. From the Rg values determined from the SANS data it is not possible to determine the size of the oligomers. Therefore, in order to compare the oligomer sizes observed in the SV experiments with the Rgvalues obtained from the SANS experiments, we performed five independent all-atom MD simulations, yielding a total simulation time was 1 μs, of twenty Aβ42 monomers inserted in a cubic box at a solute concentration of ~80 µM60. While this concentration is considerably larger than that used in the SV experiments, it should be noted that, to the best of our knowl- edge, it is lower compared to all other atomistic simulations studying Aβ aggregation. Moreover, as the current simulation results are used to provide Rg and s-values for the different oligomer sizes, their relative population as sampled in the simulations is not important. Instead, of importance is that the oligomer structures are sampled realistically. In order to check that the oligomer structures are not affected by the high peptide concentration or the use of an implicit solvent, we performed additional MD simulations of isolated dimers, trimers and tetramers using an explicit water model. The resulting oligomer structures have similar s- and Rg values and also exhibit similar structures as those obtained from the simulations involving twenty Aβ peptides. Thus, it can be concluded that our MD results are not affected by the rather high peptide concentration used in the current simulations. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 5 www.nature.com/scientificreports/ Figure 3. SV analysis at high Aβ42 concentrations. Measured data overlaid with fit data and corresponding residuals are shown for 32 μM (a), 95 μM (b) and 160 μM (c) Aβ42. Below, c(s) analysis reveals the existence of a high fraction of larger oligomeric species aside from the monomer and a comparably low fraction of the 2 to 3 S species for 32 µM (d), 95 µM (e) and 160 µM (f) Aβ42. Sedimentation was performed at 60,000 rpm, 10 °C and monitored with absorbance detection at 280 nm. Normalization was performed according to peak height of the monomeric Aβ42. The insets show the distributions with an expanded y scale. Figure 3. SV analysis at high Aβ42 concentrations. Measured data overlaid with fit data and corresponding residuals are shown for 32 μM (a), 95 μM (b) and 160 μM (c) Aβ42. Below, c(s) analysis reveals the existence of a high fraction of larger oligomeric species aside from the monomer and a comparably low fraction of the 2 to 3 S species for 32 µM (d), 95 µM (e) and 160 µM (f) Aβ42. Sedimentation was performed at 60,000 rpm, 10 °C and monitored with absorbance detection at 280 nm. Normalization was performed according to peak height of the monomeric Aβ42. The insets show the distributions with an expanded y scale. Figure 3. SV analysis at high Aβ42 concentrations. Measured data overlaid with fit data and corresponding id l h f 32 M ( ) 95 M (b) d 160 M ( ) Aβ42 B l ( ) l i l th i t Figure 4. Results from SANS scattering experiments after incubation of Aβ42 at pH 7.4. Measurement and incubation was in 50 mM Napi plus 150 mM NaCl in 100% D2O at 7 °C. (a) Exemplary SANS scattering intensity after 0.5 h. Contributions to the scattered intensity arise from aggregates larger than 50 nm resulting in a power law (black broken line), mid-size aggregates modelled as ellipsoid of revolution (blue broken line) and small oligomers (orange broken line), which are modelled by a Beaucage function. A vanishing background of monomers is modelled as a Beaucage function with fixed Rg = 1 nm. The combined fit result is shown as a black solid line. Ellipsoids have radii between 50 and 10 nm. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d (b) The radius of gyration of small oligomers from SANS analysis after incubation at concentrations of 221 μM (black), 55 µM (blue) and 22 µM (green). Figure 4. Results from SANS scattering experiments after incubation of Aβ42 at pH 7.4. Measurement and incubation was in 50 mM Napi plus 150 mM NaCl in 100% D2O at 7 °C. (a) Exemplary SANS scattering intensity after 0.5 h. Contributions to the scattered intensity arise from aggregates larger than 50 nm resulting in a power law (black broken line), mid-size aggregates modelled as ellipsoid of revolution (blue broken line) and small oligomers (orange broken line), which are modelled by a Beaucage function. A vanishing background of monomers is modelled as a Beaucage function with fixed Rg = 1 nm. The combined fit result is shown as a black solid line. Ellipsoids have radii between 50 and 10 nm. (b) The radius of gyration of small oligomers from SANS analysis after incubation at concentrations of 221 μM (black), 55 µM (blue) and 22 µM (green). Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 6 www.nature.com/scientificreports/ Figure 5. Calculated sedimentation coefficients and radii of gyration of Aβ42 oligomers obtained from MD simulations. (a) Normalized distributions of sedimentation coefficients for Aβ42 tetramers (black), pentamers (red), hexamers (blue), 12-mers (green), and 18-mers (magenta). (b) Normalized radii of gyration for the ensembles of Aβ42 tetramers (black), pentamers (red), hexamers (blue), 12-mers (green), and 18-mers (magenta). All values for tetra- to hexameric Aβ42 oligomers were calculated with the program HydroPro._ ENREF_69 Corresponding weight averaged s-values are summarized in Table 2. Figure 5. Calculated sedimentation coefficients and radii of gyration of Aβ42 oligomers obtained from MD simulations. (a) Normalized distributions of sedimentation coefficients for Aβ42 tetramers (black), pentamers (red), hexamers (blue), 12-mers (green), and 18-mers (magenta). (b) Normalized radii of gyration for the ensembles of Aβ42 tetramers (black), pentamers (red), hexamers (blue), 12-mers (green), and 18-mers (magenta). All values for tetra- to hexameric Aβ42 oligomers were calculated with the program HydroPro._ ENREF_69 Corresponding weight averaged s-values are summarized in Table 2. Figure 5. Calculated sedimentation coefficients and radii of gyration of Aβ42 oligomers obtained from MD Oligomer Tetramer Pentamer Hexamer s-value (S) 2.09 ± 0.06 2.38 ± 0.06 2.61 ± 0.07 No. of conformations 576 123 299 Table 2. Average sedimentation coefficients for Aβ42 tetramers, pentamers and hexamers based on confirmations obtained from MD simulations. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d Importantly, the SANS studies revealed no species in size between the monomer and the putative pentamer/ hexamer. First, we estimated the sedimentation coefficients of the modelled Aβ42 oligomers. Normalized distributions of the sedimentation coefficient for different oligomer orders are shown in Fig. 5a. The resulting weight averaged s-values for the calculated distribution are listed in Table 2. Tetramers displayed the lowest sedimentation coef- ficients with an average value of 2.09 ± 0.06 S, followed by pentamers with 2.38 ± 0.06 S and hexamers with an average value of 2.61 ± 0.07 S. For the current analysis we used 576 tetramers, 123 pentamers and 299 hexamers. The s-values obtained for the small oligomeric species collected in five MD simulations show a distribution, e.g., hexamers are found with s-values between 2.42 S and 2.90 S, which seems to approach a Gaussian like shape with higher sampling. The distributions reflect the heterogeneity of the simulated oligomers with regard to their sur- face area. Nevertheless the overlap between two adjacent oligomer sizes does not exceed 0.3 S units, which allows us to conclude that we identified pentamers and hexamers as smallest oligomeric species in our SV experiments. i To correlate the Rg values determined from the SANS experiments, we calculated the Rg for the ensemble of structures generated by our MD simulations (Fig. 5b). For the lowest Aβ concentration of 55 µM used in the SANS experiments, the constant presence of oligomers with Rg ~ 2.3 nm corresponds to pentamer and/or hexamer species. According to Fig. 5b, it is difficult to discriminate between pentamers and hexamers based on the Rg value only. At 221 μM the small oligomers have Rg values of around 4 nm at the beginning and after 120 h incubation we find smaller aggregates again with Rg ~ 2 nm, which probably correspond to a pentamer/hexamer (Fig. 4b). Interestingly, the largest radius of gyration calculated from SANS measurements of ~5 nm corresponds to assem- blies of 18 Aβ42 peptides, according to MD simulations. At the earliest measurement time it was not possible to discriminate between different size classes because of the strong smearing between the various species. Importantly, the SANS studies revealed no species in size between the monomer and the putative pentamer/ hexamer. mall angle neutron scattering studies of Aβ42 in solution corroborate the presence of penta- h i I SV i h fl d i SANS d i d The number of conformations is the total number before the clustering algorithm was applied. All values were calculated with the program HydroPro._ENREF_75. Table 2. Average sedimentation coefficients for Aβ42 tetramers, pentamers and hexamers based on confirmations obtained from MD simulations. The number of conformations is the total number before the clustering algorithm was applied. All values were calculated with the program HydroPro._ENREF_75. First, we estimated the sedimentation coefficients of the modelled Aβ42 oligomers. Normalized distributions of the sedimentation coefficient for different oligomer orders are shown in Fig. 5a. The resulting weight averaged s-values for the calculated distribution are listed in Table 2. Tetramers displayed the lowest sedimentation coef- ficients with an average value of 2.09 ± 0.06 S, followed by pentamers with 2.38 ± 0.06 S and hexamers with an average value of 2.61 ± 0.07 S. For the current analysis we used 576 tetramers, 123 pentamers and 299 hexamers. The s-values obtained for the small oligomeric species collected in five MD simulations show a distribution, e.g., hexamers are found with s-values between 2.42 S and 2.90 S, which seems to approach a Gaussian like shape with higher sampling. The distributions reflect the heterogeneity of the simulated oligomers with regard to their sur- face area. Nevertheless the overlap between two adjacent oligomer sizes does not exceed 0.3 S units, which allows us to conclude that we identified pentamers and hexamers as smallest oligomeric species in our SV experiments. To correlate the Rg values determined from the SANS experiments, we calculated the Rg for the ensemble of structures generated by our MD simulations (Fig. 5b). For the lowest Aβ concentration of 55 µM used in the SANS experiments, the constant presence of oligomers with Rg ~ 2.3 nm corresponds to pentamer and/or hexamer species. According to Fig. 5b, it is difficult to discriminate between pentamers and hexamers based on the Rg value only. At 221 μM the small oligomers have Rg values of around 4 nm at the beginning and after 120 h incubation we find smaller aggregates again with Rg ~ 2 nm, which probably correspond to a pentamer/hexamer (Fig. 4b). Interestingly, the largest radius of gyration calculated from SANS measurements of ~5 nm corresponds to assem- blies of 18 Aβ42 peptides, according to MD simulations. At the earliest measurement time it was not possible to discriminate between different size classes because of the strong smearing between the various species. Discussion The consistently low fraction of the small oligomeric species over a broad range of Aβ42 concentrations, which is reported by both methods, is a strong indicator of its role as an important reaction intermediate with low population.t p p p Within the accessible Aβ42 concentration range used it was impossible to shift the equilibrium towards higher fractions of the pentamer/hexamer assembly. Higher fractions are most probably prevented by a loss of the pen- tamer/hexamer to larger oligomers, once a threshold Aβ concentration has been reached. At concentrations below 10 µM, the pentamer/hexamer is the sole oligomeric species. At higher concentrations, the pentamer/hex- amer appears to be in a steady-state equilibrium with the monomer and larger oligomeric species, representing a lowly populated reaction intermediate. This classifies the pentamer/hexamer to be an on-pathway intermediate of fibril formation. i Ahmed et al. reported the characterization of a penta- or hexameric Aβ42 with structural information from solution and solid-state NMR27. They also used a low salt phosphate buffer and a similar pre-treatment of Aβ42. In contrast to our study, the oligomeric, disc-like particle they described has a size (diameter 10–15 nm, height ~2 nm) that would lead to a sedimentation coefficient of ~6 S, which is much larger than our species of ~2.6 S. Therefore our penta- to hexamer has to be a different oligomeric species. These differences might be caused by a deviating peptide conformation and/or packing. Furthermore we always observe a considerable amount of free monomer in the presence of our oligomeric species, which appears to be absent in the aforementioned study. A study on oligomeric species of Aβ based on high resolution atomic force microscopy also confirms the hexamer and 12 mer as dominant species in addition to monomers and dimers61. In earlier studies, small Aβ assemblies with 2 to 6 monomeric units have been identified mostly on the basis of SDS-PAGE analysis of either cross-linked or non-cross linked samples20, 62. In addition, a number of computational studies of Aβ oligomerization revealed the existence of several small assemblies, which considerably vary with regard to secondary structure content and overall appearance60, 63. In a study of Aβ42 fused to GroES and ubiquitin and performed at high pH in the presence of urea, Aβ42 assembled into SDS-resistant hexamers and tetramers64. This study suggests that only the most stable complexes will occur under the chosen reaction conditions. Discussion p Our SANS as well as SV results demonstrate that solutions of Aβ42 do not contain dimers, trimers or tetram- ers at detectable levels. Under the assumption of a rapid equilibrium between these small oligomers, the lower limit of detection would be increased due to broadening of the peak or missing resolution of single species to ~5% according to SV data simulations. A possible explanation for the appearance of these small species in a number of other studies would be that these species represent fragmentation products of larger species. The smallest detectable species is a penta- to hexamer. The fraction of this species is independent of the total Aβ42 concentration and close to 1% of the sedimentation boundary. Aβ42 penta-/hexamers are a recurring motif in literature and have been reported frequently over the last years (reviewed in refs 32 and 65). They appear not only to exist in solution but might also represent the Aβ42 conformation interacting with lipids or lipid bilayers66, 67. Nevertheless, we want to emphasize that our study, for the first time, provides evidence for the Aβ42 penta-/hex- amer by two solution based methods without the addition of stabilizers. Other groups that showed the presence of similar small oligomers applied the IMS-MS technique17, 68, which required the transfer of ionized molecules and molecular complexes into the gas phase, leaving space for additional events to take place. Nevertheless, it remains reasonable to assume, that, because of the concentration dependency of the aggregation, there is a low concentration at which only dimers and trimers but no larger oligomers are in equilibrium with the monomers. Lowering the total Aβ42 concentration in our experiments has been one of the strategies we followed in order to suppress the conversion of monomers into large aggregates and to increase the fraction of the small oligomeric species. However, we could not detect dimers and trimers under our experimental conditions. These species seem to be less populated than the penta-/hexameric oligomer. Additionally, it might be possible to speculate that their higher abundance in other studies is a consequence of fragmentation of larger oligomers. g q g g g Our previous studies by sedimentation velocity centrifugation revealed the power of the method to charac- terize Aβ oligomeric species in equilibrium in solution. The formerly observed gap in the size distribution could be further restricted. Discussion Our study shows evidence for the occurrence of small oligomeric assemblies built of five to six Aβ42 monomers in solution. This conclusion emerged from the application of two independent experimental approaches, which are based on different physical principles, SV analysis and SANS. The two approaches were bridged by MD sim- ulations. The experimentally determined s-values of the smallest oligomers observed in SV experiments were compared to s-values calculated for a set of different oligomers generated by MD simulations. According to this comparison, a combination of pentamers and hexamers explains the best the experimentally observed s-values. Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 7 www.nature.com/scientificreports/ From SANS measurements we calculated radii of gyration between 2 and 5 nm, which are in agreement with radii of gyration of assemblies ranging between pentamers and 18-mers calculated for the same set of MD simulated oligomers. Theoretically a sedimentation coefficient is informative on mass as well as shape of a molecular species, but since the detected oligomeric species represents only a very small fraction of the mixture the weight-average of f/f0 does not provide reliable information on mass or shape of the oligomer. Therefore, based on the experimen- tally determined s-values we calculated hydrodynamic radii assuming a tetra-, penta-, and hexamer (Supporting Eq. 1 and Supporting Table 2). Assuming that hydrodynamic radius and radius of gyration are sufficiently similar for the oligomer we concluded that the measured s-value can be best explained by a pentamer or hexamer.h These comparisons led us to a model of a penta- to hexamer for the small oligomeric Aβ42 species that would explain the best the SV and SANS results. Data interpretation was further complemented by MD simulations of Aβ42 assembly, which were performed as close as possible to the experimental conditions. Deviating buffer conditions in SANS experiments were shown to give comparable results in SV experiments (Fig. S5) with regard to oligomer formation. g Remarkably, in both the SV and SANS experiments only a small fraction of about 1 to 10% of total Aβ42 could be detected in the pentamer/hexamer state. The dominant Aβ42 species is the monomeric state and larger oli- gomers present only at higher concentrations. Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 Methods i Experimental details. Amyloid β-Proteins. Synthetic Aβ42 was purchased from Bachem as a trifluoroace- tate salt (Bachem H-1368). Aβ42 was first dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for at least 12 h in order to remove any preexisting aggregates. Before usage HFIP was removed by lyophilisation. Aliquots were stored at −80 °C until use. Repeated freeze thaw cycles were generally avoided.l Experimental details. Amyloid β-Proteins. Synthetic Aβ42 was purchased from Bachem as a trifluoroace- tate salt (Bachem H-1368). Aβ42 was first dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for at least 12 h in order to remove any preexisting aggregates. Before usage HFIP was removed by lyophilisation. Aliquots were stored at −80 °C until use. Repeated freeze thaw cycles were generally avoided.l For fluorescence detection measurements we used recombinantly produced Aβ42 with an additional cysteine residue at position 0 (Cys0). Cloning was based on a plasmid encoding wild-type Aβ(1–42) as a fusion protein with an N-terminal His6-tag, a solubilizing fusion partner (NANP)19 and a modified tobacco etch virus protease (TEV) recognition site, which was kindly provided by Finder, Glockshuber and co-workers70. Cys0-Aβ42 was labelled by maleimide chemistry with AlexaFluor488 (Molecular Probes, Thermo Fisher Scientific) including a short linker (CH2)5. The dye conjugate was further purified by SEC from unincorporated dye and is named AF488-Aβ42 (see Fig. S1 for structural information). The partial specific volume of the dye-protein conjugate was calculated according to Durchschlag71, 72 as = . v 0 7127 cm3/g; see also v values summarized (Table S1). A synthetic, covalently linked Aβ40 dimer was obtained by using Aβ40 with an additional Cys residue at the N-terminus, Cys-Aβ40 (Bachem H-7368) under oxidizing conditions. The disulphide bridge linked dimer was purified by high performance liquid chromatography (HPLC). The partial specific volume calculated for the Cys-Aβ40-dimer with the help of sednterp (vs. 20120828 BETA)73 is v = 0.7316 cm3/g at 20 °C and v = 0.7295 cm3/g at 15 °C. Sedimentation Velocity Centrifugation. AUC was performed with an Optima XL-A or ProteomLab XL-A (Beckman Coulter). The ProteomLab XL-A is equipped with standard absorbance optics and an additional fluorescence detection system (Aviv Biomedical Inc.). Samples were filled either in 12 mm titanium double sector cells with 400 µl filling volume (Nanolytics) for absorbance detection or in 3 mm titanium double sector cells with 100 µl filling volume for fluorescence measurements. Methods i An integrated spacer places the sample volume to the upper third of a cell assembly guaranteeing an optimal focus position (Nanolytics) for the fluorescence detection. For both cell assemblies quartz windows were used. Labelled or unlabelled Aβ42, pre-treated with HFIP, was dissolved in 10 mM sodium phosphate buffer (NaPi), pH 7.4 at concentrations between 0.1 to 160 µM directly prior to the SV experiments. All samples were thermally equilibrated within the centrifuge for ~2 h before start- ing the run. Sedimentation velocity runs with absorbance detection were performed either at 50,000 rpm or at 60,000 rpm, equivalent to 201,600 g or 289,000 g, respectively, at the maximum radius of 7.2 cm. At concentrations above 20 µM the runs were performed at 10 or 15 °C to suppress aggregation during centrifugation. For absorb- ance data collection a radial resolution of 30 µm was chosen together with the shortest possible scan interval, which was ~1.5 min. The detection wavelength was chosen such that sample absorbance was between 0.5 and 1.2 OD. In general, up to 25% loss of Aβ42 concentrations had been determined by comparing the signal of the first scan with the spectroscopically determined concentrations of the samples prior to filling the samples into the cells for SV measurements. This loss during the acceleration phase might be either due to incomplete dissolution, rapid aggregate formation, or attachment of Aβ42 to surfaces, i.e. glass windows. Higher recovery rates were not achievable with Aβ at neutral pH. It is a special feature of AUC that these losses can be easily quantified. h fl d b d l h d l l fi d p p y qi For the fluorescence detection system, based on laser excitation at 488 nm, the radial resolution was fixed at 20 µm and data acquisition was not done sequentially but simultaneously for all sample sectors, leading to scan intervals shorter than 1 min. Each sample sector was measured with an individually adjusted signal amplification factor resulting in 2000−3400 relative fluorescence units (RFU). The addition of carrier protein BSA, as recom- mended for fluorescence detection, was not appropriate for our system because Aβ directly interacts with BSA74. Low percentages of Tween 20 did not alter the signals and were therefore not applied either. All SV data was initially analysed using a continuous distribution c(s) with maximum entropy. www.nature.com/scientificreports/ species with an RH between 2.3 and 2.8 nm is clearly smaller. Nevertheless, our characterized oligomeric species resembles more likely a structure formerly defined as paranucleus as a building block for larger oligomeric spe- cies, like the 12- and 18 mers15, 16. In the current study, two independent, solution based methods, which do not necessitate the addition of surfactants, crosslinking or surface interactions, have been combined for the first time to investigate the smallest detectable oligomers occurring during Aβ42 aggregation. It would be desirable to verify, whether these penta-/ hexameric species exhibit the primary toxicity agent that leads to synapse failure and finally to memory loss. To address this question larger quantities of this species have to be obtained, which is our goal for future studies. Discussion We interpreted this gap as indicative for the size, i.e., s-value, range of a so called nucleus of aggregation according to the assumption of a nucleated aggregation mechanism. A nucleus would be the small- est assembly, which favours the addition of further monomers or oligomers to facilitate fibril formation versus disassembly. Therefore a nucleus is a kinetically unstable intermediate, which makes it elusive to experimental detection. The hydrodynamic radius of the nucleus has been estimated to be between 5 and 50 nm according to fluorescence correlation spectroscopy measurements performed by Garai and colleagues69. This boundary is similar to our estimated size region between 1 and 4 S, although the size of our determined penta-to hexameric Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 8 www.nature.com/scientificreports/ Methods i Thereafter the c(s)-distribution analysis with the size-distribution option “with prior probabilities” was applied as implemented in the software package sedfit (vs. 14.7 g; May 2015) (http://www.analyticalultracentrifugation.com/)75. For data analysis a res- olution of 0.05 S with a confidence level (F-ratio) of 0.95 was chosen for the appropriate s-value range. A more detailed description of the fitting process has been added to the supplement. All presented c(s) distributions have root mean square deviations of <1% of the total signal. Sedimentation coefficients are reported as s20,w values in Svedberg units with 1 S = 10−13 s, which represent the apparent sedimentation coefficient normalized to the standard conditions of 20 °C and pure water solvent. Small Angle Neutron Scattering. Proteins were dissolved in HFIP for three weeks and dried before being resolved in deuterated buffer (50 mM Napi, pH 7.4 + 150 mM NaCl in 100% D2O, filtered with Anopore Whatman, pore size 20 nm). Samples were incubated at 7 °C at rest. SANS experiments were performed at the small angle diffrac- tometer, KWS-2 of Heinz Maier-Leibnitz Zentrum (MLZ Garching, Germany) with wavelengths between 0.47 nm and 0.81 nm and detector distances from 1.1 m to 19.7 m to cover a wave vector range of 0.001 Å−1 to 0.5 Å−1 76. The wavelength spread was λ λ ∆ = . / 0 2 and measurement times from 1 to 4 h dependent on detector distance and sample scattering were used. Samples and buffer measurements for background correction were performed Scientific Reports \| 7: 2493 \| DOI:10.1038/s41598-017-02370-3 9 www.nature.com/scientificreports/ in quartz cells of 2 mm thickness together with appropriate measurements for detector sensitivity and dark cur- rent. Appropriate standard methods for evaluation and background correction were used from the software QtiKWS77. Computational details. Molecular Dynamics calculations. Five independent all-atom MD simulations of twenty Aβ42 monomers inserted in a cubic box with a side length of 350 Å and periodic boundary conditions were performed. We applied different initial velocity distributions for each simulation and simulated in total 2.5 μs, with 500 ns per simulation. We used the parallel processing MD software Gromacs 4.5.5 for performing the simulations with a leap-frog stochastic dynamics integrator, the OPLS/AA force field78–80, and implicit sol- vent using a Generalized Born model with a hydrophobic solvent accessible surface area term (GBSA)81. References Abeta toxicity in Alzheimer’s disease: globular oligomers (ADDLs) as new vaccine and drug targets. Neurochem Int 41, 345–352, doi:10.1016/S0197-0186(02)00050-5 (2002). ( ) ( ) 0. Shankar, G. M. et al. Amyloid-[beta] protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory Nat Med 14, 837–842, doi:10.1038/nm1782 (2008). ( ) 1. Mc Donald, J. M. et al. 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https://openalex.org/W3011358624	https://europepmc.org/articles/pmc7055074?pdf=render	English	null	Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose	Microbial cell factories	2,020	cc-by	15,110	© The Author(s) 2020. 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://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: 5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid- based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. Results: For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF produc- tion rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. Conclusion: We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization. Keywords: Gluconobacter oxydans, Sweetener, Chromosomal integration, 5-ketofructose, Fructose dehydrogenase the periplasm to organic acids, aldehydes, and ketones [1]. The resulting electrons are transferred via ubiqui- none to the terminal cytochrome bo3 oxidase [2] or a cyanide-insensitive bd-type oxidase [3], both of which reduce oxygen to water. Due to the distinctive properties of the membrane-bound dehydrogenases, G. oxydans has become an important workhorse for oxidative biotrans- formations in biotechnology [4–8]. The first industrial application of G. oxydans was in vitamin C production via the Reichstein-Grüssner process [9], where the sorbi- tol dehydrogenase catalyzes the oxidation of D-sorbi- tol to L-sorbose [10]. Other products whose synthesis Novel plasmid‑free Gluconobacter oxydans strains for production of the natural sweetener 5‑ketofructose ohlers2† , Chika Igwe1 , Angela Kranz2 , Matthias Pesch1 , Astrid Wirtz2 , Büchs1* and Michael Bott2* Svenja Battling1† , Karen Wohlers2† , Chika Igwe1 , Angela Kranz2 , Matthias Pesch1 , Astrid Wirtz2 , Meike Baumgart2 , Jochen Büchs1* and Michael Bott2* Svenja Battling1† , Karen Wohlers2† , Chika Igwe1 , Angela Kranz2 , Matthias Pesch1 , Astrid Wirtz2 , Meike Baumgart2 , Jochen Büchs1* and Michael Bott2* Correspondence: jochen.buechs@avt.rwth‑aachen.de; m.bott@fz‑juelich.de †Svenja Battling and Karen Wohlers contributed equally to this manuscript Battling et al. Microb Cell Fact (2020) 19:54 https://doi.org/10.1186/s12934-020-01310-7 Battling et al. Microb Cell Fact (2020) 19:54 https://doi.org/10.1186/s12934-020-01310-7 Microbial Cell Factories Open Access Backgroundh The strictly aerobic acetic acid bacterium Gluconobacter oxydans contains at least eight membrane-bound dehy- drogenases catalyzing the rapid chemo-, regio-, and ste- reoselective oxidation of sugars, alcohols, and polyols in 1 AVT‑Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, 52074 Aachen, Germany 2 IBG‑1: Biotechnology, Institute of Bio‑ and Geosciences, Forschungszentrum Jülich, 52425 Jülich, Germany Battling et al. Microb Cell Fact (2020) 19:54 Page 2 of 15 involves G. oxydans include dihydroxyacetone [11] or the anti-diabetic drug miglitol [12, 13]. cytochrome c subunit with three CXXCH motifs for covalent heme attachment and a C-terminal trans- membrane helix anchoring the entire complex to the membrane (FdhC), and a large subunit with a cova- lently bound flavin adenine dinucleotide (FAD) cofac- tor (FdhL). It lacks a signal peptide and is presumably exported via the Tat system in complex with FdhS. The corresponding genes are organized in the polycistronic fdhSCL transcription unit [29]. Plasmid-based expres- sion of the G. japonicus fdhSCL genes under the control of an adhAB promoter in a ΔadhA mutant of G. oxy- dans NBRC12528 lead to 20-fold higher FDH activity compared to G. japonicus wild-type cells, confirming the successful synthesis of FDH in this heterologous host [29].h Due to the vigorous incomplete periplasmic substrate oxidation of G. oxydans, only a small fraction of the car- bon source is taken up into the cytoplasm and enters the central carbon metabolism, which is characterized by an incomplete glycolysis and an incomplete tricarboxylic acid (TCA) cycle [14]. Glucose is metabolized predomi- nantly via the oxidative pentose phosphate pathway and to some extent via the Entner–Doudoroff pathway [15]. Pyruvate is partly converted to acetate as final product by pyruvate decarboxylase and acetaldehyde dehydrogenase [16]. As a consequence of the small fraction of substrate metabolized within the cell, the biomass yield of G. oxy- dans is quite low (about 0.1 g cell dry weight/g glucose). As this causes increased costs for biomass synthesis, met- abolic engineering was used to create G. oxydans strains with improved biomass yield on glucose by reducing or avoiding its oxidation to gluconate [16, 17]. One of the resulting strains, IK003.1, which is derived from the par- ent strain 621H, lacks both the membrane-bound and the soluble glucose dehydrogenase as well as pyruvate decar- boxylase. It has a 60% increased biomass yield on glucose and accumulates pyruvate instead of acetate [17]. The application of FDH for the targeted produc- tion of 5-KF was reported only recently [30]. A strain of G. oxydans 621H expressing the fdhSCL genes of G. japonicus under the control of the strong constitu- tive promoter P264 on the broad host range plasmid pBBR1p264-fdhSCL-ST showed good growth on fruc- tose and formed 5-KF with a yield of 89 mol% 5-KF per fructose. Moreover, when combined with a second G. oxydans strain secreting the sucrase SacC of Zymo- monas mobilis, this bacterial community was able to convert either purified sucrose or sucrose present in sugar beet extract to glucose and fructose and oxidize the latter to 5-KF with molar yields of > 90% and > 80%, respectively [30]. Most recently, 5-KF production from sucrose was reported for a G. oxydans strain with a chromosomal fdhSCL integration and plasmid-based expression of an invertase, resulting in conversion of 84 ± 2 mol% of the fructose units of sucrose into 5-KF [31]. G. oxydans carrying the expression plasmid pBBR1p264-fdhSCL-ST was also used for bioprocess development. When cultivated in a 2 L bioreactor with constant fructose feeding, 5-KF up to 489 g/L, yields up to 0.98 g 5-KF/g fructose and space-time yields up to 8.2 g/L/h were reached, demonstrating the efficiency of this 5-KF production process [26]. Excess sugar consumption is associated with obesity and various diseases such as cardiovascular diseases or type II diabetes [18–20]. Thus, the food industry aims at the replacement of nutritive sugars such as sucrose or high-fructose corn syrup by non-nutritive sugar substi- tutes such as sucralose [21]. Various artificial and natural sweeteners are already available on the market, but most of them have drawbacks. Polyols such as d-sorbitol or d-xylitol show laxative effects [18], whereas compounds such as acesulfame K or steviol glycosides have a bitter off-taste [22]. 5-Keto-d-fructose (5-KF) is considered as a potential non-nutritive natural sweetener [23], which has been found e.g. in white wine [24, 25]. It shows a sweet taste quality identical to that of fructose and has a similar intrinsic sweet threshold concentration of 16.4 mmol/L [26]. Therefore, the development of processes for the production of 5-KF has recently gained attention. Considering an industrial implementation, 5-KF pro- duction with plasmid-free strains would be desirable. Therefore, the current study aimed at the development and characterization of G. oxydans strains with genomi- cally integrated fdhSCL genes. Initially, four strains of G. oxydans IK003.1, each harboring a single copy of the fdhSCL genes at different genomic loci, were constructed and analyzed regarding 5-KF production. Based on these results, a strain carrying two genomic fdhSCL copies under control of the P264 promoter was constructed, which showed even better growth on fructose than the single-copy strains and formed 5-KF with yields of up to 0.82 g/g. G. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs Selection of genomic integration sites for the fdhSCL genes and design of integration constructs In this study, we wanted to generate plasmid-free G. oxy- dans strains for the production of 5-KF by genomic inte- gration of the FDH genes fdhSCL from G. japonicus. Because the genomic vicinity can influence the expres- sion of integrated genes, we selected four different inte- gration sites in the genome of G. oxydans IK003.1. For one of the insertions, the genes GOX2096-GOX2095 (GOX_RS11750) for a sorbitol dehydrogenase large subunit were replaced by the fdhSCL genes. GOX2096- GOX2095 represent an authentic frameshift in G. oxy- dans 621H leading to an inactive enzyme [14, 32]. Therefore, the replacement does not lead to a metabolic deficiency. In the case of the other three integration sites, the fdhSCL genes were inserted into intergenic regions (IGRs) without deleting any part of the genome. The IGRs were chosen based on the following criteria: (i) the genomic positions should be close to the origin of rep- lication to profit from a positive gene dosage effect; (ii) the positions should be located between the 3′-ends of two convergent genes to avoid an interference with the regulation of neighboring genes; (iii) the expression lev- els of the adjacent genes should vary for the three sites to test the influence of the genomic vicinity on fdhSCL expression; (iv) the chromosomal gene upstream of the fdhSCL genes should possess a terminator to enable com- parison of the three loci without readthrough from an upstream promoter. Applying those criteria and using RNAseq data [33] for the evaluation of the expression levels of the neighbouring genes, the following positions The fdhSCL integration fragments were composed of 500 bp upstream DNA of the respective insertion site followed by the strong constitutive P264 promotor, the consensus ribosome binding site (RBS) AGGAG [33], the fdhSCL genes with an ATG start codon instead of TTG for fdhS, which was shown to be beneficial for 5-KF production [29], the bidirectional 100 bp terminator region downstream of GOX0028, which led to efficient termination according to RNA seq data [33], and 500 bp downstream DNA of the respective insertion site. The integration fragments were inserted into the suicide vector pAJ63a [35] and transferred into G. oxydans IK003.1 by conjugation, followed by a two-step homologous recom- bination protocol as outlined in the Methods section. oxydans is able to oxidize fructose to 5-KF when cultivated with fructose [25] or with mannitol, which is initially oxidized to fructose [27]. However, the enzyme responsible for 5-KF formation in G. oxydans is not known yet. In contrast, fructose dehydrogenase (FDH), a membrane-bound enzyme catalyzing the periplas- mic oxidation of fructose to 5-KF with ubiquinone as electron acceptor, has been isolated and characterized from Gluconobacter japonicus NBRC3260 (formerly Gluconobacter industrius IFO3260) [28, 29]. It is a het- erotrimeric enzyme composed of a small subunit with a putative Tat signal peptide (FdhS), a Sec-secreted Battling et al. Microb Cell Fact (2020) 19:54 Page 3 of 15 Results and discussion were selected: igr1 between GOX0013 and GOX0014, igr2 between GOX0028 and GOX0029, and igr3 between GOX0038 and GOX0039. The exact integration sites were positioned directly downstream of the terminator of the upstream gene, as predicted with the online tool ARNold [34]. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs The resulting integration strains were checked via colony-PCR and named IK003.1-igr1::fdhSCL, IK003.1-igr2::fdhSCL, IK003.1-igr3::fdhSCL, and IK003.1 Δsdh::fdhSCL (Fig. 1). Shake flask cultivations with different G. oxydans IK003.1::fdhSCL strains with 18 g/L and 80 g/L fructose Shake flask cultivations with offline sampling, using the four integration strains as well as IK003.1 as reference, were performed to assess whether the integration strains produce more 5-KF than the parental strain. Cultiva- tions were performed with 18 g/L fructose (= 100 mM). Growth, fructose consumption, and 5-KF formation are Fig. 1 Scheme of the loci used for targeted integration of the fdhSCL genes into the genome of G. oxydans IK003.1. Shown are the integration sites with the flanking genes, the P264 promoter (black arrow), a consensus RBS (black rectangle), the fdhSCL genes (gray arrows), and terminators (black hairpin = native, gray hairpin = inserted). The transcription strength of the flanking genes (strong, medium, weak) and the normalized read count (given by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values in brackets) are also indicated. FPKM values were determined via RNAseq with G. oxydans 621H grown on mannitol (exponential growth phase) [33]. To differentiate the transcription levels, the FPKM values (range of 0–446,627) were grouped in the lowest 25% (0–38, weak), the median 50% (38–318, medium) and the highest 25% (318–446,627, strong) Fig. 1 Scheme of the loci used for targeted integration of the fdhSCL genes into the genome of G. oxydans IK003.1. Shown are the integration sites with the flanking genes, the P264 promoter (black arrow), a consensus RBS (black rectangle), the fdhSCL genes (gray arrows), and terminators (black hairpin = native, gray hairpin = inserted). The transcription strength of the flanking genes (strong, medium, weak) and the normalized read count (given by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values in brackets) are also indicated. FPKM values were determined via RNAseq with G. oxydans 621H grown on mannitol (exponential growth phase) [33]. To differentiate the transcription levels, the FPKM values (range of 0–446,627) were grouped in the lowest 25% (0–38, weak), the median 50% (38–318, medium) and the highest 25% (318–446,627, strong) Fig. 1 Scheme of the loci used for targeted integration of the fdhSCL genes into the genome of G. oxydans IK003.1. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs After 29 h of cultivation, when no further growth was observed, IK003.1-igr3::fdhSCL, IK003.1- igr2::fdhSCL, and IK003.1 Δsdh::fdhSCL had completely consumed the fructose, resulting in higher 5-KF con- centrations than observed for IK003.1-igr1::fdhSCL with 2.5 g/L residual fructose. The integration strains showed 15–22% higher growth rates (0.31 h−1 –0.33 h−1) than the reference strain IK003.1 (0.27 h−1) and reached higher final cell densities. Due to the lack of the fdh- SCL genes, IK003.1 cannot generate energy by periplas- mic fructose oxidation, which explains slower growth and lower biomass formation. Nevertheless, the refer- ence strain produced small amounts of 5-KF, reaching a yield of 0.04 g/g. Low 5-KF production has also previ- ously been described for G. oxydans grown on mannitol [27]. Here fructose oxidation to 5-KF was presumably catalyzed by the membrane-bound polyol dehydroge- nase mSldAB, since a side activity for fructose oxidation has been described for a Gluconobacter thailandicus mSldAB homolog [36]. All integration strains formed 5-KF, but the kinetics differed (Fig. 2b). Strain IK003.1- igr3::fdhSCL showed the fastest 5-KF production, strains IK003.1-igr2::fdhSCL and IK003.1 Δsdh::fdhSCL were somewhat slower, and IK003.1-igr1::fdhSCL was much slower than the other strains. The 5-KF yields after 29 h were comparable for the three fastest i To further investigate the four different integration sites, cultivations with online monitoring of respiratory activity in shake flasks were performed using the Respira- tion Activity MOnitoring System (RAMOS). Measuring the oxygen transfer rate (OTR) and the carbon dioxide transfer rate (CTR) provides extremely useful informa- tion about the metabolic activities and the physiological state of the microorganisms [37, 38]. 5-KF production from fructose is an oxidation reaction catalyzed by FDH and the electrons are transferred in the respiratory chain to oxygen as the final electron acceptor [26, 39]. Hence, 5-KF formation contributes to the OTR kinetics in addition to other catabolic activities of the G. oxydans strains. Cultivations with online monitoring of the respir- atory activity were performed to obtain additional infor- mation on the growth behavior of the four integration strains using IK003.1 and the plasmid-containing strain IK003.1 pBBR1p264-fdhSCL-ST as reference (Additional file 1: Fig. S1). Increasing the fructose concentration from 18 g/L to 80 g/L led to a decrease of the 5-KF yield for all four integration strains, reaching values between 0.36 g/g and 0.58 g/g (Additional file 1: Table S1). Selection of genomic integration sites for the fdhSCL genes and design of integration constructs Shown are the integration sites with the flanking genes, the P264 promoter (black arrow), a consensus RBS (black rectangle), the fdhSCL genes (gray arrows), and terminators (black hairpin = native, gray hairpin = inserted). The transcription strength of the flanking genes (strong, medium, weak) and the normalized read count (given by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values in brackets) are also indicated. FPKM values were determined via RNAseq with G. oxydans 621H grown on mannitol (exponential growth phase) [33]. To differentiate the transcription levels, the FPKM values (range of 0–446,627) were grouped in the lowest 25% (0–38, weak), the median 50% (38–318, medium) and the highest 25% (318–446,627, strong) Battling et al. Microb Cell Fact (2020) 19:54 Page 4 of 15 shown in Fig. 2. After 29 h of cultivation, when no further growth was observed, IK003.1-igr3::fdhSCL, IK003.1- igr2::fdhSCL, and IK003.1 Δsdh::fdhSCL had completely consumed the fructose, resulting in higher 5-KF con- centrations than observed for IK003.1-igr1::fdhSCL with 2.5 g/L residual fructose. The integration strains showed 15–22% higher growth rates (0.31 h−1 –0.33 h−1) than the reference strain IK003.1 (0.27 h−1) and reached higher final cell densities. Due to the lack of the fdh- SCL genes, IK003.1 cannot generate energy by periplas- mic fructose oxidation, which explains slower growth and lower biomass formation. Nevertheless, the refer- ence strain produced small amounts of 5-KF, reaching a yield of 0.04 g/g. Low 5-KF production has also previ- ously been described for G. oxydans grown on mannitol [27]. Here fructose oxidation to 5-KF was presumably catalyzed by the membrane-bound polyol dehydroge- nase mSldAB, since a side activity for fructose oxidation has been described for a Gluconobacter thailandicus mSldAB homolog [36]. All integration strains formed 5-KF, but the kinetics differed (Fig. 2b). Strain IK003.1- igr3::fdhSCL showed the fastest 5-KF production, strains IK003.1-igr2::fdhSCL and IK003.1 Δsdh::fdhSCL were somewhat slower, and IK003.1-igr1::fdhSCL was much slower than the other strains. The 5-KF yields after 29 h were comparable for the three fastest strains (0.76 g/g for IK003.1-igr3::fdhSCL and IK003.1- igr2::fdhSCL, 0.74 g/g for IK003.1 Δsdh::fdhSCL), but lower for IK003.1-igr1::fdhSCL (0.61 g/g). This experiment already showed an influence of the inte- gration site on 5-KF production with the ranking IK003.1-igr3::fdhSCL > IK003.1-igr2::fdhSCL > IK003.1 Δsdh::fdhSCL > IK003.1-igr1::fdhSCL. This ranking was later confirmed in additional experiments. shown in Fig. 2. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs Residual fruc- tose concentrations of 25 g/L to 45 g/L were detected for the integration strains (Additional file 1: Table S1). IK003.1 pBBR1p264-fdhSCL-ST reached a much higher yield of 0.88 g/g.h Fig. 2 Growth, 5-KF formation, and fructose consumption of the indicated G. oxydans strains in shake flasks. Depicted is a the cell density as OD600, b the 5-KF concentration, and c the fructose concentration determined by HPLC (method A). The strains were cultivated in 100 mL complex medium with 18 g/L fructose in 500 mL baffled shake flasks at 30 °C, 130 rpm, a shaking diameter of 50 mm, and 85% humidity. Shown are mean values of biological duplicates The kinetics of OTR, CTR, and the respiratory quotient (RQ = CTR/OTR) observed for the six strains (Addi- tional file 1: Fig. S1a–c) are likely caused by differences in the fructose oxidation rate, as a consequence of varying fdhSCL expression levels, which will be discussed below. The plasmid-containing strain IK003.1 pBBR1p264- fdhSCL-ST presumably has the highest FDH activity and showed the highest OTR. The maximal total oxygen consumption (TOC) of 220 mmol/L was reached already after 19 h (Additional file 1: Fig. S1d). Due to the rapid oxidation of fructose to 5-KF, less fructose is available for uptake and intracellular oxidation, resulting in the lowest total carbon dioxide evolution (TCE) of 50 mmol/L for IK003.1 pBBR1p264-fdhSCL-ST (Additional file 1: Fig. S1e). The reference strain IK003.1 had only a very low activity for fructose conversion to 5-KF and a very low maximal TOC of 80 mmol/L. The CTR was also quite low for IK003.1, because the strain may be energy-lim- ited in the absence of FDH-based respiration, resulting Fig. 2 Growth, 5-KF formation, and fructose consumption of the indicated G. oxydans strains in shake flasks. Depicted is a the cell density as OD600, b the 5-KF concentration, and c the fructose concentration determined by HPLC (method A). The strains were cultivated in 100 mL complex medium with 18 g/L fructose in 500 mL baffled shake flasks at 30 °C, 130 rpm, a shaking diameter of 50 mm, and 85% humidity. Shown are mean values of biological duplicates Battling et al. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs Microb Cell Fact (2020) 19:54 Page 5 of 15 0 10 20 30 40 50 60 0 6 12 18 24 30 0 100 200 300 400 500 Oxygen transfer rate (OTR) (mmol/L/h) 0 mM MES 50 mM MES 100 mM MES 150 mM MES Total oxygen consumption (TOC) (mmol/L) Time (h) b a c 0 mM MES 50 mM MES 100 mM MES 150 mM MES 0 20 40 60 80 Fructose, 5-Ketofructose (g/L) Yield (g/g) * * * * * * 0 1 2 3 4 5 pH 0.0 0.2 0.4 0.6 0.8 1.0 Fig. 3 Cultivation of G. oxydans IK003.1-igr1::fdhSCL in a RAMOS device with 80 g/L fructose and different MES buffer concentrations. Depicted is a the residual fructose concentration (red), the 5-KF concentration (green), the yield g/g (grey), and the pH (blue), b the oxygen transfer rate (OTR) and c the total oxygen consumption (TOC). Cultivations were performed at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm in complex medium with 80 g/L fructose and MES buffer at concentrations of 0 mM, 50 mM, 100 mM and 150 mM. Shown are mean values of duplicates and the concentrations determined for fructose and 5-KF in the two experiments as well as the resulting yields are shown as stars 0 10 20 30 40 50 60 0 6 12 18 24 30 0 100 200 300 400 500 Oxygen transfer rate (OTR) (mmol/L/h) 0 mM MES 50 mM MES 100 mM MES 150 mM MES Total oxygen consumption (TOC) (mmol/L) Time (h) b a c 0 mM MES 50 mM MES 100 mM MES 150 mM MES 0 20 40 60 80 Fructose, 5-Ketofructose (g/L) Yield (g/g) *** * * * * 0 1 2 3 4 5 pH 0.0 0.2 0.4 0.6 0.8 1.0 in a maximal TCE of only 75 mmol/L. The 5-KF produc- tion of the four integration strains was slower than in the plasmid-based strain, resulting in a lower OTR. A maxi- mal TOC of 250 mmol/L was reached for the integration strains at the end of the cultivation, except for IK003.1- igr1::fdhSCL. At the same time, more fructose was taken up and catabolized within the cell, resulting in higher CTR. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs Shown are mean values of duplicates and the concentrations determined for fructose and 5-KF in the two experiments as well as the resulting yields are shown as stars gff Depicted is a the residual fructose concentration (red), the 5-KF concentration (green), the yield g/g (grey), and the pH (blue), b the oxygen transfer rate (OTR) and c the total oxygen consumption (TOC). Cultivations were performed at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm in complex medium with 80 g/L fructose and MES buffer at concentrations of 0 mM, 50 mM, 100 mM and 150 mM. Shown are mean values of duplicates and the concentrations determined for fructose and 5-KF in the two experiments as well as the resulting yields are shown as stars for unbuffered cultivation to 4–4.7 for the cultivations with MES buffer (Fig. 3a). Fructose was completely con- sumed in the buffered cultures, resulting in a significantly increased 5-KF formation of up to 71 g/L and an increase of the yield from 0.56 g/g for the unbuffered culture to 0.86 g/g for the culture with 50 mM MES (Fig. 3a). At 100 mM and 150 mM MES, the 5-KF concentration and the yield were lower than at 50 mM MES, but the final OD600 was higher (4.1 for 0 mM MES, 4.9 for 50 mM MES, 5.4 for 100 mM MES, 5.9 for 150 mM MES). Higher pH values thus favor biomass formation and decrease 5-KF production. Selection of genomic integration sites for the fdhSCL genes and design of integration constructs The maximal TCE is two to four times higher for all integration strains in comparison with the plasmid- containing strain (Additional file 1: Fig. S1e). The kinetics of the RQ reflects these differences (Additional file 1: Fig. S1c). ) Another parameter that probably influences the kinet- ics of OTR and CTR is the pH value. Strain IK003.1 forms pyruvate (pKa 2.49) instead of acetate (pKa 4.78), leading to a stronger acidification of the medium. In the experiment shown in Additional file 1: Fig. S1, pyru- vate formation is dependent on the rate of intracellular fructose catabolism and thus should be reflected by the CTR value. Consequently, the integration strains should show a stronger acidification than the plasmid-contain- ing strain and IK003.1, which was confirmed by the pH values measured after 29 h of cultivation (Additional file 1: Table S1). The pH influences the FDH activity and thus fructose consumption. The pH optimum for FDH is around 4 and activity decreases slightly at higher pH val- ues, but strongly at lower pH, with no activity observed at pH 3 [29]. It is therefore likely that the OTR and CTR kinetics observed for the integration strains with a slow decrease after the maximum (Additional file 1: Fig. S1) and the incomplete fructose consumption (Additional file 1: Table S1) is caused by inhibition of FDH and other enzymes, such as those of the pentose phosphate path- way [40], due to acidification. Fig. 3 Cultivation of G. oxydans IK003.1-igr1::fdhSCL in a RAMOS d i ith 80 /L f t d diff t MES b ff t ti Fig. 3 Cultivation of G. oxydans IK003.1-igr1::fdhSCL in a RAMOS device with 80 g/L fructose and different MES buffer concentrations. Depicted is a the residual fructose concentration (red), the 5-KF concentration (green), the yield g/g (grey), and the pH (blue), b the oxygen transfer rate (OTR) and c the total oxygen consumption (TOC). Cultivations were performed at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm in complex medium with 80 g/L fructose and MES buffer at concentrations of 0 mM, 50 mM, 100 mM and 150 mM. Influence of buffering on growth and 5‑KF production during cultivation of IK003.1‑igr1::fdhSCL with 80 g/L fructose in a RAMOS deviceh The decreasing pH during the cultivation had presum- ably a negative influence on growth and product forma- tion during the cultivation. Therefore, it was investigated whether pH control has a positive effect on growth and product formation. CaCO3 is sometimes used in shake flask cultivations for buffering, but it has several disad- vantages including turbidity of the medium [41]. There- fore, 2-(N-morpholino)ethansulfonic acid (MES) buffer with a pKa value of 6.1 was used [42]. Strain IK003.1- igr1::fdhSCL was chosen to compare cultivations with- out buffer and with 50 mM, 100 mM, and 150 mM MES buffer. The OTR kinetics of cultivations with the different MES concentrations showed clear differences (Fig. 3b). The maximal OTR of all MES-buffered cultures was about 50 mmol/L/h and thus 2.5-fold higher than for the unbuffered culture. At 100 mM and 150 mM MES, the OTR slowed down compared to 50 mM MES. This is most likely due to the higher osmolality of the medium, which increased from 0.56 Osmol/kg for unbuffered medium to 0.78 Osmol/kg for medium with 150 mM MES. The negative influence of osmolality on growth of G. oxydans has already been described [41, 43]. f The addition of MES buffer had a strongly positive effect on 5-KF formation (Fig. 3). As expected, the pH value at the end of the cultivation increased from 3.3 Battling et al. Microb Cell Fact (2020) 19:54 Battling et al. Microb Cell Fact (2020) 19:54 Page 6 of 15 0 20 40 60 80 0 20 40 60 0 6 12 18 24 30 0.0 0.5 1.0 IK003.1 IK003.1 pBBR1p264-fdhSCL-ST IK003.1-igr1::fdhSCL IK003.1-igr2::fdhSCL IK003.1-igr3::fdhSCL sdh::fdhSCL Oxygen transfer rate (OTR) (mmol/L/h) b Carbon dioxide transfer rate (CTR) (mmol/L/h) c Respiratory quotient (RQ) Time (h) d a 0 20 40 60 80 100 Fructose, 5-Ketofructose (g/L) Yield (g/g) IK003.1 IK003.1 pBBR1p264-fdhSCL-ST IK003.1-igr1::fdhSCL IK003.1-igr2::fdhSCL IK003.1-igr3::fdhSCL IK003.1 sdh::fdhSCL 0 1 2 3 4 5 6 7 8 9 OD600 ** * * 0.0 0.2 0.4 0.6 0.8 1.0 Fig. 4 Cultivation of the indicated G. oxydans strains in a RAMOS device with 80 g/L fructose and 150 mM MES. Comparison of different G. oxydans strains during cultivation in a RAMOS device with 80 g/L fructose and 150 mM MES bufferff Based on the positive effect of MES buffering on 5-KF production by IK003.1-igr1::fdhSCL, these condi- tions were used to compare the four different integra- tion strains among each other, with the parent strain IK003.1, and with the plasmid-containing strain IK003.1 pBBR1p264-fdhSCL-ST (Fig. 4). Fig. 4 Cultivation of the indicated G. oxydans strains in a RAMOS Fig. 4 Cultivation of the indicated G. oxydans strains in a RAMOS device with 80 g/L fructose and 150 mM MES. Depicted is a the residual fructose concentration and the 5-KF concentration (determined by HPLC method B), the yield g/g, and the optical density (OD600) after 29 h, b the oxygen transfer rate (OTR), c the carbon dioxide transfer rate (CTR), and d the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h). The strains were cultivated in complex medium with 80 g/L fructose and 150 mM MES at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates and the values determined for optical density, the concentrations of fructose and 5-KF, and the resulting yields in the two experiments are shown as stars Fig. 4 Cultivation of the indicated G. oxydans strains in a RAMOS device with 80 g/L fructose and 150 mM MES. Depicted is a the residual fructose concentration and the 5-KF concentration (determined by HPLC method B), the yield g/g, and the optical density (OD600) after 29 h, b the oxygen transfer rate (OTR), c the carbon dioxide transfer rate (CTR), and d the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h). The strains were cultivated in complex medium with 80 g/L fructose and 150 mM MES at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates and the values determined for optical density, the concentrations of fructose and 5-KF, and the resulting yields in the two experiments are shown as stars As expected from the previous experiments, all strains except for IK003.1 consumed fructose completely and the final pH values varied between 4.3 and 4.6. 5-KF forma- tion by the four integration strains was very similar (57– 59 g/L), corresponding to yields of 0.71–0.75 g/g (Fig. 4a). Influence of buffering on growth and 5‑KF production during cultivation of IK003.1‑igr1::fdhSCL with 80 g/L fructose in a RAMOS deviceh 0 20 40 60 80 0 20 40 60 0 6 12 18 24 30 0.0 0.5 1.0 IK003.1 IK003.1 pBBR1p264-fdhSCL-ST IK003.1-igr1::fdhSCL IK003.1-igr2::fdhSCL IK003.1-igr3::fdhSCL sdh::fdhSCL Oxygen transfer rate (OTR) (mmol/L/h) b Carbon dioxide transfer rate (CTR) (mmol/L/h) c Respiratory quotient (RQ) Time (h) d b b Influence of buffering on growth and 5‑KF production during cultivation of IK003.1‑igr1::fdhSCL with 80 g/L fructose in a RAMOS deviceh Depicted is a the residual fructose concentration and the 5-KF concentration (determined by HPLC method B), the yield g/g, and the optical density (OD600) after 29 h, b the oxygen transfer rate (OTR), c the carbon dioxide transfer rate (CTR), and d the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h). The strains were cultivated in complex medium with 80 g/L fructose and 150 mM MES at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates and the values determined for optical density, the concentrations of fructose and 5-KF, and the resulting yields in the two experiments are shown as stars 0 20 40 60 80 0 20 40 60 0 6 12 18 24 30 0.0 0.5 1.0 IK003.1 IK003.1 pBBR1p264-fdhSCL-ST IK003.1-igr1::fdhSCL IK003.1-igr2::fdhSCL IK003.1-igr3::fdhSCL sdh::fdhSCL Oxygen transfer rate (OTR) (mmol/L/h) b Carbon dioxide transfer rate (CTR) (mmol/L/h) c Respiratory quotient (RQ) Time (h) d a 0 20 40 60 80 100 Fructose, 5-Ketofructose (g/L) Yield (g/g) IK003.1 IK003.1 pBBR1p264-fdhSCL-ST IK003.1-igr1::fdhSCL IK003.1-igr2::fdhSCL IK003.1-igr3::fdhSCL IK003.1 sdh::fdhSCL 0 1 2 3 4 5 6 7 8 9 OD600 * * 0.0 0.2 0.4 0.6 0.8 1.0 ** The kinetics of TOC is shown in Fig. 3c. As expected from the OTR kinetics, the TOC values of the MES- containing cultures were much higher than that of the unbuffered culture. Increasing MES concentrations correlated with increased TOC values, which can be explained by the fact that higher MES concentration led to higher biomass formation and thus a higher fraction of fructose was metabolized intracellularly, leading to more reducing equivalents per fructose than its simple oxida- tion to 5-KF. Despite the increased lag phase, the positive effect of MES buffer regarding pH, TOC, OD600, and yield pre- dominates. Therefore, subsequent shake flasks experi- ments were conducted with 150 mM MES buffer, which led to the highest TOC and the highest pH at the end of the cultivation. This was important for further experi- ments with fructose concentrations up to 210 g/L per- formed in shake flasks (Additional file 1: Fig. S5, Tab. S2). Comparison of different G. oxydans strains during cultivation in a RAMOS device with 80 g/L fructose and 150 mM MES bufferff Compared to the unbuffered cultures (Additional file 1: Table S1) this was an increase of 1.2- to 2-fold. The plas- mid-containing strain formed 65 g/L 5-KF corresponding to a yield of 0.84 g/g.hi The final OD600 of the integration strains ranged between 6.9 and 8.3 (Fig. 4a). In view of the comparable 5-KF formation of the four strains, these differences were unexpected and apparently are a consequence of the dif- ferent genomic integration sites. The plasmid-containing strain had a much lower final OD600 of only 3.3 compared to the integration strains. As discussed above, this is caused by the higher copy number resulting in more fdh- SCL transcript, resulting in faster fructose oxidation and a lower amount of fructose entering the cell. Additionally, a longer period of fructose oxidation by FDH also pro- vides a longer period of energy supply for biomass forma- tion from components of the yeast extract. Once fructose has been exhausted, growth is no longer sustained.h reached its OTR maximum of 36 mmol/L/h already after 13 h, whereas the integration strains reached their maxi- mum of ~ 55 mmol/L/h after 17 h –19 h (Fig. 4b). CTR was very low for the plasmid-based strain and much higher for the integration strains. This is in line with a higher intracellular fructose catabolism and a presum- ably higher consumption of yeast extract components (Fig. 4c). For strain IK003.1, both OTR and CTR were retarded compared to the integration strains. These kinetics are due to slow intracellular oxidation of fruc- tose. In line with these data, the RQ value for IK003.1 was the highest of all strains (about 0.9), whereas the RQ of the plasmid-based strain was very low with a value of The statements made above are supported by the OTR and CTR kinetics (Fig. 4b, c). The plasmid-based strain Battling et al. Microb Cell Fact (2020) 19:54 Page 7 of 15 Page 7 of 15 of 0.86 g/g was reached for the cultivation with 160 g/L fructose (Additional file 1: Fig. S5 and Table S2). about 0.2. The integration strains showed RQ values in the range of 0.6–0.8 (Fig. 4d). i In a previous study, a 5-KF production process in a 2 L fermenter with the plasmid-based strain G. oxydans 621H pBBR1p264-fdhSCL-ST was described. This strain carried the same plasmid as IK003.1 pBBR1p264-fdh- SCL-ST. G. Scale up and fermentation in 2 L fermenter with IK003.1‑igr3::fdhSCL An important step for the industrial implementation of 5-KF production with a plasmid-free strain is the design and establishment of a suitable fermentation process. As a first step, a scale up from shake flasks to a 2 L fermenter was performed with strain IK003.1-igr3::fdhSCL, which was the best performing integration strain so far. To allow a direct comparison between shake flask (RAMOS system) and fermenter, the same medium, initial pH, and temperature were used for both cultivation devices. The shake flask was filled with inoculated medium by sterile transfer of a sample of the fermenter culture to ensure optimal comparability. In shake flask cultivations with 80 g/L fructose, no oxygen limitation was detected in the RAMOS devices. Hence, for a successful scale-up, oxy- gen limitation in the fermenter had to be avoided. For that reason, the dissolved oxygen tension (DOT) was kept above 30% by adjusting the agitation speed, whereas the aeration rate in the fermenter was kept constant at 1 vessel volume per minute (vvm). To verify the success- ful scale-up, online data (OTR, CTR, RQ) and offline data (OD600, pH, fructose and 5-KF concentration) were measured and compared. The results are shown in Addi- tional file 1: Fig. S2. No significant differences in both, online and offline data, were found. Additional fermen- tations were performed as first optimization steps and confirmed the consistency of online and offline data (Additional file 1: Fig. S3 and Fig. S4). Hence, the scale up to a 2 L fermenter was successful and the process could be further optimized. 0 10 20 30 40 50 60 0 3 6 9 12 0 20 40 60 80 100 0 6 12 18 24 30 0 40 80 120 160 Oxygen transfer rate (OTR), Carbon dioxide transfer rate (CTR) (mmol/L/h) OTR CTR OD OD600 b DOT Dissolved oxygen tension (DOT) (%) Fructose 5-Ketofructose 5-Ketofructose, Fructose (g/L) Time (h) c d 300 600 900 1200 Agitation Agitation speed (rpm) AF DOT control 5.0 5.2 5.4 5.6 5.8 pH pH 0.0 0.5 1.0 1.5 2.0 RQ Respiratory quotient (RQ) a Fig. 5 Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with pH control. Comparison of different G. oxydans strains during cultivation in a RAMOS device with 80 g/L fructose and 150 mM MES bufferff oxydans 621H is not optimized for biomass formation from glucose and produces acetate rather than pyruvate. A batch fermentation with 150 g/L fructose was performed, reaching a 5-KF yield of 0.87 g/g [26]. To compare our genomic integration strain IK003.1- igr3::fdhSCL with this strain, a 2 L batch fermentation was performed under similar conditions (Fig. 5). An ini- tial fructose concentration of 150 g/L was used to allow a direct comparison. Due to pH control by KOH addition, MES buffer was not included in the medium. The pH was set to 5, promoting both growth of G. oxydans and FDH activity. The DOT was maintained above 30% and anti- foam was added twice during the cultivation. The OTR reached a maximum of 53 mmol/L/h after approx. 20 h and decreased sharply after 27 h, indicating the complete consumption of fructose (Fig. 5a). The OD600 increased This experiment revealed that when using buffered con- ditions, the fdhSCL integration strains approached the 5-KF titer and the yield of the strain with plasmid-based fdhSCL expression. With respect to the four genomic integration sites, the differences in 5-KF titer and yield were rather small, but IK003.1-igr3::fdhSCL showed the fastest growth followed by IK003.1 Δsdh::fdhSCL and IK003.1-igr2::fdhSCL. Scale up and fermentation in 2 L fermenter with IK003.1‑igr3::fdhSCL Depicted is a the oxygen and carbon dioxide transfer rates (OTR and CTR), the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h), b optical density (OD600) and pH, c dissolved oxygen tension (DOT) and agitation speed, antifoam (AF) agent addition, and period of DOT control (indicated by arrows), d fructose and 5-KF concentrations. The cultivation was performed in complex medium with 150 g/L fructose with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1200 rpm), aeration rate (Q) = L/min, T = 30 °C, pHcontrol ≥ 5 0 10 20 30 40 50 60 0 3 6 9 12 0 20 40 60 80 100 0 6 12 18 24 30 0 40 80 120 160 Oxygen transfer rate (OTR), Carbon dioxide transfer rate (CTR) (mmol/L/h) OTR CTR OD OD600 b DOT Dissolved oxygen tension (DOT) (%) Fructose 5-Ketofructose 5-Ketofructose, Fructose (g/L) Time (h) c d 300 600 900 1200 Agitation Agitation speed (rpm) AF DOT control 5.0 5.2 5.4 5.6 5.8 pH pH 0.0 0.5 1.0 1.5 2.0 RQ Respiratory quotient (RQ) a Higher product concentrations are desirable for an industrial application. To achieve higher 5-KF titers, the fructose concentration in batch fermentations was increased. To identify the optimal initial fructose con- centration for the cultivation of IK003.1-igr3::fdhSCL, an experiment in shake flasks was performed using the RAMOS device and fructose concentrations between 80 g/L and 210 g/L. IK003.1-igr3::fdhSCL completely consumed fructose up to 210 g/L and the highest yield Fig. 5 Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with pH control. Depicted is a the oxygen and carbon dioxide transfer rates (OTR and CTR), the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h), b optical density (OD600) and pH, c dissolved oxygen tension (DOT) and agitation speed, antifoam (AF) agent addition, and period of DOT control (indicated by arrows), d fructose and 5-KF concentrations. The cultivation was performed in complex medium with 150 g/L fructose with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1200 rpm), aeration rate (Q) = L/min, T = 30 °C, pHcontrol ≥ 5 Fig. 5 Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with pH control. Shake flask cultivation of double integration strain IK003.1::fdhSCL2h The results described above were obtained with integra- tion strains containing a single genomic fdhSCL copy and a resulting lower expression rate compared to plasmid- based fdhSCL expression (see below). To further improve the potential for plasmid-free 5-KF production, the dou- ble integration strain IK003.1::fdhSCL2 was constructed using the two most potent integration sites igr3 and igr2 for fdhSCL integration. The performance of this strain was compared to the parent single integration strains IK003.1-igr2::fdhSCL and IK003.1-igr3::fdhSCL and to the plasmid-based strain IK003.1 pBBR1p264-fdhSCL-ST in a shake flask experiment. Growth curves, fructose con- sumption, and 5-KF formation are shown in Fig. 6. ig. 6 Growth, 5-KF formation, and fructose consumption of th Fig. 6 Growth, 5-KF formation, and fructose consumption of the indicated G. oxydans strains. Depicted are a the growth as OD600, b the 5-KF and c the fructose concentration (determined by HPLC method A). The strains were cultivated in 100 mL complex medium with 18 g/L fructose in 500 mL baffled shake flasks at 30 °C, 130 rpm, a shaking diameter of 50 mm and 85% humidity. Shown are mean values of biological duplicates g The growth rate of strain IK003.1::fdhSCL2 (0.32 h−1) was close to that of the plasmid-based strain (0.33 h−1) and slightly higher than that of the single integration strains (0.28–0.29 h−1). The final OD600 after 30 h of the double integration strain (3.5) was lower than that of IK003.1-igr2::fdhSCL (4.1) and IK003.1-igr3::fdhSCL (4.1) and higher than that of the plasmid-based strain (2.3). Most importantly, the double integration strain IK003.1::fdhSCL2 was clearly faster than the single inte- gration strains with respect to fructose consumption and 5-KF formation, but still slower than the plasmid-based strain. In a further experiment, the IK003.1::fdhSCL2 strain was compared with the parent single integra- tion strains in a RAMOS cultivation device with 80 g/L fructose and 150 mM MES buffer (initial pH 6.0). As shown in Additional file 1: Fig. S6, the OTR kinetics con- firmed faster fructose consumption by the double inte- gration strain, reaching maximal OTR values at about 12 h, whereas the single integration strains reached the maximum after about 17 h. Under these conditions, IK003.1::fdhSCL2 reached a final 5-KF yield of 0.82 g/g, close to that of IK003.1 pBBR1p264-fdhSCL (0.84 g/g) obtained in the experiment shown in Fig. 4.h consumption and 5-KF formation. Scale up and fermentation in 2 L fermenter with IK003.1‑igr3::fdhSCL Depicted is a the oxygen and carbon dioxide transfer rates (OTR and CTR), the respiratory quotient (RQ, shown for OTR values above 5 mmol/L/h), b optical density (OD600) and pH, c dissolved oxygen tension (DOT) and agitation speed, antifoam (AF) agent addition, and period of DOT control (indicated by arrows), d fructose and 5-KF concentrations. The cultivation was performed in complex medium with 150 g/L fructose with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1200 rpm), aeration rate (Q) = L/min, T = 30 °C, pHcontrol ≥ 5 Battling et al. Microb Cell Fact (2020) 19:54 Battling et al. Microb Cell Fact (2020) 19:54 Page 8 of 15 a b c Fig. 6 Growth, 5-KF formation, and fructose consumption of the indicated G. oxydans strains. Depicted are a the growth as OD600, b the 5-KF and c the fructose concentration (determined by HPLC method A). The strains were cultivated in 100 mL complex medium with 18 g/L fructose in 500 mL baffled shake flasks at 30 °C, 130 rpm, a shaking diameter of 50 mm and 85% humidity. Shown are mean values of biological duplicates over time and reached a final value of 9.2 (Fig. 5b), which is 1.7-fold higher compared with G. oxydans 621H pBBR1p264-fdhSCL-ST [26]. This difference is presum- ably caused by a somewhat higher fraction of fructose metabolized within the IK003.1 pBBR1p264-fdhSCL-ST cells and possibly also by the genetic alterations intro- duced into strain IK003.1 [17]. Most importantly, the 5-KF yield IK003.1-igr3::fdhSCL was 0.84 g/g and thus only slightly smaller than the 5-KF yield of 0.87 g/g deter- mined for the plasmid-based strain [26]. over time and reached a final value of 9.2 (Fig. 5b), which is 1.7-fold higher compared with G. oxydans 621H pBBR1p264-fdhSCL-ST [26]. This difference is presum- ably caused by a somewhat higher fraction of fructose metabolized within the IK003.1 pBBR1p264-fdhSCL-ST cells and possibly also by the genetic alterations intro- duced into strain IK003.1 [17]. Most importantly, the 5-KF yield IK003.1-igr3::fdhSCL was 0.84 g/g and thus only slightly smaller than the 5-KF yield of 0.87 g/g deter- mined for the plasmid-based strain [26]. a b c b Shake flask cultivation of double integration strain IK003.1::fdhSCL2h In the plasmid-based strain, the copy number of the fdhSCL genes is presum- ably above 20, as the copy number of the parent vector was determined to be 23 ± 6 in G. oxydans ∆hsdR [44]. In this case, the increase in fdhSCL copy number did not lead to tenfold higher rates of fructose consumption and 5-KF production, showing that there were other factors that limit fructose oxidation. Analysis of fdhSCL expression by RT‑qPCRf To validate that different fdhSCL transcription levels are responsible for the phenotypic differences of single inte- gration strains, reverse transcription quantitative PCR (RT-qPCR) was performed with the single integration strains, the double integration strain, and the plasmid- based strain using RNA from cells in the exponential growth phase. The gap gene (GOX0508), encoding glyc- eraldehyde 3–phosphate dehydrogenase, was tested as reference. It has been used as reference gene for RT- qPCR in G. oxydans before [45, 46]. However, with this reference gene we failed to detect the difference between single integration strains and the double integration strain, suggesting that gap expression is not constant under the experimental conditions employed and thus not a suitable reference gene (data not shown). We there- fore used the gene for a ribosomal protein as reference, The properties of IK003.1::fdhSCL2 showed that dou- bling of the copy number of the fdhSCL genes and thus presumably a higher rate of fdhSCL transcription is sufficient to significantly increase the rate of fructose Battling et al. Microb Cell Fact (2020) 19:54 Page 9 of 15 applicable to the differences observed here for the four single integration strains, since all four sites are close to the origin and contain the same DNA sequence. Genome architecture and DNA supercoiling are known to influ- ence bacterial gene expression with DNA gyrase playing an important role by introducing negative supercoils or relaxing positive supercoils introduced by RNA polymer- ase [47, 49, 50]. In Escherichia coli, an increase in gyrase cleavage sites was found downstream of highly tran- scribed operons [51]. Furthermore, nucleoid-associated proteins influence DNA folding and gene expression [52, 53]. Such mechanisms, which have not been studied at all in G. oxydans, are likely to contribute to the variation in fdhSCL expression at the four different integration sites. namely GOX0264 encoding the ribosomal protein L35. The results obtained for the fdhSCL/GOX0264 mRNA ratios are shown in Fig. 7, normalized to the mRNA ratio of the plasmid-based strain (5.03), which was set as 1. The fdh mRNA ratio of the four single integration strains varied between 3.5% and 6.6% of the value meas- ured for the plasmid-based strain, which fits with a copy number of about 20 for pBBR1p264 in G. oxydans [44]. Conclusions In this study, four different integration sites for heterolo- gous genes were identified in the genome of G. oxydans, all enabling successful expression. The integration into the intergenic region of convergent genes flanked by ter- minators is thus a favorable option for future engineering studies requiring genomic expression of foreign genes. The position-dependent effects on gene expression observed in our experiments suggests that a compari- son of different integration sites might be worthwhile. Chromosomal fdhSCL expression should enable stable expression and in fact we never observed loss of the abil- ity for 5-KF production during growth for up to 30 gen- erations and handling of the strains over several months. Furthermore, chromosomal expression avoids the neces- sity to use antibiotics required for plasmid-based fdh- SCL expression. Plasmid loss can be an issue in the case of plasmid-based expression, however, no evidence for this has been observed in the case pBBR1p264-fdhSCL- ST. Another advantage of genomic fdhSCL expression is that it facilitates further metabolic engineering of the strains, for example toward utilization of alternative sub- strates. As doubling the genomic copy number of the fdhSCL genes correlated with twofold increased 5-KF synthesis rates, additional fdhSCL integrations could further increase 5-KF production. Also, the use of a pro- moter stronger than P264 could further enhance fdhSCL expression. Besides genetic optimization, process optimi- zation is an obvious method to improve 5-KF production. In summary, our study provided the efficient G. oxydans strain IK003.1::fdhSCL2 for plasmid-free 5-KF produc- tion that will serve as basis for future strain and process development. Despite the usage of the same promoter and termi- nator structures, fdhSCL expression in the four differ- ent integration sites varied, indicating that the genomic vicinity of the target gene had an influence on the expres- sion. It has been reported that RNA polymerase activ- ity increases with proximity to the origin of replication [47] and that transcription speed is influenced by the codon composition [48]. However, both factors are not Fig. 7 Relative fdhSCL transcript levels for the indicated G. oxydans strains as determined by RT-qPCR. Depicted is the transcript ratio fdhSCL/GOX0264 normalized to the transcript ratio of the plasmid-based strain IK003.1 pBBR1p264-fdhSCL-ST, which was set as 1.0. Shown are the mean values of at least three biological replicates and technical duplicates, with standard deviation as error bars Fig. 7 Relative fdhSCL transcript levels for the indicated G. Conclusions oxydans strains as determined by RT-qPCR. Depicted is the transcript ratio fdhSCL/GOX0264 normalized to the transcript ratio of the plasmid-based strain IK003.1 pBBR1p264-fdhSCL-ST, which was set as 1.0. Shown are the mean values of at least three biological replicates and technical duplicates, with standard deviation as error bars Analysis of fdhSCL expression by RT‑qPCRf In the double integration strain, the fdh mRNA ratio was about twice as high (14% of the level of the plasmid- based strain), roughly corresponding to the sum of the values measured for IK003.1-igr2::fdhSCL and IK003.1- igr3::fdhSCL (12%). These results confirm the differ- ent expression levels expected from the fdhSCL copy numbers of the different strains. The fdhSCL transcrip- tion levels observed for the different integration strains correlated with the 5-KF production levels in the order IK003.1::fdhSCL2 > IK003.1-igr3::fdhSCL > IK003.1- igr2::fdhSCL > IK003.1 Δsdh::fdhSCL > IK003.1- igr1::fdhSCL. Strains, plasmids and oligonucleotides japonicus NBRC3260 with a C-terminal Strep-tag II-encoding sequence fused to the 3′-end of fdhL [30] pAJ63a KmR, FUS, derivative of pk18mobGII, integration vector for Δupp based counter-selection [35] pAJ63a-igr1::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter between GOX0013 and GOX0014 (GOX_RS01200 and GOX_RS01205) This work pAJ63a-igr2::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter between GOX0028 and GOX0029 (GOX_RS01280 and GOX_RS01285) This work pAJ63a-igr3::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter between GOX0038 and GOX0039 (GOX_RS01330 and GOX_RS01335) This work pAJ63a Δsdh::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter with simultaneous deletion of GOX2095-6 (GOX_RS11750) This work Table 1 Strains and plasmids used in this study Strain or plasmid Relevant characteristics Source or references BBR1p264-fdhSCL-ST pBBR1MCS-2 derivative containing the promoter region of GOX0264 upstream of the fdhSCL genes of G. japonicus NBRC3260 with a C-terminal Strep-tag II-encoding sequence fused to the 3′-end of fdhL AJ63a KmR, FUS, derivative of pk18mobGII, integration vector for Δupp based counter-selection AJ63a-igr2::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter between GOX0028 and GOX0029 (GOX_RS01280 and GOX_RS01285) AJ63a Δsdh::fdhSCL KmR, FUS, derivative of pAJ63a for integration of the fdhSCL genes under the control of the P264 promoter with simultaneous deletion of GOX2095-6 (GOX_RS11750) Genomics (Ebersberg, Germany) and are shown in Addi- tional file 1: Table S3. Generation of fdhSCL integration strains To generate the fdhSCL integration strains, derivatives of pAJ63a [35] containing the fdhSCL genes of G. japoni- cus with promoter and terminator and flanked by about 500 bp DNA regions up- and downstream of the selected integration site were constructed. The required DNA fragments were amplified from suitable templates with the Phusion High Fidelity PCR Master Mix (New Eng- land Biolabs, Frankfurt am Main, Germany). The fdhSCL fragment with an ATG start codon for fdhS instead of the native TTG start codon was amplified from pBBR1p264- fdhSCL-ST [30] with the oligonucleotides RBS-ATG-fdh- SCL-fwd, containing a consensus RBS, and fdhSCL-rev [33]. For all integration constructs, a 508 bp fragment covering the strong promoter of GOX0264 was ampli- fied from pBBR1p264 [44] using the oligonucleotides P264-fwd and P264-rev-RBS-overlap with an overlap to the RBS and fdhS. A 100 bp bidirectional terminator region downstream of GOX0028 containing an overlap to fdhL was amplified with the oligonucleotides Term- GOX0028-fwd-fdhL-overlap and Term-GOX0028-rev Strains, plasmids and oligonucleotides All strains and plasmids used in this study are listed in Table 1. Oligonucleotides were obtained from Eurofins Battling et al. Microb Cell Fact (2020) 19:54 Page 10 of 15 Table 1 Strains and plasmids used in this study Strain or plasmid Relevant characteristics Source or references E. coli DH5α F– endA1 Φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 relA1 hsdR17(rK–mK +) deoR supE44 thi-1 gyrA96 phoA λ–, strain used for cloning [57] S17-1 ΔrecA, endA1, hsdR17, supE44, thi-1, tra + , strain used for conjugation of G. oxydans [62] G. oxydans IK003.1 G. oxydans 621H Δupp ΔgdhS::sdhCDABE Δpdc::ndh ΔgdhM::sucCD [17] IK003.1-igr1::fdhSCL IK003.1 with the fdhSCL genes of G. japonicus under the control of the P264 promoter integrated between GOX0013 and GOX0014 (GOX_RS01200 and GOX_RS01205) This work IK003.1-igr2::fdhSCL IK003.1 with the fdhSCL genes of of G. japonicus under the control of the P264 promoter integrated between GOX0028 and GOX0029 (GOX_RS01280 and GOX_RS01285) This work IK003.1-igr3::fdhSCL IK003.1 with the fdhSCL genes of G. japonicus under the control of the P264 promoter integrated between GOX0038 and GOX0039 (GOX_RS01330 and GOX_RS01335) This work IK003.1 Δsdh::fdhSCL IK003.1 with the fdhSCL genes of G. japonicus under control of the P264 promoter replacing the genes GOX2095-6 (GOX_RS11750) encoding an inactive sorbitol dehydrogenase (authentic genomic frameshift) This work IK003.1::fdhSCL2 IK003.1-igr2::fdhSCL with an additional copy of the fdhSCL genes of G. japonicus under the control of the P264 promoter integrated between GOX0038 and GOX0039 This work Plasmids pBBR1p264-fdhSCL-ST pBBR1MCS-2 derivative containing the promoter region of GOX0264 upstream of the fdhSCL genes of G. Cultivation in a respiration activity monitoring system (RAMOS) Cultivation in a respiration activity monitoring system (RAMOS) from genomic DNA of strain IK003.1, which was isolated with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Two individual flanking regions of about 500 bp up- and downstream the respective integration sites were amplified from G. oxydans IK003.1 genomic DNA using specific oligonucleotide pairs with overlaps to the pAJ63a backbone, digested with PstI and KpnI, the promotor region and the terminator region to assemble all fragments via Gibson assembly [56]. The resulting fusion constructs were used to transform E. coli DH5α by the RbCl method [57]. Plasmids of positive clones were isolated using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and verified by sequencing (Eurofins Genomics, Ebersberg, Germany). Then the plasmids were transferred into the donor strain E. coli S17-1 for integration into G. oxydans IK003.1 via conjugation as described previously [17]. Positive clones, containing genomically integrated fdhSCL genes but not the vector backbone, were selected with 60 µg/mL 5-fluorouracile [35]. Positive clones were checked via colony PCR using a forward primer that binds upstream the upstream flank- ing region and a reverse primer, binding downstream of the downstream flanking region. Since the generation of the fdhSCL double integration strain turned out to be more challenging than the single integration strains, an adapted protocol was applied. IK003.1-igr2::fdhSCL was conjugated with E. coli S17–1 pAJ63a-igr3::fdhSCL according to the above mentioned protocol, while recom- bination media for the second recombination step and the agar plates for selecting the positive clones contained fructose instead of mannitol, as a strain with two chro- mosomal fdhSCL copies was assumed to show improved growth on fructose in comparison to a strain with a single fdhSCL copy. The double integration strain was obtained after several attempts. Cultivation in a respiration activity monitoring system (RAMOS) Online monitoring of the respiratory activity in shake flask cultivations was performed using the Respira- tion Activity MOnitoring System (RAMOS) devel- oped at the chair of biochemical engineering (RWTH Aachen University) [37, 38]. Commercial versions of the RAMOS device can be acquired from Kühner AG (Birs- feld, Switzerland) or HiTec Zang GmbH (Herzogen- rath, Germany). Eight 250 mL shake flasks (unbaffled) were cultivated in parallel with an initial filling volume of 10 mL, 350 rpm shaking frequency and 50 mm shak- ing diameter (Climo-Shaker ISF1-X, Kuhner, Birsfelden, Switzerland). The aeration rate was set to 10 mL/min (1 vvm). Cultivation in a respiration activity monitoring system (RAMOS) Each flask is equipped with a partial pressure sensor for oxygen and a differential pressure sensor to determine oxygen and carbon dioxide transfer rates (OTR and CTR). The respiratory quotient (RQ) is the quotient of CTR and OTR. [37, 38]. For strain mainte- nance, glycerol stocks were used. Cells cultivated in com- plex medium with mannitol were harvested during the exponential growth phase, centrifuged and re-suspended in fresh preculture medium with 200 g/L glycerol. The glycerol stocks were stored at − 80 °C. Precultures were inoculated with 100 µL glycerol stock suspension (OD600 = 2.4) and cultivated at 30 °C for 11 h to 19 h. Main cultures were inoculated from pre-cultures starting with an OD600 of 0.1. Preculture cells were cen- trifuged for 3 min at 16,214g and room temperature, and resuspended in main culture medium. Samples for offline analysis were taken from Erlenmeyer flasks operated at the same conditions in parallel to the online measure- ment. If necessary, 2-(N-morpholino) ethansulfonic acid (MES) buffer (pH 6, adjusted with 3 M KOH) was added in different concentrations to main cultures. Media composition G. oxydans strains were cultivated in complex medium containing 5 g/L yeast extract (Karl Roth GmbH, Karlsruhe, Germany or BD Biosciences, Heidelberg, Ger- many), 2.5 g/L MgSO4 × 7 H2O, 1 g/L (NH4)2SO4, 1 g/L KH2PO4. The initial pH was adjusted to 6 with KOH [54]. The media were supplemented with 50 µg/mL cefoxitin and 10 µM thymidine, and for plasmid-carrying strains 50 µg/mL kanamycin was added. For the precultures, 40 g/L mannitol was added as carbon source and main cultures were conducted with different fructose concen- trations as indicated in the respective experiments. E. coli strains were cultivated in lysogeny broth-based media [55] at 37 °C and 130 rpm, and 50 µg/mL kanamycin was added for plasmid-carrying strains. Battling et al. Microb Cell Fact (2020) 19:54 Battling et al. Microb Cell Fact (2020) 19:54 Page 11 of 15 Page 11 of 15 Analysis of gene expression by RT‑qPCRl The optical density of cultures at 600 nm (OD600) was measured either with a Genesys 20 photometer (Thermo Scientific, Darmstadt, Germany) or with an Ultraspec 2100 UV–Visible Spectrophotometer (biochrom, Hol- liston MA, USA). The photometers have a linear range between 0.1 and 0.3 and samples were diluted with 0.9% (w/v) NaCl if necessary. The pH values were determined with a HI221 Basic pH (Hanna Instruments Deutschland GmbH, Vöhringen, Germany), which was calibrated using two standard buffer solutions at pH 4 and 7. The osmolality was determined using the cryoscopic Osmom- eter OSMOMAT® 030 (Genotec, Berlin, Germany). Strains were cultivated in shake flasks as described above and harvested in the exponential growth phase (after 7.25 h). 35 mL culture were mixed with 15 g ice, centri- fuged for 5 min at 5.000g and 4 °C and the resulting cell pellet was shock-frozen in liquid nitrogen and stored at − 80 °C. RNA was isolated using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) including the following steps: cell disruption in a Precellys 24 homogenizer (Bertin, Frankfurt am Main, Germany), chloroform extraction, isopropanol precipitation, an on-column DNA digest on RNeasy mini spin columns, with RNase-free DNase (Qia- gen, Hilden, Germany). Purified RNA was subjected to a second DNA digest using the Ambion™ DNAfree™ DNA Removal Kit (Thermo Scientific, Waltham, MA USA). RNA was checked for integrity after the first or second DNase digest to exclude strong RNA degradation using a High Sensitivity RNA ScreenTape in the TapeStation 2200 (Agilent, Santa Clara, CA USA). Final RNA concen- trations were determined using the Colibri microvolume spectrometer (Titerek-Berthold, Pforzheim, Germany). Fructose consumption and 5-KF production were ana- lyzed by HPLC of culture supernatants. 1 mL culture was centrifuged at 17,000g for 10 min and the superna- tant was frozen at -20 °C until further analysis. Thawed samples were heated for 60 min at 60 °C (to avoid double peaks, probably caused by the existence of 5-ketofruc- tose in an equilibrium of the keto and the germinal diol form [26]), filtered (0.2 µm syringe filter, Whatman™, GE Healthcare, Freiburg, Germany) and diluted with deion- ized water. Cultivation in 2 L bioreactors For biological replicates, individual 10 mL-precultures in 100 mL unbaffled shake flasks with complex medium containing 40 g/L mannitol were inoculated from agar plates and incubated for about 24 h at 30 °C and 250 rpm. Cells from these precultures were harvested for 15 min at 20 °C and 5.000g, washed once in the main culture medium and used to inoculate 100 mL main cultures in 500 mL baffled shake flasks to an OD600 (optical den- sity at 600 nm) of 0.15 in complex medium with 18 g/L (100 mM) fructose. Main cultures were incubated at 30 °C and 130 rpm with a shaking diameter of 50 mm and 85% humidity (Shaker ISF1-X, Kuhner, Birsfelden, Switzerland). Fermentation experiments were performed in a 2 L Sar- torius BIOSTAT® Bplus stirred tank reactor (Sartorius, Goettingen, Germany). The temperature was set to 30 °C and aeration was set to 1 L/min with a filling volume of 1 L (1 vvm). The fermenter was equipped with a six blade rusthton turbine and 4 baffles. During cultivation, the dissolved oxygen tension (DOT) was measured using a VisiFerm™ DO 225 pO2 sensor (Hamilton, Hoechst, Germany). The DOT was controlled at ≥ 30% by auto- matically adjusting the stirring rate between 500 rpm and 1500 rpm. The pH value was measured using a pH sensor (EasyFerm Plus K8 200, Hamilton, Hoechst, Germany). If necessary, pH was controlled with a 3 M KOH solu- tion. Oxygen and carbon dioxide in the exhaust gas were measured using a DASGIP GA4 exhaust gas analyser Battling et al. Microb Cell Fact (2020) 19:54 Page 12 of 15 Page 12 of 15 (DASGIP, Eppendorf, Jülich, Germany). 0.5 mL antifoam agent Plurafac LF 1300 (BASF, Ludwigshafen, Germany) was added at the beginning of each experiment and when needed to prevent foaming. During fermentation, sam- ples were taken from the bioreactor for offline analysis. Volume change by KOH titration and sampling were con- sidered for mass balancing. (40 × 8 mm, CS-Chromatographie Service, Langerwehe, Germany), the separating column Organic Acid Resin (250 × 8 mm, CS-Chromatographie Service, Langer- wehe, Germany), and refraction index detector RID-20A (Shimadzu, Duisburg, Germany). The flow rate was set to 0.8 mL/min and an injection volume of 20 µL sample was used for analysis. 5 mM H2SO4 was used as mobile phase. Cultivation in 2 L bioreactors Eluted components were detected and quantified using standard curves prepared with fructose and 5-KF solutions of known concentrations (0.032 g/L to 10 g/L). Evaporation during cultivations in the RAMOS device was determined gravimetrically and was taken into account for determination of fructose and 5-KF concen- trations. Yields were calculated by dividing the produced 5-KF by the total fructose concentration and are indi- cated in g/g. The performance of scale-up from shake flasks to the fermenter was investigated by cultivating simultaneously in both cultivation systems. For this purpose, media com- position, pH, temperature and oxygen supply (aeration rate) were kept constant for both cultivation systems. The shake flasks were filled with medium from a fermenter. Cultivation broth was sterilely transferred from the fer- menter into shake flasks to ensure that media composi- tion, pH and optical density were identical [58–60]. Analysis of gene expression by RT‑qPCRl Depicted is (a) the oxygen and carbon dioxide transfer rates (OTR and CTR), (b) optical density (OD600) and pH, (c) dis- solved oxygen tension (DOT) and agitation speed, and (d) fructose and 5-ketofructose concentrations. The cultivation was performed with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1500 rpm), aeration rate (Q) = 1 L/min, T = 30 °C. Figure S4. Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with 80 g/L fructose and pH control. Depicted is (a) the oxygen and carbon dioxide transfer rates (OTR and CTR), (b) optical density (OD600) and pH, (c) dissolved oxygen tension (DOT) and agitation speed, and (d) fructose and 5-ketofructose concentra- tions. The cultivation was performed with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1500 rpm), aeration rate (Q) = 1 L/ min, T = 30 °C. Figure S5. Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a RAMOS device with different fructose concentrations and 150 mM MES (initial pH of 6). Depicted is the oxygen transfer rate during growth with the indicated concentrations of fructose in complex medium at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates. Figure S6. Cultivation of the indicated G. oxydans strains in a RAMOS device with 80 g/L fructose and 150 mM MES (initial pH of 6). Depicted is (a) the 5-ketofructose con- centration (HPLC method B), the yield g/g, the OD600 and the final pH after 29 h and (b) the oxygen transfer rate (OTR). Cultivations were performed in complex medium with 80 g/L fructose and 150 mM MES buffer at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates. p To set up RT-qPCR, primers were designed using the online tool Primer3plus [61] and default qPCR set- tings with a target PCR product of 100–120 bp and an annealing temperature of 60 °C. Three different primer pairs for fdhSCL, two binding in fdhS and one in fdhC, one primer pair for GOX0264, and three primer pairs for gap (GOX0508) were designed and tested for PCR specificity. Analysis of gene expression by RT‑qPCRl Sugars were quantified using the same 5-KF standard with two different methods: HPLC method A: A 10 µL sample was measured in an Agilent LC-1100 system (Agilent, Santa Clara, CA, USA) equipped with a Carbo-Ca Guard Catridge (Phenomenex, Aschaffenburg, Germany) using a Rezex RCM-Monosaccharide 300, 7.8 mm column (Phenomenex, Aschaffenburg, Germany) for separation at 60 °C with water as eluent at a flow rate of 0.6 mL/min. 5-KF and fructose were detected with a refraction index detector (35 °C) [27]. 5-KF and fructose were calibrated in a range of 0.1 to 8 g/L, with retention times of 12.7 and 14.6 min, respectively. HPLC method B: An HPLC system of Shimadzu (Duisburg, Germany) was equipped with a precolumn Organic Acid Resin cDNA was generated with SuperScript™ III Reverse Transcriptase (Thermo Scientific, Braunschweig, Ger- many) with the following protocol. 10 ng RNA, 150 ng random primers (Invitrogen, Thermo Scientific, Braun- schweig, Germany) and 1 µL of 10 mM dNTP mix (Inv- itrogen, Thermo Scientific, Braunschweig, Germany) in a total volume of 14 µL were incubated for 5 min at 65 °C and cooled for 1 min at 4 °C. Subsequently, 4 µL 5× first strand buffer, 1 µL 0.1 mM DTT, and 1 µL SuperScript III reverse transcriptase were added and incubated for 5 min at 25 °C, 60 min at 50 °C and finally for 5 min at 70 °C for inactivation of reverse transcriptase. For all samples, additionally a no amplification control was generated with water instead of reverse transcriptase to exclude Battling et al. Microb Cell Fact (2020) 19:54 Page 13 of 15 genomic DNA contaminations in RT-qPCR. RT-qPCR was performed with a qTower instrument (Analytik Jena, Jena, Germany) using KAPA SYBR FAST qPCR Master Mix (Roche Diagnostics, Mannheim, Germany). 2 µL of cDNA or no template control were used for RT-qPCR. For each sample, at least three biological replicates with two technical replicates were measured. RAMOS system. Fermentation was performed with 1 L filling volume in a L fermenter, DOT was kept ≥ 30% by variation of the agitation speed (500–1250 rpm), aeration rate (Q) = 1 L/min, 30 °C. Figure S3. Cultiva- tion of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with 100 mM MES and 80 g/L fructose. Abbreviations KF K f 5-KF: 5-Keto-d-fructose; CTR: Carbon dioxide transfer rate; DOT: Dissolved oxy- gen tension; DNA: Deoxyribonucleic acid; FAD: Flavin adenine dinucleotide; FDH: Fructose dehydrogenase; FPKM: Fragments Per Kilobase of transcript per Million mapped reads; HPLC: High-performance liquid chromatography; IGRs: Intergenic regions; MES: 2-(N-Morpholino)ethansulfonic acid; OD600: Optical density at 600 nm; ori: Origin of replication; OTR: Oxygen transfer rate; RAMOS: Respiration activity MOnitoring System; RBS: Ribosome binding site; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; RQ: Respiratory quotient; TCA: Tricarboxylic acid cycle; TCE: Total carbon dioxide evolution; TOC: Total oxygen consumption; vvm: Vessel volume per minute. Funding This work was financially supported by the Bundesministerium für Bildung und Forschung (BMBF) within Project No. 031B0370 by grants given to Michael Bott (031B0370B) and Jochen Büchs (031B0370C). Analysis of gene expression by RT‑qPCRl To optimize RT-qPCR conditions, all primer pairs were compared in a gradient from 57.1 to 63.9 °C regarding PCR specificity via agarose gel electrophore- sis and melting curve analysis. The optimal temperature was determined to be 57.1 °C. Subsequently, the PCR efficiency was determined by running a RT-qPCR with an 8-step 1:10 dilution series of cDNA. Ct values were determined using qPCRsoft 3.1 (Analytik Jena). The opti- mal primer pairs were selected to quantitatively amplify cDNA fragments of fdhC (q-fdhC-fwd + q-fdhC-rev, PCR efficiency of 103.2%), of GOX0264 (q-GOX0264- fwd + q-GOX0264-rev, 103%), and of GOX0508 (q-gap- fwd2 + q-gap-rev2, 91.8%) Relative transcript levels were calculated from Ct values, considering the primer effi- ciencies, and normalized to the transcript levels of the plasmid strain. Supplementary information Supplementary information acc org/10.1186/s12934-020-01310-7. Supplementary information accompanies this paper at https://doi. org/10.1186/s12934-020-01310-7. Supplementary information accompanies this paper at https://doi. org/10.1186/s12934-020-01310-7. Authors’ contributions SB designed and performed characterization and optimization experiments in shake flasks with online monitoring (RAMOS), scale up and fermentation experiments, analyzed the data and drafted the manuscript. KW constructed the integration strains, designed and performed the shake flask cultivations and the RT-qPCR, analyzed the data and drafted the manuscript. CI performed the scale up experiments. AK selected the integration sites. MP performed some of the shake flask cultivations with online monitoring. AW set up HPLC method A. MBo, JB and MBa supervised the study, assisted in data inter- pretation and participated in drafting the manuscript. All authors read and approved the final manuscript. Additional file 1: Table S1. Offline data for cultivation of the indicated G. oxydans strains with 80 g/L fructose in a RAMOS device shown in Fig. S1. Table S2. Offline date for the cultivation of G. oxydans IK003.1-igr3::fdhSCL in a RAMOS device with different fructose concentrations shown in Fig. S5. Table S3. Oligonucleotides used in this study. Figure S1. Cultivation of the indicated G. oxydans strains with 80 g/L fructose in a RAMOS device with online monitoring. Depicted are (a) the oxygen transfer rate (OTR) (b) the carbon dioxide transfer rate (CTR) (c) the respiratory quotient (RQ shown for OTR values above 5 mmol/L/h), (d) the total oxygen consumption (TOC) and (e) the total carbon dioxide evolution (TCE). The strains were cultivated in complex medium with 80 g/L fructose at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates. Figure S2. Scale-up of batch fermentation of G. oxydans IK003.1-igr3::fdhSCL from shake flasks (RAMOS device) to a 2 L fermenter with 80 g/L fructose and 150 mM MES. Depicted are (a) oxygen transfer rate (OTR) and carbon dioxide transfer rate (CTR), (b) growth as OD600 and pH, (c) dissolved oxygen tension (DOT), agitation speed during fermentation, addition of antifoam agent (AF), and period of DOT control (indicated by arrows) and (d) fructose and 5-ketofructose concentration as determined by HPLC (method B). Cultivations were performed in complex medium with 80 g/L fructose prepared in the fermenter. The shake flask experiment was started with a sterile sample from the fermenter at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm using the Availability of data and materials The datasets supporting the conclusions of this article are included within the article and the additional file (Additional file1: Tables S1, S2, and S3, Figures S1, S2, S3, S4, S5, and S6). The datasets supporting the conclusions of this article are included within the article and the additional file (Additional file1: Tables S1, S2, and S3, Figures S1, S2, S3, S4, S5, and S6). Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. References 2002;60:233–42. 5. Deppenmeier U, Hoffmeister M, Prust C. Biochemistry and biotechno- logical applications of Gluconobacter strains. Appl Microbiol Biotechnol. 2002;60:233–42. 28. 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https://openalex.org/W2953378399	https://www.ruproctology.com/jour/article/download/1111/1111	Russian	null	MALIGNANT TUMORS OF THE ENDOMETRIUM, BRAIN, THYROID IN THE FAMILY WITH ADENOMATOUS POLYPOSIS	Koloproktologiâ	2,018	cc-by	3,035	ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ ТЕЛА МАТКИ, ГОЛОВНОГО МОЗГА, ЩИТОВИДНОЙ ЖЕЛЕЗЫ В СЕМЬЕ С АДЕНОМАТОЗНЫМ ПОЛИПОЗОМ Шелыгин Ю.А.1, Потапов А.А.2, Кузьминов А.М.1, Вышегородцев Д.В.1, Мурусидзе Н.А.2, Чичеватов Д.А.3, Пономарева Е.Е.3, Майновская О.А.1, Шубин В.П.1, Цуканов А.С Шелыгин Ю.А. , Потапов А.А. , Кузьминов А.М. , Вышегородцев Д.В.1, Мурусидзе Н.А.2, Чичеватов Д.А.3, р уру Пономарева Е.Е.3, Майновская О.А.1, Шубин В.П.1, Цуканов А.С.1 1 ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, г. Москва 2 ФГАУ «Национальный медицинский исследовательский центр нейрохирургии им. академика Н.Н.Бурденко» Минздрава России, г. Москва 3 ГБУЗ «Областной онкологический диспансер», г. Пенза MALIGNANT TUMORS OF THE ENDOMETRIUM, BRAIN, THYROID IN THE FAMILY WITH ADENOMATOUS POLYPOSIS Shelygin Yu.A.1, Potapov A.A.2, Kuzminov A.M.1, Vyshegorodtsev D.V.1, Murusidze N.A.2, Chichevatov D.A.3, Ponomareva E.E.3, Maynovskaya O.A.1, Shubin V.P.1, Tsukanov A.S.1 1 State Scientific Center of Coloproctology, Moscow, Russia 2 N.N. Burdenko Neurosurgical Institute of the Russian Academy of Medical Sciences, Moscow, Russia 3 Penza Regional Oncology Hospital, Penza, Russia [Key words: familial adenomatous polyposis, Türko syndrome, thyroid cancer, uterine cancer, APC gene] [Key words: familial adenomatous polyposis, Türko syndrome, thyroid cancer, uterine cancer, APC gene] Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru Семейный аденоматоз толстой кишки (САТК) явля- ется вторым по частоте наследственным колорек- тальным раковым синдромом (1 % от всех случаев заболевания), уступая только синдрому Линча [5]. САТК – аутосомно-доминантный наследственный синдром, в большинстве случаев обусловленный герминальной мутацией в гене APС (Adenomatous Polyposis Coli), продукт которого принимает уча- стие в регуляции транскрипции, апоптозе, клеточ- ной адгезии, а также контроле клеточного цикла [2,8]. Клинически данное заболевание проявляется поражением толстой кишки множеством аденом [3]. При этом полипы имеют прогрессивный рост и, при отсутствии своевременного хирургическо- го лечения, риск развития рака толстой кишки составляет 100 % (Рис. 1). Достаточно часто САТК сопровождается тяжелыми метаболическими нару- шениями. В настоящее время операцией выбора при хирургическом лечении САТК при отсутствии противопоказаний является колэктомия с резек- цией прямой кишки и формированием тонкоки- шечного резервуара. В последующем все пациенты с САТК нуждаются в пожизненном клиническом мониторинге. Это обусловлено тем, что в последние годы появились сообщения о риске развития рака не только в анальном канале, сохраненной аналь- ной транзиторной зоне, но и в тонкокишечном резервуаре [13]. В 10-15 % случаев среди больных САТК встреча- ются десмоидные опухоли (десмоидные фибромы) брыжейки тонкой кишки и передней брюшной стенки. Чаще эти опухоли встречаются у женщин. Существует мнение, что провоцирующим момен- том, приводящим к развитию десмом, являет- ся хирургическая травма. Однако до конца этот вопрос остается малоизученным. Десмоидные фибромы занимают промежуточное положение между злокачественными и доброкачественными опухолями и проявляют локальные агрессивные свойства [11]. При САТК существует риск развития злокачествен- ных новообразований различных органов: двенад- 69 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ ТЕЛА МАТКИ, ГОЛОВНОГО МОЗГА, ЩИТОВИДНОЙ ЖЕЛЕЗЫ В СЕМЬЕ С АДЕНОМАТОЗНЫМ ПОЛИПОЗОМ цатиперстная кишка (3-10 %), щитовидная железа (2 %), головной мозг (1-2 %) и др. [12]. Известно, что молодые женщины (моложе 35 лет) с САТК подвергаются высокому риску (в 160 раз выше общепопуляционного) развития рака щитовидной железы [9]. Необходимо отметить, что именно лока- лизация мутации в гене APC обусловливает разви- тие определенных внекишечных новообразований. Так, мутации в гене APC у европейских больных с десмоидными фибромами, в основном, локали- зуются в участке с 1310 по 2011 кодоны, а у паци- ентов с раком щитовидной железы – в участке со 140 по 1309 кодоны [4,6]. Достоверной корре- ляции между нахождением мутации в гене APC и развитием опухоли головного мозга у пациентов c САТК не установлено из-за редкости такого рода больных. нервной системы [14]. Позднее было замечено, что «синдром Тюрко» гетерогенен, охватывая как минимум два подтипа. Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru Первый из них характери- зуется герминальной мутацией в одном из генов системы репарации неспаренных оснований ДНК, таких как PMS2 или MLH1, и представляет собой появление глиобластомы у пациентов с синдромом Линча (наследственный неполипозный рак тол- стой кишки). Для второго подтипа синдрома Тюрко характерно наличие у пациента медуллобластомы и герминальной мутации в гене APC [10]. Рак щитовидной железы у пациента с САТК был впервые описан в 1949 году Crail H.W. Интересно, что в этой же семье встретился случай опухоли головного мозга [7]. Поскольку подобные семьи в мире встречаются крайне редко, особый инте- рес представляет наше собственное клиническое наблюдение, характеризующееся различными вне- кишечными проявлениями САТК в одной семье. В данной статье мы приводим описание этой семьи. Больная Д., 18 лет, история болезни 2764-14, проживающая в Пензенской области, поступила в ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России 30.04.2014 г. с жалобами на периодические боли в животе, частый жидкий стул, до 5 раз, с приме- сью крови и слизи. Эти жалобы пациентка отме- чала в течение 1 года. При обследовании по месту жительства, в ГБУЗ «Пензенская областная кли- ническая больница им. Н.Н. Бурденко» был уста- новлен диагноз – семейный аденоматоз толстой кишки. Больная была направлена в ФГБУ «ГНЦ колопроктологии им. А.Н.Рыжих» Минздрава России, где прошла комплексное обследование. Выполнена колоноскопия. Установлено, что в сле- пой кишке имеется 10 плоских полипов до 0,3 см в диаметре, на илеоцекальном клапане два полипа до 0,5 см. В восходящей кишке около 10 полипов диаметром 0,5 см на одну гаустру, в поперечной В 1959 году Turcot J. с соавт. описали семью, вклю- чающую двух братьев и сестру с САТК, у которых развились злокачественные опухоли центральной Рисунок 1. Эндоскопическое исследование толстой кишки. Рак на фоне САТК Рисунок 1. Эндоскопическое исследование толстой кишки. Рак на фоне САТК Рисунок 2. Сиквенс фрагмента гена APC (мутация c.3183_3187delACAAA указана стрелкой) Рисунок 2. Сиквенс фрагмента гена APC (мутация c.3183_3187delACAAA указана стрелкой) 70 КОЛОПРОКТОЛОГИЯ, 2018, №1 (63) ободочной кишке около 15 полипов на гаустру диаметром до 1,0 см. В левых отделах число поли- пов значительно увеличивается. В нисходящей кишке до 15 полипов на одну гаустру диаметром до 3,0 см. В сигмовидной кишке до 20 полипов на гаустру от 1,5 до 3,0 см в диаметре. В прямой кишке 35 полипов. В нижнеампулярном отделе прямой кишки ворсинчатая опухоль 3,5 см в диаметре. При гастроскопии (07.05.14) выявлено полиповидное образование луковицы двенадцатиперстной кишки 0,4 см. Гистологическое исследование № 40846: фрагмент слизистой двенадцатиперстной кишки со структурами тубулярной аденомы с умеренной дисплазией. Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru Следует отметить, что диагноз семейный аде- номатоз толстой кишки был установлен в ГБУЗ «Пензенская областная клиническая больница имени Н.Н. Бурденко» в 2010 году. От предложен- ного хирургического лечения пациентка отказа- лась. В дальнейшем, с октября 2014 года появи- лись выделения крови из половых путей. В ГБУЗ «Областной онкологический диспансер» Минздрава Пензенской области установлен диагноз «рак тела матки». 26.12.2014 г. выполнена операция – тоталь- ная гистерэктомия. Результат гистологического заключения № 162 (31.12.14): высокодифференци- рованная аденокарцинома, ограниченная предела- ми эндометрия, (stlaT1aN0M0). Роста опухоли в цер- викальном канале нет. Послеоперационный период протекал без осложнений и пациентке вновь было рекомендовано хирургическое лечение по поводу САТК, от которого она воздержалась. Следует отметить, что диагноз семейный аде- номатоз толстой кишки был установлен в ГБУЗ «Пензенская областная клиническая больница имени Н.Н. Бурденко» в 2010 году. От предложен- ного хирургического лечения пациентка отказа- лась. В дальнейшем, с октября 2014 года появи- лись выделения крови из половых путей. В ГБУЗ «Областной онкологический диспансер» Минздрава Пензенской области установлен диагноз «рак тела матки». 26.12.2014 г. выполнена операция – тоталь- ная гистерэктомия. Результат гистологического заключения № 162 (31.12.14): высокодифференци- рованная аденокарцинома, ограниченная предела- ми эндометрия, (stlaT1aN0M0). Роста опухоли в цер- викальном канале нет. Послеоперационный период протекал без осложнений и пациентке вновь было рекомендовано хирургическое лечение по поводу САТК, от которого она воздержалась. Здесь следует напомнить, что наследственная мутация в гене APC у пациентки локализуется в кодоне 1061, патогенные наследственные вари- анты в котором, согласно данным зарубежных исследователей, могут приводить к развитию рака щитовидной железы [6]. Особый интерес представляет семейный анам- нез нашей пациентки. Ее мать, пациентка Ж., 38 лет, (история болезни 6878-15), поступила на лечении в ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России 12.11.2015 г. с жалобами на наличие одно- ствольной илеостомы, выделение крови из пря- мой кишки. Из анамнеза известно, что пациентка Ж. была оперирована в «НИИ нейрохирургии им. Н.Н. Бурденко» РАМН (история болезни 3594-09) в возрасте 31 года по поводу опухоли правой гемис- Особый интерес представляет семейный анам- нез нашей пациентки. Ее мать, пациентка Ж., 38 лет, (история болезни 6878-15), поступила на лечении в ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России 12.11.2015 г. с жалобами на наличие одно- ствольной илеостомы, выделение крови из пря- мой кишки. Из анамнеза известно, что пациентка Ж. была оперирована в «НИИ нейрохирургии им. Н.Н. Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru При компьютерной томографии голов- ного мозга признаков объемных образований не выявлено. При молекулярно-генетическом исследова- нии у больной выявлена мутация в гене APC – c.3183_3187delACAAA (p.Gln1062X) (Рис. 2). Таким образом, на основании всех полученных данных у пациентки установлен диагноз – семей- ный аденоматоз толстой кишки, классическая форма (мутация c.3183_3187delACAAA в гене APC). Больной 23.05.14 г. выполнено хирургическое вмешательство в объеме колэктомии с резекцией прямой кишки, демукозацией сохраненной части прямой кишки, формированием J-образного тон- кокишечного резервуара с наложением резервуа- роректального анастомоза, превентивной илеосто- мии по Торнболлу (Рис. 3). С целью реконструкции слизистой оболочки сохраненной части прямой кишки была применена биотехнология, о кото- рой мы сообщали ранее [1]. Послеоперационный период протекал без осложнений. Через 6 месяцев, 20.11.14 г. пациентке выполнена реконструктивно- пластическая операция в объеме внутрибрюшного закрытия илеостомы. Осложнений после операции не было, и пациентка была выписана под наблюде- ние врача по месту жительства. При наблюдении за пациенткой в течение одного года отмечен хоро- ший функциональный результат. Частота дневной дефекации 4-6 раз. Ночная дефекация отсутствует. Признаков анальной инконтиненции нет, пациент- ка удерживает все компоненты кишечного содер- жимого. Рисунок 3. Макропрепарат толстой кишки паци- ентки Д. (И.Б. № 2764-14) В июне 2015 года пациентка впервые обнаружи- ла опухолевидный узел в области щитовидной железы. Несмотря на то, что данное опухолевид- ное образование быстро увеличивалось в раз- мерах, пациентка за медицинской помощью не Рисунок 3. Макропрепарат толстой кишки паци- ентки Д. (И.Б. № 2764-14) Рисунок 5. Микропрепарат пациентки Ж. Опухоль мозжечка – медуллобластома. Увеличение × 100. Окраска гематоксилином и эозином Рисунок 4. Микропрепарат пациентки Д. Фолликулярный рак щитовидной железы. Увеличение × 100. Окраска гематоксилином и эози- ном Рисунок 4. Микропрепарат пациентки Д. Фолликулярный рак щитовидной железы. Увеличение × 100. Окраска гематоксилином и эози- ном Рисунок 5. Микропрепарат пациентки Ж. Опухоль мозжечка – медуллобластома. Увеличение × 100. Окраска гематоксилином и эозином 71 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ ТЕЛА МАТКИ, ГОЛОВНОГО МОЗГА, ЩИТОВИДНОЙ ЖЕЛЕЗЫ В СЕМЬЕ С АДЕНОМАТОЗНЫМ ПОЛИПОЗОМ обращалась. В декабре 2015 года она обратилась в Государственное бюджетное учреждение здраво- охранения «Областной онкологический диспансер» Министерства здравоохранения Пензенской обла- сти. 12.02.2016 г. произведена тиреоидэктомия с паратрахеальной лимфодиссекцией. Результат гистологического заключения № 1710/16 от 17.02.2016 г. – фолликулярный рак с инвазией в капсулу железы, в прилежащих тканях паратра- хеальной клетчатки опухолевого роста не выявлено (Рис. 4). Послеоперационный период протекал без осложнений. ф р р у ление опухоли. Послеоперационный период без осложнений. Результат гистологического исследо- вания № 6526-30/09: медуллобластома (Рис. 5). Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru Бурденко» РАМН (история болезни 3594-09) в возрасте 31 года по поводу опухоли правой гемис- В феврале 2015 года у больной развились явле- ния кишечной непроходимости с метаболически- ми нарушениями, обусловленные раком сигмовид- ной кишки на фоне САТК. 12.02.2015 г. больная была оперирована по месту жительства, в г. Пенза выполнена субтотальная резекция толстой кишки с сохранением прямой и части сигмовидной кишки и формированием одноствольной илеостомы по Бруку. Послеоперационный период протекал бла- гополучно, и пациентка была выписана. Однако уже в октябре 2015 года она отметила выделение крови из прямой кишки и с этими жалобами обра- тилась к колопроктологу по месту жительства. При осмотре отмечено прогрессирование роста поли- пов прямой кишки, и пациентка направлена ФГБУ «ГНЦ колопроктологии им. А.Н.Рыжих» Минздрава России. Рисунок 6. Макропрепарат сигмовидной и прямой кишки больной Ж. (И.Б. 6878-15) Больная была обследована: при эндоскопическом исследовании установлено, что протя женность сохраненной части прямой кишке 20 см, в отклю- ченной прямой кишке около 35 полипов диаметром 0,5-2,5 см на широких и суженных основаниях. В среднеампулярном отделе прямой кишки опу- холь ворсинчатого вида, занимающая до полови- ны окружности кишки. 18.11.2015 г. пациентке выполнено хирургическое вмешательство в объ- еме брюшно-анальной резекции прямой кишки, реконструкции илеостомы. Гистологическое иссле- дование № 37877-916: на расстоянии 3 см от зуб- чатой линии обнаружена изъязвленная умерен- нодифференцированная аденокарцинома, про- растающая в мышечный слой кишечной стенки; на расстоянии 11 см от зубчатой линии еще одна Рисунок 6. Макропрепарат сигмовидной и прямой кишки больной Ж. (И.Б. 6878-15) 72 КОЛОПРОКТОЛОГИЯ, 2018, №1 (63) изъязвленная умереннодифференцированная аде- нокарцинома, прорастающая в поверхностные отделы мышечного слоя кишечной стенки (Рис. 6). Послеоперационный период без осложнений. На сегодняшний день обе пациентки находятся под наблюдением врачей по месту жительства. Признаков прогрессирования злокачественного процесса не выявлено. исследование наследственной предрасположенно- сти к разным формам полипоза толстой кишки. / Ю.А.Шелыгин, В.Н.Кашников, С.А.Фролов и соавт. // Колопроктология. – 2013. – т. 1. – № 43. – с. 9-14. исследование наследственной предрасположенно- сти к разным формам полипоза толстой кишки. / Ю.А.Шелыгин, В.Н.Кашников, С.А.Фролов и соавт. // Колопроктология. – 2013. – т. 1. – № 43. – с. 9-14. изъязвленная умереннодифференцированная аде- нокарцинома, прорастающая в поверхностные отделы мышечного слоя кишечной стенки (Рис. 6). Послеоперационный период без осложнений. На сегодняшний день обе пациентки находятся под наблюдением врачей по месту жительства. Признаков прогрессирования злокачественного процесса не выявлено. 4. Bertario, L. Multiple approach to the exploration of genotype-phenotype correlations in familial adenomatous polyposis. / L.Bertario, A.Russo, P.Sala et al. // Journal of clinical oncology. – 2003. Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru – № 21. – p. 1698-1707. р Таким образом, приведенное нами клиническое наблюдение свидетельствует о том, что семейный аденоматоз толстой кишки является крайне тяже- лым наследственным заболеванием, обусловлива- ющим развитие не только колоректального рака, но и возникновение злокачественных новообразо- ваний в других органах. В представленном ранее зарубежном исследовании у членов одной семьи на фоне САТК был установлен рак толстой кишки, щитовидной железы и опухоль головного мозга. В нашем сообщении кроме злокачественных опу- холей указанных органов мы впервые наблюда- ли также и рак тела матки. Наше наблюдение свидетельствует о том, что всем больным САТК, а также их родственникам, имеющим наследствен- ную мутацию в гене APC, необходимо проведение пожизненного клинического мониторинга в специ- ализированных медицинских центрах. При этом обследование должно преследовать цель не только мониторинга состояния анального канала, тонко- кишечного резервуара и отделов толстой кишки, сохраненных после хирургического лечения САТК, но и других органов на предмет выявления опухо- лей на ранних стадиях их развития. 5. Bulow, S. Results of national registration of familial adenomatous polyposis. / S.Bulow // Gut. – 2003. – № 52. – p. 742-746. 6. Cetta, F. Germline mutations of the APC gene in patients with familial adenomatous polyposis- associated thyroid carcinoma: results from a European cooperative study. / F.Cetta, G.Montalto, M.Gori et al. // Journal of clinical endocrinology and metabolism. – 2000. – № 85. – p. 286-292. // Journal of clinical endocrinology and metabolism. – 2000. – № 85. – p. 286-292. 7. Crail, H.W. Multiple primary malignancies arising in the rectum, brain and thyroid. Report of a case. / H.W.Crail // US Navy Med Bull. – 1949. – № 49. – p. 123-128. 7. Crail, H.W. Multiple primary malignancies arising in the rectum, brain and thyroid. Report of a case. / H.W.Crail // US Navy Med Bull. – 1949. – № 49. – p. 123-128. 8. Fearnhead, N.S. The ABC of APC. / N.S.Fearnhead, M.P.Britton, W.F.Bodmer // Human molecular genetics. – 2001. – № 10. – p. 721-733. M.P.Britton, W.F.Bodmer // Human molecular genetics. – 2001. – № 10. – p. 721-733. 9. Groen, E.J. Extra-intestinal manifestations of familial adenomatous polyposis. / E.J.Groen, A.Roos, F.L.Muntinghe et al. // Ann. Surg.Oncol. – 2008. – №15. – p. 2439-2450. 9. Groen, E.J. Extra-intestinal manifestations of familial adenomatous polyposis. / E.J.Groen, A.Roos, F.L.Muntinghe et al. // Ann. Surg.Oncol. – 2008. – № 15. – p. 2439-2450. 10. Hamilton, S.R. The molecular basis of Turcot’s syndrome. Адрес для переписки: Цуканов Алексей Сергеевич, ФГБУ «ГНЦК им. А.Н.Рыжих» Минздрава России, ул. Саляма Адиля, д. 2, Москва, 123423; e-mail: tsukanov81@rambler.ru / S.R.Hamilton, B.Liu, R.E.Parsons et al. // N. Engl. J. Med. – 1995. – № 332. – p. 839-847. 11. Kartheuser, A. Restorative proctocolectomy and ileal pouch-anal anastomosis for familial adenomatous polyposis revisited. / A.Kartheuser, P.Stangherlin, D.Brandt et al. // Familial Cancer. – 2006. – № 5. – p. 241-260. ЛИТЕРАТУРА 1. Коган, Е.А. Морфологические аспекты кле- точной реконструкции слизистой оболочки пря- мой кишки при хирургическом лечении семей- ного аденоматоза толстой кишки. / Е.А.Коган, Д.В.Вышегородцев, Н.М.Файзуллина и соавт. // Российский журнал Гастроэнтерологии, Гепатологии, Колопроктологии. – 2013. – т. 23. – № 6. – с. 73-79. 2. Цуканов, А.С. Мутации в гене APC у россий- ских пациентов с классической формой семей- ного аденоматоза толстой кишки. / А.С.Цуканов, Н.И.Поспехова, В.П.Шубин и соавт. // Генетика. – 2017. – № 3. – с. 356-363. 3 Шелыгин Ю А Молекулярно генетическое 1. Коган, Е.А. Морфологические аспекты кле- точной реконструкции слизистой оболочки пря- мой кишки при хирургическом лечении семей- ного аденоматоза толстой кишки. / Е.А.Коган, Д.В.Вышегородцев, Н.М.Файзуллина и соавт. // Российский журнал Гастроэнтерологии, Гепатологии, Колопроктологии. – 2013. – т. 23. – № 6. – с. 73-79. 12. Syngal, S. 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Молекулярно-генетическое 73 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ ТЕЛА МАТКИ, ГОЛОВНОГО МОЗГА, ЩИТОВИДНОЙ ЖЕЛЕЗЫ В СЕМЬЕ С АДЕНОМАТОЗНЫМ ПОЛИПОЗОМ
https://openalex.org/W2507551267	https://europepmc.org/articles/pmc5342469?pdf=render	English	null	Mutational load and mutational patterns in relation to age in head and neck cancer	Oncotarget	2,016	cc-by	7,259	www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 43 Mutational load and mutational patterns in relation to age in head and neck cancer Research Paper: Gerotarget (Focus on Aging) www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 43 Mutational load and mutational patterns in relation to age in head and neck cancer Stefano Meucci1, Ulrich Keilholz1, Ingeborg Tinhofer2 and Oliva A. Ebner1 1 Charité Comprehensive Cancer Center, Charité University Hospital, Charitéplatz, Berlin, Germany 2 Department of Radiation Oncology and Radiotherapy, Charité University Hospital Berlin, Translational Radiation Oncology Research Laboratory, Charitéplatz, Berlin, Germany Research Paper: Gerotarget (Focus on Aging) www.impactjournals.com/oncotarget/ Stefano Meucci1, Ulrich Keilholz1, Ingeborg Tinhofer2 and Oliva A. Ebner1 1 Charité Comprehensive Cancer Center, Charité University Hospital, Charitéplatz, Berlin, Germany 2 Department of Radiation Oncology and Radiotherapy, Charité University Hospital Berlin, Translational Radiation Oncology Research Laboratory, Charitéplatz, Berlin, Germany Stefano Meucci1, Ulrich Keilholz1, Ingeborg Tinhofer2 and Oliva A. Ebner1 1 Charité Comprehensive Cancer Center, Charité University Hospital, Charitéplatz, Berlin, Germany 2 Department of Radiation Oncology and Radiotherapy, Charité University Hospital Berlin, Translational Radiation Oncology Research Laboratory, Charitéplatz, Berlin, Germany Correspondence to: Ulrich Keilholz, email: ulrich.keilholz@charite.de Correspondence to: Stefano Meucci, email: stefano.meucci@charite.de Keywords: head and neck squamous cell carcinoma; aging; somatic mutations; sequencing; genomics; Gerotarget Received: December 21, 2015 Accepted: July 23, 2016 Published: August 16, 2016 ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is a cancer with well-defined tumor causes such as HPV infection, smoking and drinking. Using The Cancer Genome Atlas (TCGA) HNSCC cohort we systematically studied the mutational load as well as patterns related to patient age in HNSCC. To obtain a homogenous set we excluded all patients with HPV infection as well as wild type TP53. We found that the overall mutational load is higher in patients of old age. Through unsupervised hierarchical clustering, we detected distinct mutational clusters in very young as well as very old patients. In the group of old patients, we identified four enriched pathways (“Axon Guidance”, “ECM-Receptor Interaction”, “Focal Adhesion” and “Notch Signaling”) that are only sporadically mutated in the other age groups. Our findings indicate that the four pathways regulate cell motility, tumor invasion and angiogenesis supposedly leading to less aggressive tumors in older age patients. Importantly, we did not see a strict pattern of genes always mutated in older age but rather an accumulation of mutations in the same pathways. Our study provides indications of age-dependent differences in mutational backgrounds of tumors that might be relevant for treatment approaches of HNSCCs patients. Patients selection and cohort features To create a homogenous set of tumors we used a subset of the TCGA cohort (Figure 1). The original patient cohort was selected by two criteria, the first (i) excluded 36 HPV(+) patients (HPV classification according to the TCGA publication). As expected, the HPV-positive phenotype was strongly associated with the oropharyngeal site and the patient age mean was lower than for the HPV-negative patients. The second selection criteria (ii) excluded 40 TP53 wild type patients, which left the final selected cohort of 203 HPV-negative/TP53- mutated patients. The original TCGA data set showed a higher rate (86%) of TP53 mutations among HPV-negative samples than have been previously reported, while only 1 out of 36 HPV-positive cases had a non-synonymous TP53 mutation. Our selection rendered 203 patients with a total amount of 29.860 single nucleotide polymorphisms (SNPs) distributed on 11.489 genes. While some differences in tumor behavior in older patients have been recorded, no study so far systematically explored the relationship between genetic tumor background and age in HNSCC. A recent study performed on the extensive data set available on The Cancer Genome Atlas (TCGA) portal showed the age-related accumulation of somatic mutations in diverse human tissues [19]. However, it is still an open question whether differences in mutations between the ages are random coincidence or follow distinct patterns. Therefore, this study aims to explore the specific age-mutation relationship in tumors of HNSCC patients to determine if age-related genetic parameters have to be considered in the disease prognosis and treatment decision. To investigate a possible connection between patterns and frequency of genetic mutations and patient age, we used the recently published TCGA study on HNSCCs of 279 patients. Statistical hypothesis tests showed that the patient selection does not lead to a biased distribution of patients features such as age, smoking and alcohol consumption. Whereas, the hypergeometric test performed on the tumor localization distribution showed an enrichment in Larynx tumor (p < 0.05) and a depletion in Oropharynx tumor (p < 0.05) in the selected cohort (Table S1). HPV infection shows an age bias as a relevant impact on the mutational background of the tumor. In previous studies the mutation rate of HPV-positive tumors was lower than that found in HPV-negative HNSCC, consistent with recent epidemiologic studies that establish biological differences between HPV-positive and HPV- negative disease [20]. Patients selection and cohort features The major biologic difference between HPV-positive and - negative tumors, however, concerns p53, which in its role as a guardian of the genome influences multiple genes. In general, patients with HPV- positive tumors have non-mutated TP53, however, HPV itself inhibits p53 function. Conversely, HPV-negative tumors frequently harbour TP53 mutations. A comparison of the original TCGA cohort and our subset can be found in Table 1. Furthermore, the results of the investigation of TNM and overall staging at diagnosis in relation to the age showed a smaller size of the primary tumor, a decrease of lymph node metastasis and a higher percentage of “localized cancers” and “first stage of locally advanced cancers” in old patients (Table S1). The youngest patients group showed the same statistics as the oldest, however the results are not comparable due to the significant difference between the two age group ranges. Thus, in order to select a homogenous patient cohort for investigating possible age-related differences, we only considered the 203 patients from the TCGA-cohort which were HPV negative and carried a TP53 mutation. This homogenous subset of patients allowed for a systematic study of the age influence on mutation load and spectrum without introducing a heterogeneity caused by HPV or p53. Of course, it would have been equally interesting to study the other subclasses of HNSCCs. However, since only few patients belonged into these subsets, a statistical sound investigation would not have been possible within the current TCGA cohort. RESULTS RESULTS adolescence or young adult age, and smoking cessation is associated with the diagnosis of the malignancy [14,15]. In addition, several studies report HPV-negative HNSCCs mostly occurring in smokers [16-18]. Therefore the age at diagnosis of HNSCC patients is very closely correlated to the duration of smoking. INTRODUCTION incidence in HPV positive tumors, in oropharyngeal tumors, is exceeding 50% in current cohorts [4-6], and these tumors have been associated with a favorable clinical outcome [7, 8]. Approximately 80% of HNSCCs are HPV- negative (HPV-), the majority of them contain a mutation in TP53 and are characterized by many numerical genetic changes (high chromosome instability). In the remaining cases, characterized by a lower number of numerical genetic changes, p53 seems not to be inactivated [3]. Head and neck squamous cell carcinomas (HNSCCs) affect 600,000 patients per year worldwide [1]. HNSCCs are characterized by phenotypic, etiological, biological and clinical heterogeneity and can originate from the paranasal sinuses, nasal cavity, oral cavity, pharynx and larynx. The major known risk factors of HNSCC are consumption of tobacco and alcohol, as well as human papillomaviruses (HPV) infection [2]. Multiple studies have elucidated the specific genetic background of HNSCC, establishing subclasses of tumors alongside HPV infection and/ or TP53 mutations. Due to the heterogeneity of study cohorts, the estimated percentages are relatively high variable. Overall, approximately 20% of HNSCCs contain transcriptionally active human papillomavirus (HPV+), and mainly TP53 wild type, which however is inactivated by the viral E6 and E7 oncogenes [3]. The Tumor characteristics vary between patients of different ages. Elderly patients are mainly diagnosed with a lower incidence of regional lymph node metastasis at diagnosis, often associated with a less aggressive tumor phenotype [9, 10]. Yet, whether older patients have similar or shorter survival is up for debate and shows controversial results in different studies [11-13]. Ageing related physiological alterations and the duration of active smoking should be simultaneously taken into account. As shown in lung cancer the onset of smoking is usually in www.impactjournals.com/oncotarget Oncotarget 69188 Increase of mutation frequency with age When looking at the mutations in each patient, a wide range in numbers along with a single extreme value in one patient (age 69) was observed (Figure S1). i A significant rise (p < 0.01) in the average number of mutations in different genes was observed with increasing age (Figure 2), which was also true if the patient with the extreme number of mutations was removed. Interestingly, we repeated the mutation load analysis excluding silent mutations and multiple mutations in one gene, to have an additional overview of disruptive mutations with increasing age. Although the number of mutations in each patient decrease drastically, the same significant p-value www.impactjournals.com/oncotarget Oncotarget 69189 O t 69190 ww impactjournals com/oncotarget Figure 1: CONSORT diagram of original and selected patient cohort. Figure 1: CONSORT diagram of original and selected patient cohort. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 69190 Table 1: Comparison of TCGA cohort and selected patients Characteristics TCGA (279 patients) Selected cohort (203 Patients) Age (years) Median 61 62 Range 19-90 19-87 Smoking history Yes 220 159 No 52 37 Unknown 7 7 Primary tumor location Oral Cavity 172 129 Larynx 72 64 Oropharynx 33 9 Hypopharynx 2 1 HPV/ p53 status HPV + / p53 + 35 0 HPV + / p53 - 1 0 HPV - / p53 + 40 0 HPV - / p53 - 203 203 was detected (Figure S2, list of mutation frequencies in Table S2). Thus, the number of mutated genes found in a tumor was higher in older patients than in younger patients, designating a quantitative difference in mutational load between patient ages irrespective of whether this signified a pure stochastic increase or the accumulation of disease relevant mutations. Interestingly, there were only five genes with recurrent mutations, three of which are well known players (TP53, CDKN2A, PIK3CA) and two are pseudogenes (RPSAP58, WASH3P). l of mutated genes. Unsupervised hierarchical clustering based on gene mutation frequencies was performed for patients pooled into age groups of decades aside two groups of the very young (pooled age 19-40) and very old (pooled ages 81 to 87). A minimum difference of 0.15 in mutation frequency between at least two age groups was considered for cluster analysis (Figure S3). This cut-off allowed for a comprehensive set of mutational patterns within the specific age groups including less frequently mutated genes. In order to investigate the influence of patients features such the tumor localization and the smoking consumption on the increasing of the average number of mutations with age, we repeated the regression analysis for each subset. Although it´s necessary to consider the different number of patients in each subset and the difficulty on the acquisition of smoking habits (possible false-positive/-negative), both smokers and non-smokers subsets showed a significant correlation (linear regression as well as Spearman’s rank correlation analysis), therefore the smoke history surprisingly seems not to influence the age-related mutational load of our sub-cohort. Whereas, the mutational load of our sub-cohort as well as the original cohort are mostly influenced by the location of the cancers, only oral cavity tumors showed a significant correlation with age (Table S1). www.impactjournals.com/oncotarget The results showed two relevant clusters of frequently mutated genes, in particular, 39 genes in very young patients and 108 genes in very old patients (Table S3 with cut clusters A and B from Figure 3, unmarked heatmap Figure S3). Both the young as well as the old group, each consisting of 11 patients, displayed prominent differences to middle age groups. The genes of the two specific clusters were tested for KEGG pathway enrichments using the online David Gene Ontology tool [21]. The old age gene cluster showed six statistically significant enriched pathways, two of which, the “Axon Guidance” (p < 0.003) and “ECM-Receptor Interaction” (p < 0.04) pathways, stood out due to their role in angiogenesis processes and cell / tissue architecture maintenance respectively (Table S4). Six “Axon Guidance” genes (SEMA5A, DCC, PLXNB2, UNC5D, ITGB1, EPHA2) and four of the “ECM-Receptor Interaction” pathway (LAMA2, LAMA4, TNC, ITGB1) were significantly enriched. In contrast, no enriched pathways were found for the young age gene cluster. To see if the very old and very young patients were homogenous groups or comprised of smaller subgroups we looked at both in more detail. Further division of the Relationship of mutational patterns and age As a next step, we wanted to investigate whether this increase was a mere accumulation of random mutations or whether we could find age-specific patterns www.impactjournals.com/oncotarget Oncotarget 69191 Note that we included six genes from the old ages clusters (CDKN2A, CSMD3, FAT1, NOTCH1, PIK3CA, TTN) that did make the 0.15 cutoff, but were in fact frequently mutated in most other ages as well. However, since the enrichment analysis showed significant p-values for both the set with as well as without the six genes we kept them in our gene set. i young age group into two fractions comprising ages 19 to 35 (7 patients) and 36 to 40 (4 patients) did yield an enrichment of the TGFB pathway for the latter age group (Figure S3, Table S4). This enrichment, however, was mainly based on the mutations of the enriched genes in one single patient (age 39). In addition, TGFβRII and TGFb1, two main players of the TGFB pathway and responsible for more aggressive tumors in HNSCCs were not mutated in the young ages at all [22]. We therefore did not interpret this finding as an age-specific enrichment. The “87” age group showed six significantly enriched pathways among which are again “ECM- Receptor Interaction” (p < 0.006, 11 genes) and “Axon Guidance” (p < 0.006, 13 genes), and in addition “Notch Signalling” (p < 0.007, 7 genes) as well as “Focal Adhesion” (p < 0.05, 15 genes) (Table S4). The 47 genes specifically mutated in the “81-85” group did not yield any enrichment. However, when combining all highly mutated genes in this group, “Axon Guidance” was enriched as well yet with a slightly elevated p-value (p = 0.056, 4 genes) (Table S4). The overlap of the two old age groups is only three genes (PLXNB2, SEMA5A, UNC5D), yet genes of the same families (such as Ephrins, Slits and Rho-associated protein kinases) were mutated in both age groups. Overall, the old age groups only revealed a certain degree of homogeneity, with the very old patients (age 87) showing distinct pathway enrichment. However, the high number of overlapping genes as well as the commonly Old age specific clusters and pathway enrichment The old age group, on the other hand, showed distinct clusters and enrichment when split into smaller fractions of ages 81 to 85 (7 patients) and 86 to 87 (4 patients, all age 87). Figure 4 shows a major cluster of 542 genes in the “87” age class (Cluster D) and two gene clusters in the “81-85” age group (Cluster A and B, unmarked heatmap Figure S5). The latter consisted of 47 specific mutations and 59 genes overlapping with the “87” age group, indicating a common genetic background of tumors in elderly patients (Table S3). Figure 2: Correlation of average mutations and age considering one mutation per gene. Upper graph number of patients in the respective age group. Lower graph, average number of mutations for all patients in the respective age groups. Linear regression analysis was done using F-statistics and shows a significant increase in mutations in older patients (p = 0.000161, adjusted r2 = 0.24). Grey area around regression line indicates 95% confidence interval. Regression without the patient having an extreme number of mutations (TCGA-D6-6516, see Figure S1) also yields a significant connection between average mutations and age p = 0.000291, r2 = 0.22 (data not shown). Figure 2: Correlation of average mutations and age considering one mutation per gene. Upper graph number of patients in the respective age group. Lower graph, average number of mutations for all patients in the respective age groups. Linear regression analysis was done using F-statistics and shows a significant increase in mutations in older patients (p = 0.000161, adjusted r2 = 0.24). Grey area around regression line indicates 95% confidence interval. Regression without the patient having an extreme number of mutations (TCGA-D6-6516, see Figure S1) also yields a significant connection between average mutations and age p = 0.000291, r2 = 0.22 (data not shown). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 69192 enriched pathways fostered the idea of specific mutational trends happening at the old age rather than one specific mutational pattern. addition to genes mutated at a high frequency we found a group of 10 genes mutated at lower frequencies in the “81-85” group (Figure 5). Mapping of “Axon Guidance” pathway genes to all age groups As the mapping of all genes to the “Axon Guidance” pathway had shown, we were missing mutated genes of a pathway that did not make the 0.15 frequency cutoff. However, since our results suggested that mutations in old patients did not necessarily follow a strict common pattern and different genes were affected, we did not expect all mutations in a pathway to occur at high frequencies. At the same time we had seen a common trend for both old age groups with impact of mutations on similar gene families. Thus we decided to merge the highly mutated genes of both separate age groups for a more comprehensive picture of affected pathways. We mapped all genes of the “Axon Guidance” pathway (according to the KEGG database) to our list of mutation frequencies to see if the pathway is affected in other age groups as well without being particularly enriched (Figure 5, Table S5). From the total 127 genes of the “Axon Guidance” pathway 99 mapped to our data. As the heatmap showed, mutations in this pathway were present in other ages as well, yet at a lower frequency than in the two old age groups. Again, the trend of increased pathway alterations towards old ages was visible. In Figure 3: Unsupervised hierarchical clustering of gene mutation frequencies of specific age groups with pooled young and old ages. Patients were grouped into age groups of very young (ages 19-40) and very old (ages 81-87) with 10-year bins in between, then clustered according to the mutations frequencies of the quantified genes. Only genes with a minimum frequency difference of 0.15 Figure 3: Unsupervised hierarchical clustering of gene mutation frequencies of specific age groups with pooled young and old ages. Patients were grouped into age groups of very young (ages 19-40) and very old (ages 81-87) with 10-year bins in between, then clustered according to the mutations frequencies of the quantified genes. Only genes with a minimum frequency difference of 0.15 between at least two of the age groups are displayed. Black boxes indicate extracted genes for A., age group 81 to 87 and B., age group 19 to 40 (for genes see Table S3). Figure 3: Unsupervised hierarchical clustering of gene mutation frequencies of specific age groups with pooled young and old ages. Mapping of “Axon Guidance” pathway genes to all age groups Patients were grouped into age groups of very young (ages 19-40) and very old (ages 81-87) with 10-year bins in between, then clustered according to the mutations frequencies of the quantified genes. Only genes with a minimum frequency difference of 0.15 between at least two of the age groups are displayed. Black boxes indicate extracted genes for A., age group 81 to 87 and B., age group 19 to 40 (for genes see Table S3). www.impactjournals.com/oncotarget Oncotarget 69193 Overall, we identified 24 genes mutated in the “Axon Guidance” pathway among the 81-87 group of patients. 14 genes resulted from the pathway enrichment analysis and 10 genes were additionally revealed by the pathway heatmap clusters (Table S9). Even though 79% (19 out of 24) carried a single mutation showing the tumor heterogeneity, in the end 82% of the old patient group “81-87” (9 out of 11) reported a disruption in the Axon Guidance pathway. Furthermore, 82% (9 out of 11) of the patients showed mutations in “ECM-Receptor Interaction” pathway and all patients reported mutations in “Focal Adhesion” pathway. In particular we identified 21 and 34 significantly mutated genes respectively, which overlapped in 20 genes (Table S9). Interestingly we found several mutated genes of the same protein families, six laminins (LAMA1, LAMA2, LAMA3, LAMA4, LAMC1, LAMB1), three integrins (ITGA11, ITGA2, ITGB1) and eight collagen mutate genes (COL1A1, COL11A1, Pathway enrichment analysis was performed on a consistent group of 589 mutated genes, many of which were hidden by the 0.15 clustering threshold when calculating the mutation frequency of the two old ages groups together, rather than separately.i The result showed 10 significantly enriched pathways (Table S4), two of which had been found enriched before, namely “Axon Guidance” (p < 0.004, 14 genes) and “ECM-Receptor Interaction” (p < 0.001, 13 genes). In addition, “Notch Signaling” (p < 0.002, 8 genes) and “Focal Adhesion” (p < 0.009, 18 genes) were selected for detailed analysis due to their important role on cell growth, cell / tissue architecture and cell motility. When mapping all pathway genes to our data, we saw in all three the same pattern as in the “Axon Guidance” pathway of sporadic mutations that occur in all ages, yet the frequency of mutated genes was much higher in the old age groups of 81 to 87 (Figure S4-S6, Table S6-S8). Mapping of “Axon Guidance” pathway genes to all age groups Figure 4: Unsupervised hierarchical clustering of gene mutation frequencies of specific age groups with separate old ages. Patients were grouped into age groups of very young (ages 19-40), decades in-between and two separate old ages groups (ages 81-85 and 87), then clustered according to the mutations frequencies of the quantified genes. Only genes with a minimum frequency difference of 0.15 between two of the age groups are displayed. Black boxes indicate extracted genes for A., age group 81 to 85 upper cluster (shared with ages 86 to 87), B., age group 81 to 85 lower cluster (separate genes from ages 86 to 87), C., two clusters of ages 19 to 40 and D., ages 86 to 87 (for genes see Table S3). Figure 4: Unsupervised hierarchical clustering of gene mutation frequencies of specific age groups with separate old ages. Patients were grouped into age groups of very young (ages 19-40), decades in-between and two separate old ages groups (ages 81-85 and 87), then clustered according to the mutations frequencies of the quantified genes. Only genes with a minimum frequency difference of 0.15 between two of the age groups are displayed. Black boxes indicate extracted genes for A., age group 81 to 85 upper cluster (shared with ages 86 to 87), B., age group 81 to 85 lower cluster (separate genes from ages 86 to 87), C., two clusters of ages 19 to 40 and D., ages 86 to 87 (for genes see Table S3). www.impactjournals.com/oncotarget Oncotarget 69194 COL29A1, COL6A3, COL27A1, COL11A1, COL4A5, COL4A4), crucial in ECM-signaling processes and focal adhesions, as well as six ephrin genes (EPHA2, EPHA3, EPHA5, EPHA6, EPHA8, EPHB6), which have a central role in “Axon Guidance” signalling processes. Lastly, 64% of the patients (7 out of 11) displayed mutations in “Notch-Signaling” pathway. We found 10 mutated genes, three of which were NOTCH genes (NOTCH1, NOTCH3, NOTCH4). genetic events in the elderly such as prolonged tobacco exposure and ageing-related genomic instability. To provide better insight into the underlying mechanisms, we therefore investigated whether a mere accumulation of random mutations or distinctive mutational patterns are related to patient age. Unsupervised clustering of the selected cohort showed distinct clusters of genes expressed with a higher mutation frequency for the very young and very old ages. Mapping of “Axon Guidance” pathway genes to all age groups While the young age group did not reveal pathway enrichment, the old age group showed enrichment of KEGG pathways, including “ECM-Receptor Interaction ” and “Axon Guidance”. Further division of the old age group into ages 81 to 85 and age 87 showed several genes of the same pathways shared by the two groups. Which when clustered together showed ten enriched pathways. Besides “Axon Guidance” and “ECM-Receptor Interaction”, “Notch-Signaling” and “Focal Adhesion” were of special interest to us. When mapping all genes of the respective pathways to our data, we saw mutations in all patients, however no evident clusters were present other than for ages 81 to 87. These results indicate that while the specific mutational patterns might only exist in very subtle ways due to the heterogeneity of the tumors, Altogether, we only saw a few genes recurrently mutated and thus no age specific mutational pattern. However, even though not always the same genes were affected, the mutations accumulated in the same pathways, indicating a common trend in elderly patient to have similar functions of the cell changed by mutations. DISCUSSION In the present study, we evaluated the somatic DNA single nucleotide polymorphisms (SNPs) landscape of a selected HNSCC patient cohort in relation to ageing processes. The average number of mutations for each patient showed a significant rise (p < 0.01) with increasing age. Multiple factors participate to the accumulation of Figure 5: Unsupervised hierarchical clustering of mutation frequencies of genes involved in the “Axon Guidance” pathway (according to the KEGG database). Patients were grouped into age groups of very young (ages 19-40), decades in- between and two separate old ages groups (ages 81-85 and 86 to 87), then clustered according to the mutation frequencies of all quantified genes of the “Axon Guidance” pathway. Genes that did not make the original cut off of 0.15 difference in mutation frequency but are found mutated in this pathway in age group 81-85 are LRRC4C, DPYSL2, EPHA8, LIMK1, NCK1, NGEF, RAC1, ROBO2, ROCK1 and SLIT2. Figure 5: Unsupervised hierarchical clustering of mutation frequencies of genes involved in the “Axon Guidance” pathway (according to the KEGG database). Patients were grouped into age groups of very young (ages 19-40), decades in- between and two separate old ages groups (ages 81-85 and 86 to 87), then clustered according to the mutation frequencies of all quantified genes of the “Axon Guidance” pathway. Genes that did not make the original cut off of 0.15 difference in mutation frequency but are found mutated in this pathway in age group 81-85 are LRRC4C, DPYSL2, EPHA8, LIMK1, NCK1, NGEF, RAC1, ROBO2, ROCK1 and SLIT2. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 69195 [41, 42]. Therefore mutations on upstream proteins like laminins and integrins again suggested a decreased cellular motility in HNSCC. we could see an increase in mutations in distinct pathways over the ages with a peak in the very old fraction. Next, we looked at the involved genes in more detail, starting with the “Axon Guidance” pathway. y NOTCH signalling is a highly conserved pathway that plays distinct roles during tissue homeostasis, proliferation and apoptosis [46]. Even though NOTCH1 is one of the most frequently mutated genes in HNSCC, there are contradictory studies about its influence on tumor development [20]. Loss-of-function mutations in the NOTCH1 gene have been detected in a significant proportion of patients [47]. On the other side it is known that NOTCH activation can enhance proliferation, inhibit apoptosis and promote angiogenesis [45, 46]. Thus, while we reported high mutation frequencies for 10 genes of the notch pathway (inter alia NOTCH1, NOTCH3, NOTCH4), we couldn´t make assumptions about the exact impact of this finding. p y Although “Axon Guidance” represents a key stage in the formation of neuronal networks, recent studies also linked this pathway to regulation of angiogenesis processes (endothelial cell migration, proliferation and vessel formation). Involved proteins are Netrins, Slit proteins, Semaphorins, Ephrins and their cognate receptors (e.g. UNC5, ROBO1-4), all of which are frequently mutated in the elderly groups [24, 25]. The expression of Eph receptors, five of which were mutated in our data, is frequently elevated in different types of malignant tumors possibly resulting in increased cellular motility, tumor cell invasion and metastasis [26, 27]. One of these, EPHA2, is overexpressed in squamous cell carcinoma of oral tongue [27] and was also protruding in our mutational analysis. Moreover, studies have proven that blocking EphA receptor signaling decreases tumor vascular density, volume and cell proliferation in vivo [28- 31]. Since we found many of these genes to be mutated in the old age group, we postulated a correlation between “Axon Guidance” aberrations and the HNSCC features of elderly patients which may result in decreased cellular motility and tumor cell invasion. Concluding, we found a proportional increase of the mutation frequency rate in relation to the age of the HNSCC patients. This increase, however, did not follow distinct mutational patterns but rather an accumulation of mutations in specific pathways. www.impactjournals.com/oncotarget The results of this pathway analysis suggested a reduced tumor invasiveness and metastasis in older patients, which was underlined by the tumor staging at diagnosis. This distinct mutational background might be relevant for treatment approaches decisions and should therefore be taken under closer consideration in future studies. y For the “ECM-Receptor Interaction” pathway, we identified 21 frequently mutated genes, including six laminins, three integrins and eight collagens. These molecules are structurally and functionally involved in interactions at the extracellular matrix (ECM) which lead to a direct or indirect control of cellular activities such as cell migration, differentiation, proliferation, and apoptosis [32, 33]. A re-expression of the laminin α2 and α4 chains, which were significantly mutated in our old patients group, could be shown in adult hyperproliferative, dysplastic and carcinomatous lesions [31, 32]. Several studies showed Laminin 332 (LAMA3, LAMB3 and LAMC2) to be highly expressed in HNSCC and foster tumor invasiveness, an effect that is reversed when the laminins are repressed by microRNA-29s [36-40]. Several other integrins composed by the Integrin β1 chain, highly mutated in patients of old age, have been identified as crucial for tumor cell invasion and angiogenesis [38, 39]. Altogether, the many mutations in this pathway again suggested a decreased cell migration and tumor invasion in old patients.i Clustering and pathway enrichment analysis Unsupervised hierarchical clustering based on gene mutation frequencies was performed for different age groups. Genes were clustered according to Euclidian distance measure using the method “complete”. Genes from age group specific clusters were extracted and tested using the online David Gene Ontology tool [21]. The full list of identified genes was used as background for enrichment calculation. In reverse, all genes of an enriched KEGG pathway were mapped to our list of mutation frequencies to investigate the number and distribution of mutated genes in the respective pathway within all ages of our dataset. 8. El-Mofty SK, Lu DW. Prevalence of human papillomavirus type 16 DNA in squamous cell carcinoma of the palatine tonsil, and not the oral cavity, in young patients: a distinct clinicopathologic and molecular disease entity. Am J Surg Pathol. 2003; 27(11): 1463–1470. doi:10.1097/00000478- 200311000-00010. 9. Koch WM, Patel H, Brennan J, Boyle JO, Sidransky D. Squamous cell carcinoma of the head and neck in the elderly. Arch Otolaryngol Head Neck Surg. 1995; 121(3): 262–265. doi:10.1016/j.bjoms.2005.05.011. 10. Ershler WB. Why tumors grow more slowly in old people. J Natl Cancer Inst. 1986; 77(4): 837–839. REFERENCES first evaluation of the mutational rate related to age we considered all mutations, including silent mutations as well as multiple mutations in one gene. For all subsequent analysis, silent mutations were removed and multiple mutations in the same gene were only considered once per patient to prevent statistical overestimations. 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010; 127(12): 2893– 2917. doi:10.1002/ijc.25516. 2. Keck MK, Zuo Z, Khattri A, Stricker TP, Brown CD, Imanguli M, Rieke D, Endhardt K, Fang P, Bragelmann J, DeBoer R, El-Dinali M, Aktolga S, et al. Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-HPV subtypes. Clin Cancer Res. 2014; 21(4): 870–881. doi:10.1158/1078-0432.CCR- 14-2481. Mutation frequency and age of patients The primary statistical hypothesis was, that there would be an increase in the number of mutations with increasing age. The relations between total average mutation frequencies and patient age groups (in years) were calculated by linear regression using F-statistics. A two-sided p-value of below 0.05 (Pr(>\|t\|)) was considered significant. The frequency of mutation of a specific gene was calculated using the sum of mutations in a specific age group divided by the number of patients in the respective age group. Additional Spearman’s rank correlation analysis was performed to identify the genes whose mutation frequency correlates with the age (Table S10). 3. Leemans CR, Braakhuis BJM, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011; 11(1): 9–22. doi:10.1038/nrc2982. 4. Fakhry C, Cohen E. The rise of HPV-positive oropharyngeal cancers in the United States. Cancer Prev Res. 2015; 8(1): 9–11. doi:10.1158/1940-6207.CAPR-14-0425. 5. Field N, Lechner M. Exploring the implications of HPV infection for head and neck cancer. Sex Transm Infect. 2015; 0(0): 2014–2016. doi:10.1136/sextrans-2014-051808. 6. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, Jiang B, Goodman MT, Sibug-Saber M, Cozen W, Liu L, Lynch CF, Wentzensen N, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol. 2011; 29(32): 4294– 4301. doi:10.1200/JCO.2011.36.4596. All data analysis was done using R [52] unless stated otherwise. Acquisition and processing of data The clinical data of the TCGA patient set was derived via download from the TCGA data matrix (https://tcga-data.nci.nih.gov/tcga/dataAccessMatrix. htm). Somatic mutations of HNSCCs from the TCGA study was derived by download from the cBio Portal [18, 19]. The somatic mutations of the 279 HNSCC patients detected by exome sequencing within the TCGA project [16] were merged with the clinical data from TCGA to combine genomic mutations with the age information of all patients. Entries without official gene names were removed. Since we were interested in patients with a HPV negative genomic background, we excluded all patients with a positive HPV status according to the standards of the TCGA publication. As HPV determination has certain limitations [16], it remained uncertain, whether all tumors classified as HPV negative were truly negative, or whether diagnostic sensitivity may have misclassified some cases. Therefore, we selected for patients with at least one mutation in TP53 for our study, as TP53 mutations are generally not found in HPV positive tumors. For a The clinical data of the TCGA patient set was derived via download from the TCGA data matrix (https://tcga-data.nci.nih.gov/tcga/dataAccessMatrix. The “Focal Adhesion” pathway was significantly enriched in the 81-87 age group as well. We identified 34 genes, 20 of which overlap with the “ECM-Receptor Interaction” pathway, in particular laminins and integrins. The latter regulate kinases, such as the focal adhesion kinase (FAK), which is crucial for the attachment and signal transduction between cells and the ECM [43]. 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https://openalex.org/W1977083964	http://digital.bl.fcen.uba.ar/Download/paper/paper_19326203_v2_n10_p_Strier.pdf	English	null	Turing Patterns Inside Cells	PloS one	2,007	cc-by	4,681	INTRODUCTION More specifically, we show the existence of cell-sized Turing patterns in a model of glycolysis, using realistic parameter values and equal diffusion coefficients of ATP and ADP. In this paper we overcome the drawbacks of these previous works by showing that the full 5-variable Selkov model describing the PFK- controlled steps of the glycolytic pathway supports Turing patterns for realistic reaction rates and equal diffusivities of ATP and ADP. We show that Turing structures of subcellular size (10 mm) may be found by increasing the glycolytic flux and the enzyme concentration while keeping fixed the set of kinetic constants that give good agreement with the classic experimental results on homogeneous oscillations in The general concept behind Turing patterns involves a combi- nation of short-range activation and large-range inhibition [9]. Several reaction-diffusion models have been proposed in the literature to explain, among others, coat patterns in mammals [9], skin patterns in fish [10] or shell patterns in mollusks [11]. However, none of these models identifies the ‘‘morphogens’’ involved and some of the reaction schemes lack a biological motivation. This gives almost unrestricted freedom to choose kinetic parameters and diffusion ratios. Thus, the astonishing visual similarities often observed between the predicted and the real patterns might be unrelated to the actual biological phenomena. Academic Editor: Nick Monk, University of Nottingham, United Kingdom Received August 22, 2007; Accepted September 23, 2007; Published October 17, 2007 One of the early attempts to look for an example of a symmetry breaking mechanism in biology was due to I. Prigogine et al. [12]. They analyzed a spatially extended version of the (adiabatically reduced) 2-variable Selkov model of glycolysis, derived in [13] (see also [14,15]). Namely, they added diffusion terms to each of the two differential equations describing the time evolution of the concen- trations of ATP and ADP. It followed from their study that Turing patterns could only exist provided that ATP and ADP diffused at sufficiently unequal rates. This condition seems difficult to be met a priori, given the similarities between ATP and ADP. More recently, Hasslacher et al. [16] presented numerical simulations of Turing Copyright: 2007 Strier, Ponce Dawson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. INTRODUCTION pattern formation in a closely related model [17] that were obtained assuming that ATP diffused 25 times faster than ADP. The authors gave a qualitative justification for their choice of diffusivities making an analogy between the role of enzymes in the glycolytic pathway and that of the immobile color indicators used for visualization in open chemical reactors [7,8] which effectively reduce the transport rate of one of the species involved in the reactions [18]. However, the argument that applied to the latter case cannot be translated to the case of the glycolytic pathway without a deeper analysis. Firstly, enzymes affect both the dynamics of ATP and ADP. Thus, the rescaling of diffusion coefficients may occur in such a way so as to prevent the formation of patterns. Second, when the 2-variable reaction-diffusion model is obtained by the adiabatic reduction of the larger set of equations in which the enzyme dynamics is explicitly considered [19], the elimination of the fast variables (the enzymes) introduces changes of the same order of magnitude in both the diffusion and the reaction terms [20]. Thus, both changes need to be considered simultaneously in order to analyze Turing pattern formation in this setting. Finally, this theoretical discussion remains meaningless unless reasonably sized patterns can be shown to exist for realistic values of the reaction rates and of the free diffusivities. Concentration gradients inside the cytosol are a vital piece of the cell’s machinery. They underlie morphogenesis [1], and are involved in cell migration [2], cell growth [3], cell division [4], and the mechanical responses that follow fertilization, among other functions [5]. Stationary concentration gradients can spontane- ously emerge from a homogeneous background in the presence of instabilities. In 1952 Alan Turing proposed a hypothetical sequential route for cell differentiation, based on a reaction- diffusion process [6]. Starting from a spatially uniform state he proved that stable inhomogeneities in ‘‘morphogen’’ concentration could spontaneously emerge through a diffusion-driven symmetry- breaking instability. His highly idealized model stimulated a large amount of work which eventually led to the observation of Turing patterns in open chemical reactors [7,8]. Nevertheless, the occurrence of Turing patterns has not been unequivocally proven for a biochemical reaction in which reaction rates, concentrations and diffusion coefficients are within realistic physiological values. We do so in this paper. Turing Patterns Inside Cells Damia´n E. Strier1, Silvina Ponce Dawson2* 1 Service de Chimie Physique and Center for Nonlinear Phenomena and Complex Systems, Universite´ Libre de Bruxelles, Brussels, Belgium, 2 Departamento de Fı´sica, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Pabello´n I, University of Buenos Aires, Buenos Aires, Argentina Concentration gradients inside cells are involved in key processes such as cell division and morphogenesis. Here we show that a model of the enzymatic step catalized by phosphofructokinase (PFK), a step which is responsible for the appearance of homogeneous oscillations in the glycolytic pathway, displays Turing patterns with an intrinsic length-scale that is smaller than a typical cell size. All the parameter values are fully consistent with classic experiments on glycolytic oscillations and equal diffusion coefficients are assumed for ATP and ADP. We identify the enzyme concentration and the glycolytic flux as the possible regulators of the pattern. To the best of our knowledge, this is the first closed example of Turing pattern formation in a model of a vital step of the cell metabolism, with a built-in mechanism for changing the diffusion length of the reactants, and with parameter values that are compatible with experiments. Turing patterns inside cells could provide a check-point that combines mechanical and biochemical information to trigger events during the cell division process. Citation: Strier DE, Ponce Dawson S (2007) Turing Patterns Inside Cells. PLoS ONE 2(10): e1053. doi:10.1371/journal.pone.0001053 Citation: Strier DE, Ponce Dawson S (2007) Turing Patterns Inside Cells. PLoS ONE 2(10): e1053. doi:10.1371/journa Received August 22, 2007; Accepted September 23, 2007; Published October 17, 2007 Academic Editor: Nick Monk, University of Nottingham, United Kingdom * To whom correspondence should be addressed. E-mail: silvina@df.uba.ar PLoS ONE \| www.plosone.org RESULTS It is this activating step of the allosteric enzyme which provides the mechanism responsible for the instabilities. We assume that ATP and ADP diffuse while the enzyme and its complexes are immobile, due to the larger mass. We also assume that, initially, all concentrations are spatially uniform. Using mass action kinetics, the reaction-diffusion equations describing this model can be written, in dimensionless form, as: Ls1 Lt ~n{(1zK1)s1u1zK1u2zd1+2s1, Ls2 Lt ~a u2{cK3sc 2u3zcK3u1 {gs2zd2+2s2, e du1 dt ~u2{s1u1z K3 1zK1 sc 2u3{u1 , e du2 dt ~s1u1{u2, ð2Þ ð2Þ where the dimensionless concentrations are: where the dimensionless concentrations are: s1:kz1½S1=(kz2zk{1), s2:(kz3=k{3)1=c½S2, u1:½ESc 2=e0, u2: S1ESc 2 =e0 u3~1{(u1zu2), ð3Þ s1:kz1½S1=(kz2zk{1), s2:(kz3=k{3)1=c½S2, kz2&178 s{1, 1zK1 kz1 &30 mMs, kz3 k{3 1=2 &2:9\|10{3 mM{1: ð6Þ ð3Þ ð6Þ with e0, the total concentration of enzyme which remains constant and uniform throughout the evolution. The other quantities are with e0, the total concentration of enzyme which remains constant and uniform throughout the evolution. The other quantities are These numbers imply that, at the Hopf bifurcation, the stationary homogeneous solution of Eqs. (2) occurs at [S1],150 mM and [S2],145 mM, and that the dimensional period of the oscillations is T,2.7 min, which agrees with the experimentally determined values. We show the oscillatory behavior of [ATP] and [ADP] close to this bifurcation in Fig. 1 (a). These numbers imply that, at the Hopf bifurcation, the stationary homogeneous solution of Eqs. (2) occurs at [S1],150 mM and [S2],145 mM, and that the dimensional period of the oscillations is T,2.7 min, which agrees with the experimentally determined values. We show the oscillatory behavior of [ATP] and [ADP] close to this bifurcation in Fig. 1 (a). g: kz2zk{1 e0kz1kz2 n2, n: n1 kz2e0 , e: e0kz1kz2 (kz2zk{1)2 , a: kz2zk{1 kz1 kz3 k{3 1=c , K1: k{1 kz2 , K3: k{3 kz2 : ð4Þ Given these previous estimates, we explore the behavior of Eqs. (2), in the spatially extended case, varying the parameters n, g, and e and keeping the purely kinetic constants, a, K1 and K3 fixed at the previously mentioned values. We obtain the set of parameter values for which the homogeneous stationary solution of Eqs. (1) is unstable against spatially inhomogeneous perturbations (i.e., the Turing space) by analyzing the dispersion relation of the linearized evolution equations. We show in Fig. RESULTS The 5-variable Selkov model reads [13]: ? n1 S1zESc 2 < kz1 k{1 S1ESc 2, S1ESc 2 ? kz2 ESc 2zS2 ? n2 , cS2zE < kz3 k{3 ESc 2, ð1Þ ð1Þ We now constrain the values of the various parameters of Eqs. (2) according to previous estimations and measurements [13,15,26,21,22,23]. Under the experimental conditions of [22], in which experiments are done using yeast extracts, the values of n1 and n2 at which the oscillations start are n1,5.8 mMs21 and n2,0.04 s21 [15]. The enzyme is very diluted in these experi- ments, with e0* between 3 and 10 mM, the average concentrations of ATP and ADP are [S1] = 630 mM and [S2] = 150 mM, respectively, while the period of the oscillations, T, is between 3 and 5 min [15]. It has been shown unequivocally that the transition to oscillatory behavior in glycolysis is due to a Hopf bifurcation [24]. This means that the transition can be generically encountered by varying only one parameter. Equations (2) have a single homogeneous fixed point solution that depends on several parameters, which indeed undergoes a Hopf bifurcation. Thus, there are various ways by which this bifurcation can be reached. Among them, we choose the set of parameter values that are compatible with the observed frequency of oscillations and ATP and ADP concentrations. In particular, we find that at g = g* = 0.15, n = n<0.0041, e = e = 1026, a = 15, K1 = 1500, K3 = 1, a Hopf bifurcation occurs for the set of Eqs. (2) in the spatially homogeneous case. Using the definitions of these dimensionless quantities in terms of dimensional ones and the experimentally determined values, n1,5.8 mMs21 and n2,0.04 s21, we obtain that the total amount of enzyme at the Hopf bifurcation is e0 = e0,7.9 mM and that the various reaction rates satisfy where E is the free enzyme (PFK), which can form the complexes, ES2 c and S1ES2 c; the substrate, S1 (ATP) is supplied by an external source at the rate, n1, and the product, S2 c (ADP) is removed by a first order reaction at rate n2[S2]. From scheme (1) it is clear that the enzyme is inactive unless it has c product molecules bound, forming the complex ES2 c. As done in [13], we set c = 2 all throughout this paper. Turing Patterns inside Cells The dimensionless space and time coordinates are obtained by dividing the dimensional ones by an arbitrary length-scale, L, and yeast extracts [21,22,23,24,14]. The emergence of these patterns can be traced back to the differential interactions of ATP and ADP with PFK and its complexes. We then conclude that the key enzymatic step responsible for glycolytic oscillations may also provide a robust mechanism for the formation of steady state inhomogeneities in the concentration of ATP and ADP at the cellular and supracellular level. T: kz2zk{1 e0kz1kz2 , ð5Þ ð5Þ respectively. Assuming that the diffusion coefficients of ATP (D1) and ADP (D2) are equal (D;D1 = D2), we take L;10(DT)1/2. Using the diffusion coefficients in vivo, D,150 mm2 s21 [25], we obtain L = 122 mm (T s21)K. while the dimensionless diffusion coefficients are d1 = d2 = 0.01. The relatively small amount of enzyme used in experiments implies that e%1. For this reason, a quasi-steady state approximation of Eqs. (2) was analyzed in [13] considering, furthermore, spatially uniform concentrations. INTRODUCTION Funding: This research is supported by UBACyT X099, CONICET PIP 5131, PICT 17- 21001 of ANPCyT (Argentina) and Santa Fe Institute. Sponsors played no role in the design and conduct of this study. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Interests: The authors have declared that no competing interests exi To whom correspondence should be addressed. E-mail: silvina@df.uba.ar * To whom correspondence should be addressed. E-mail: silvina@df.uba.ar PLoS ONE \| www.plosone.org October 2007 \| Issue 10 \| e1053 October 2007 \| Issue 10 \| e1053 1 Issue 10 \| e1053 Turing Patterns inside Cells Turing Patterns inside Cells Turing Patterns inside Cells PLoS ONE \| www.plosone.org RESULTS 1(b) the (dimensionless) linear growth rate, q, of the unstable modes as a function of the square of the (dimensionless) wavenumber, k, for n = 0.03, g = 1.215, e = 0.0003. There is a bounded band of unstable ð4Þ PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org October 2007 \| Issue 10 \| e1053 October 2007 \| Issue 10 \| e1053 2 Turing Patterns inside Cells Figure 1. Behaviors predicted by the 5-variable Selkov model for different parameter values. (a) Glycolytic oscillations in S1 (solid curve) and S2 (dashed curve) for g = 0.15, n = 0.00345, e = 1026, a = 15, K1 = 1500, K3 = 1. (b) Linear growth rate of the unstable modes as a function of the square of the wavenumber, k for g = 1.215, n = 0.03, e = 0.0003, a = 15, K1 = 1500, K3 = 1, and d1 = d2 = 0.01. Inset: Evolution of [S1] in the spatially homogeneous case for the same parameter values. (c) Turing space (shadowed domain) as a function of the (dimension- less) input and output rates of ATP (n) and ADP (g), for the same parameter values as in (b). (d) Predicted value of the wave-length of the most unstable mode at each point in the Turing space of (c). doi:10.1371/journal.pone.0001053.g001 Figure 2. Turing pattern obtained with the 5-variable Selkov model in two space dimensions. Stationary pattern in [ATP] achieved after 10 min from an initial condition randomly distributed around the Turing-unstable fixed point. White corresponds to [ATP] = 2.47 mM and black to 1.1 mM. The simulation was done for g = 1.3, n = 0.0175, e = 0.0005, a = 15, K1 = 1500, K3 = 1, and d1 = d2 = 0.01. doi:10.1371/journal.pone.0001053.g002 Figure 2. Turing pattern obtained with the 5-variable Selkov model in two space dimensions. Stationary pattern in [ATP] achieved after 10 min from an initial condition randomly distributed around the Turing-unstable fixed point. White corresponds to [ATP] = 2.47 mM and black to 1.1 mM. The simulation was done for g = 1.3, n = 0.0175, e = 0.0005, a = 15, K1 = 1500, K3 = 1, and d1 = d2 = 0.01. doi:10.1371/journal.pone.0001053.g002 Figure 1. Behaviors predicted by the 5-variable Selkov model for different parameter values. Conlusions In this paper we have provided the first closed example of Turing pattern formation in a model of a vital piece of a cell’s real biochemistry, with a built-in mechanism for the change of the morphogens diffusion length, and with parameter values that are compatible with experiments. Our results suggest that the pattern of enzyme regulation that gives rise to the glycolytic oscillations may also provide the basis for the formation of stationary spatial structures both at the cellular and supracellular level. The model we have used is highly idealized and cannot account for certain observations. However, we think that some of its basic dynamical features should be common to those of more sophisticated models [22,28] and of the real system. In particular, it does reproduce the oscillations that are observed for dilute enzyme concentrations [22,23], and has helped the finding of rotating spirals in vitro [29]. There are studies that show that the Turing and Hopf bifurcations are intimately related in systems with immobile species [30]. Thus, we expect all these models to share the existence of a Turing bifurcation at large enough (immobile) enzyme concentration and a Hopf bifurcation at lower ones. Our study has shown that the interactions involved in the PFK catalyzed step of the glycolytic pathway change the ‘‘effective’’ diffusion coefficients of ATP and ADP in the necessary direction for Turing pattern formation. These results could be tested in the type of open reactors that have especially been conceived for the investigation of spatio-temporal dynamics in glycolysis [31]. We finally integrate numerically Eqs. (2) in a square domain of side 8L = 84.5 mm with periodic boundary conditions, using a finite-difference scheme on a 1506150 square grid. Fig. 2 shows the resulting pattern in S1 after 10 min have passed since the initial situation, at which the concentrations are given by the homoge- neous stationary solution ([S1] = 2.4 mM and [S2] = 71 mM), with 10% added noise. The typical size of the pattern agrees with the critical wavelength of the linear stability analysis, lc <12 mm. The simulation shows that Eqs. (2) support stable Turing patterns with equal diffusion coefficients of ATP and ADP. The existence of these patterns can be understood in terms of the reduced set of equations obtained in the quasi-steady state approximation [19,20,27]. DISCUSSION Conlusions modes for k?0, while for k = 0 (see the inset) the fixed point is stable. We show in Fig. 1(c) the Turing space on the (n,g) plane for e = 0.0003. As it may be observed, patterns may exist for larger values of n1 as n2 also gets larger, i.e., as the glycolytic flux increases. A similar picture holds on the (e,g) plane (data not shown): no pattern is possible at low enzyme concentration (if e#1024). We show in Fig. 1(d) the wave-length of the most unstable mode, lc, when g and n are varied in the shaded region of Fig. 1(c). We then conclude that the characteristic size gets smaller as the rate of product removal, n2, becomes larger. We observe that, for the parameter values considered, the length-scale is always less than 23 mm, so that it can fit inside a typical cell. Using the definition of e with the value of K1 = 1500 and the previous rate constant estimates (Eqs. (6)) obtained at the Hopf bifurcation, which we assume remain fixed, we conclude that e0$800 mM for the Turing instability to occur. As we discuss later, the patterns arise due to the effective rescaling of the diffusion coefficients of ADP and ATP that the enzyme produces. This rescaling gets smeared out as e0 decreases, leading, in turn, to the disappearance of the patterns. modes for k?0, while for k = 0 (see the inset) the fixed point is stable. We show in Fig. 1(c) the Turing space on the (n,g) plane for e = 0.0003. As it may be observed, patterns may exist for larger values of n1 as n2 also gets larger, i.e., as the glycolytic flux increases. A similar picture holds on the (e,g) plane (data not shown): no pattern is possible at low enzyme concentration (if e#1024). We show in Fig. 1(d) the wave-length of the most unstable mode, lc, when g and n are varied in the shaded region of Fig. 1(c). We then conclude that the characteristic size gets smaller as the rate of product removal, n2, becomes larger. We observe that, for the parameter values considered, the length-scale is always less than 23 mm, so that it can fit inside a typical cell. Using the definition of e with the value of K1 = 1500 and the previous rate constant estimates (Eqs. RESULTS (a) Glycolytic oscillations in S1 (solid curve) and S2 (dashed curve) for g = 0.15, n = 0.00345, e = 1026, a = 15, K1 = 1500, K3 = 1. (b) Linear growth rate of the unstable modes as a function of the square of the wavenumber, k for g = 1.215, n = 0.03, e = 0.0003, a = 15, K1 = 1500, K3 = 1, and d1 = d2 = 0.01. Inset: Evolution of [S1] in the spatially homogeneous case for the same parameter values. (c) Turing space (shadowed domain) as a function of the (dimension- less) input and output rates of ATP (n) and ADP (g), for the same parameter values as in (b). (d) Predicted value of the wave-length of the most unstable mode at each point in the Turing space of (c). doi:10.1371/journal.pone.0001053.g001 scheme, ATP cannot bind to the immobile activated complex unless c = 2 ADP molecules are already bound. Thus, the effective diffusivity of ADP –which plays the role of the activator of the step– is reduced by a larger amount than that of ATP. DISCUSSION Conlusions (6)) obtained at the Hopf bifurcation, which we assume remain fixed, we conclude that e0$800 mM for the Turing instability to occur. As we discuss later, the patterns arise due to the effective rescaling of the diffusion coefficients of ADP and ATP that the enzyme produces. This rescaling gets smeared out as e0 decreases, leading, in turn, to the disappearance of the patterns. ACKNOWLEDGMENTS This paper is dedicated to B. Hasslacher who recently passed away and whose pioneering ideas were a key factor for the development of the present work. We also acknowledge useful conversations with G. B. Mindlin, J.E. Pearson and A. Goldbeter. REFERENCES 1. Lawrence PA (1992) The Making of a Fly: the genetics of animal design. Boston: Blackwell Scientific Publications. 16. Hasslacher B, Kapral R, Lawniczak A (1993) Molecular Turing structures in the biochemistry of the cell. Chaos 3: 7–13. y 17. Richter PH, Rehmus P, Ross J (1981) Control and dissipation in oscillatory chemical engines. Prog Theor Phys 66: 385–405. 2. Xu H-T, Yuan X-B, Guan C-B, Duan S, Wu C-P, et al. (2004) Calcium signaling in chemorepellant Slit2-dependent regulation of neuronal migration. Proc Natl Acad Sci USA 101: 4296–4301. g g y 18. Lengyel I, Epstein I (1991) Modeling of Turing Structures in the Chlorite— Iodide—Malonic Acid—Starch Reaction System. Science 251: 650–652. g g y 18. Lengyel I, Epstein I (1991) Modeling of Turing Structures in t Iodide—Malonic Acid—Starch Reaction System. Science 251: 6 3. 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Annu Rev Biochem 40: 237–258. 7. Castets V, Dulos E, Boissonade J, De Kepper P (1990) Experimental evidence of a sustained standing Turing-type nonequilibrium chemical pattern. Phys Rev Lett 64: 2953–2956. 23. Boiteux A, Goldbeter A, Hess B (1975) Control of Oscillating Glycolysis of Yeast by Stochastic, Periodic, and Steady Source of Substrate: A Model and Experimental Study. Proc Natl Acad Sci USA 72: 3829–3833. 8. Ouyang Q, Swinney H (1991) Transition from a uniform state to hexagonal and striped Turing patterns. Nature 352: 610–612. striped Turing patterns. Nature 352: 610–612. 24. Conlusions The effective diffusion coefficients of ATP and ADP that are obtained in this approximation are different due to the different interaction of ATP and ADP with the enzymes that belong to the pathway. In this We have also found that the patterns can fit inside a typical cell and that the time it takes for the patterns to form is relatively short (of the order of minutes). The formation of Turing patterns in this biochemical pathway could then be related to organizing centers in eukaryotic cells, playing a role during cell division [4]. The fact that the Turing patterns have an intrinsic length-scale implies that there could be zero, one or several spots of high [ATP] inside the cell, depending on the relationship between the cell and the pattern sizes (see Fig. 2). In particular, there are two properties October 2007 \| Issue 10 \| e1053 PLoS ONE \| www.plosone.org 3 Turing Patterns inside Cells evolved after the appearance of the first eukaryotic cell. The pathway should be old and highly conserved. The glycolytic pathway shares both properties. which imply that the number of high [ATP] spots could change during the cell division cycle. First, the pattern size decreases as the strength of the glycolitic flux increase. This flux must increase during interphase for biosynthesis and growth. Thus, the pattern size might become small enough to fit inside the cell only after the glycolytic flux has increased sufficiently. The change of the cell size also acts in the same direction, allowing more ‘‘room’’ for more high [ATP] spots to fit as the cell grows during interphase. In this way, the change in the number of high [ATP] spots as the cell grows could provide a check-point that combines mechanical information (cell size) and biochemistry to trigger the subsequent chain of events in the cell division process. If a key stage of the replicating machine of every eukaryotic cell strongly relies on a metabolic pathway, it would be unlikely that such pathway had Author Contributions Conceived and designed the experiments: SP. Performed the experiments: DS. Analyzed the data: DS. Wrote the paper: SP DS. Conceived and designed the experiments: SP. Performed the experiments: DS. Analyzed the data: DS. Wrote the paper: SP DS. REFERENCES Dano S, Sørensen PG, Hynne F (1999) Sustained oscillations in living cells. Nature 402: 320–322. 9. Murray JD (1989) Mathematical Biology. 12 York: Springer. 10. Kondo S, Asai R (1995) A Reaction-Diffusion Wave on the Skin of the Marine Angelfish Pomacanthus. Nature 376: 765–767. 25. Bezrukov SM, Vodyanoy I, Parsegian VA (1994) Counting polymers moving through a single ion channel. Nature 370: 279–281. 11. Meinhardt H (1999) On pattern and growth. In: Chaplain MAJ, Singh GD, McLachlan JC, eds (1999) On Growth and Form: Spatio-temporal Pattern Formation in Biology. London: John Wiley & Sons. pp 129–148. g g 26. Pye EK, Chance B (1966) Proc Nat Acad Sci USA 55: 888–894. 27. Strier DE (2002) PhD thesis, Universidad de Buenos Aires. 28. Goldbeter A, Lefever R (1972) Dissipative structures for an allosteric model. Biophys J 12: 1302–1315. 12. Prigogine I, Lefever R, Goldbeter A, Herschkowitz-Kaufman M (1969) Symmetry Breaking Instabilities in Biological Systems. Nature 223: 913–916. p y 29. Mair T, Muller SC (1996) Traveling NADH and proton waves during oscillatory glycolysis in vitro. J Biol Chem 271: 627–630. 13. Selkov EE (1968) On the Mechanism of Single-Frequency Self-Oscillations in Glycolysis. I. A Simple Kinetic Model. Eur J Biochem 4: 79–86. 13. Selkov EE (1968) On the Mechanism of Single-Frequency Self- Glycolysis. I. A Simple Kinetic Model. Eur J Biochem 4: 79–86. 30. Pearson JE, Bruno W (1992) Pattern Formation in an N+Q Component Reaction-Diffusion System. Chaos 2: 513–524. 14. Goldbeter A (1997) Biochemical Oscillations and Cellular Rhythms: The Molecular Bases of Periodic and Chaotic Behaviour. Cambridge: Cambridge University Press. y 31. Bagyan S, Mair T, Dulos E, Boissonade J, DeKepper P, et al. (2005) Glycolytic oscillations and waves in an open spatial reactor: Impact of feed-back regulation of phosphofructokinase. Biophys Chem 116: S. 67–76. 15. Selkov EE (1968) Mol Biol 2: 252–266 (English Translation). 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https://openalex.org/W3033941268	https://europepmc.org/articles/pmc7286131?pdf=render	English	null	MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing	Frontiers in microbiology	2,020	cc-by	14,231	MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing Leon Tribolet1†, Emily Kerr2†, Christopher Cowled1, Andrew G. D. Bean1, Cameron R. Stewart1, Megan Dearnley3 and Ryan J. Farr3* 1 Health and Biosecurity, Australian Animal Health Laboratory, Commonwealth Scientiﬁc and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia, 2 Institute for Frontier Materials, Deakin University, Geelong, VIC, Australia, 3 Diagnostics, Surveillance and Response (DSR), Australian Animal Health Laboratory, Commonwealth Scientiﬁc and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia In the pursuit of improved diagnostic tests for infectious diseases, several classes of molecules have been scrutinized as prospective biomarkers. Small (18–22 nucleotide), non-coding RNA transcripts called microRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential, due to their role in numerous diseases, previously established methods for quantitation and their stability within bioﬂuids. Despite efforts to identify, characterize and apply miRNA signatures as diagnostic markers in a range of non-infectious diseases, their application in infectious disease has advanced relatively slowly. Here, we outline the beneﬁts that miRNA biomarkers offer to the diagnosis, management, and treatment of infectious diseases. Investigation of these novel biomarkers could advance the use of personalized medicine in infectious disease treatment, which raises important considerations for validating their use as diagnostic or prognostic markers. Finally, we discuss new and emerging miRNA detection platforms, with a focus on rapid, point-of-care testing, to evaluate the beneﬁts and obstacles of miRNA biomarkers for infectious disease. In the pursuit of improved diagnostic tests for infectious diseases, several classes of molecules have been scrutinized as prospective biomarkers. Small (18–22 nucleotide), non-coding RNA transcripts called microRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential, due to their role in numerous diseases, previously established methods for quantitation and their stability within bioﬂuids. Despite efforts to identify, characterize and apply miRNA signatures as diagnostic markers in a range of non-infectious diseases, their application in infectious disease has advanced relatively slowly. Here, we outline the beneﬁts that miRNA biomarkers offer to the diagnosis, management, and treatment of infectious diseases. Investigation of these novel biomarkers could advance the use of personalized medicine in infectious disease treatment, which raises important considerations for validating their use as diagnostic or prognostic markers. Finally, we discuss new and emerging miRNA detection platforms, with a focus on rapid, point-of-care testing, to evaluate the beneﬁts and obstacles of miRNA biomarkers for infectious disease. Edited by: Thomas Dandekar, Julius Maximilian University of Würzburg, Germany Reviewed by: Andrew John Percy, Cambridge Isotope Laboratories, Inc., United States Alina Deshpande, Los Alamos National Laboratory (DOE), United States Keywords: microRNAs, infectious disease, diagnostics, biomarkers, personalized medicine Correspondence: Ryan J. Farr ryan.farr@csiro.au Correspondence: Ryan J. Farr ryan.farr@csiro.au REVIEW REVIEW published: 03 June 2020 doi: 10.3389/fmicb.2020.01197 published: 03 June 2020 doi: 10.3389/fmicb.2020.01197 INFECTIOUS DISEASE DIAGNOSTICS: CURRENT OUTLOOK †These authors have contributed equally to this work Infectious diseases account for a large proportion of morbidity and 15% of total global mortality (WHO, 2018a). Due to the ease of global travel and potential for global health epidemics, there is a critical need for the early diagnosis of infected individuals to assist in containing the spread of disease. For many diseases early intervention and expedient treatment is required for a favorable prognosis. Sometimes clinical presentation or case history is suﬃciently diagnostic, however, choosing an appropriate course of treatment often relies upon the outcome of laboratory-based diagnostic testing. The clinical diagnostic landscape for the detection of infectious diseases is diverse and includes either looking for the pathogen or looking for the host’s response to the pathogen. Conventional laboratory tests include in vitro culture and isolation, protein-based assays (for example ELISA and serology), microscopy (histological, pathological, and morphological assays), mass spectrometry (Kullolli et al., 2014) and molecular diagnostics which uses nucleic acid-based assays (such as quantitative (q)PCR and sequencing). Many of these methods require a substantial amount of time to perform and are reliant on intensive sample preparation, expert users, and suﬀer from technical limitations. Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Received: 04 March 2020 Accepted: 12 May 2020 Published: 03 June 2020 Keywords: microRNAs, infectious disease, diagnostics, biomarkers, personalized medicine Tribolet L, Kerr E, Cowled C, Bean AGD, Stewart CR, Dearnley M and Farr RJ (2020) MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing. Front. Microbiol. 11:1197. doi: 10.3389/fmicb.2020.01197 Citation: Tribolet L, Kerr E, Cowled C, Bean AGD, Stewart CR, Dearnley M and Farr RJ (2020) MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing. Front. Microbiol. 11:1197. doi: 10.3389/fmicb.2020.01197 Diagnostic tests might perform within acceptance criteria yet give the wrong answer, resulting in either a false positive or false negative. This can be due to low sensitivity, low speciﬁcity, and/or June 2020 \| Volume 11 \| Article 1197 1 Frontiers in Microbiology \| www.frontiersin.org MicroRNA Biomarkers of Infectious Diseases Tribolet et al. not collecting a representative sample from the patient. In some cases, infections can be localized to speciﬁc cell types or tissues and a systemic sample, such as blood, may not contain detectable levels of the pathogen required for detection. In contrast, antibodies may be easily detected, however, antibody responses may take weeks to months to manifest, with the speciﬁcity of these responses sometimes hampered by cross- reactivity (Lanciotti et al., 2008). Antibody testing can be suitable for population level disease testing and analysis, to monitor epidemic/pandemic spread and to ascertain which patients have had prior exposure to a speciﬁc pathogen. Additionally, certain viruses, including rabies (Ito et al., 2016) and cytomegalovirus (Patro, 2019), eﬃciently modulate or evade the immune response, further complicating their detection. Tests for some pathogens are also ineﬀective during the early phases of infection. For example, the gold-standard diagnostic test for rabies (lyssavirus) infection is a ﬂuorescent antibody test that can only be performed on post-mortem brain tissue. Other classes of diagnostic molecules include other nucleic acids such as messenger RNA (mRNA) for the detection of urothelial carcinoma (Urquidi et al., 2016), proteins such as procalcitonin (PCT) interleukin-6 (IL- 6) and C-reactive protein (CRP) for sepsis (Lai et al., 2020) the presence of metabolites such as creatinine in serum or urea for renal disease (Stevens et al., 2006) or even presence of volatile compounds in breath analytics are being tested as cancer diagnostics (Markar et al., 2016). from chronic lymphocytic leukemia. Shortly after this, miRNA expression was found to change during tumorigenesis (Michael et al., 2003), and could be successfully used to classify multiple human cancers (Lu et al., 2005). Since then, studies have examined the potential of miRNAs as biomarkers of diabetes (Farr et al., 2013, 2015a,b), Alzheimer’s Disease (Lugli et al., 2015), and numerous other non-infectious conditions. Citation: Meta- analyses of multiple cancer studies strongly support a role for miRNAs as diagnostic, providing the ability to identify a disease (Zhi et al., 2015) or prognostic, identifying the likelihood of developing speciﬁc disease outcomes (Schmitz et al., 2016) biomarkers. Additionally, miRNAs may extend the clinical utility of current proteins or metabolite-based tests. For instance, a recent paper demonstrated that miR-29a and miR-335 in combination with matrix metalloprotease protein-2 (MMP2), proved to be a superior diagnostic in breast cancer to the current carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) tests that are widely used (Ali Ahmed et al., 2020). ) y ( ) Recently, several reports describe changes in circulating miRNAs in response to infectious diseases, raising the possibility for a new diagnostic tool against infections. MicroRNAs have been identiﬁed as potential biomarkers of infections caused by a range of pathogens, including Hendra virus (Stewart et al., 2013), HIV (Biswas et al., 2019), tuberculosis (Zhang et al., 2013), malaria (Li et al., 2018), including diﬀerentiating complicated and uncomplicated P. vivax malaria (Kaur et al., 2018) and Ebola (Duy et al., 2016). In some studies, changes in miRNA proﬁles were observed early in disease onset, before the pathogen could be directly detected and prior to the onset of seroconversion (Stewart et al., 2013; Biswas et al., 2019). MicroRNAs have also been implicated in inﬂuenza infections (Scheller et al., 2019) and rhinoviruses (Hasegawa et al., 2018). Therefore, the potential for miRNA diagnostics with other respiratory viruses, such as the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, is not to be understated. An identiﬁed and validated COVID-19 miRNA signature could provide diﬀerential diagnosis to identify COVID-19 infections from other infections with similar presenting symptoms such as inﬂuenza, rhinoviruses or other coronaviruses. Additionally, as mentioned above miRNAs have prognostic potential, to identify the likelihood of severe vs. mild disease outcomes and also to identify asymptomatic infections (Hou et al., 2017), which are problematic in epidemic or pandemic spread and disease tracking. In the search for new diagnostic biomarkers to circumvent these issues, many diﬀerent classes of molecules have been studied. Amongst the most promising are microRNAs (miRNAs), small (18–22nt) non-coding RNA molecules found within all bodily ﬂuids and tissues and most cell types (Halushka et al., 2018), which play an essential role in post-transcriptional regulation of gene expression. Citation: There are approximately 2,600 miRNAs in the human genome thus far according to the online miRNA repository, miRbase (version 221) and approximately 2,000 miRNAs in circulation (Juzenas et al., 2017). MicroRNAs are named with the miR preﬁx followed by an identifying number. If there are extremely similar sequences, additional suﬃxes (letters or numbers) are provided. Older nomenclature can also refer to a miRNA as the guide (used to identify target mRNA) or passenger (denoted with a ‘∗’ suﬃx) miRNA. As more studies demonstrated that both strands can be functional (Jin et al., 2015; Gao et al., 2020), this was replaced with the use of -3p and -5p suﬃxes, denoting the 3′ or 5′ end of the miRNA precursor. For an in-depth review of miRNA biogenesis and function, please see Saliminejad et al. (2019). The potential utility of these molecules as diagnostic biomarkers is enhanced by their presence and stability in a wide range of biological ﬂuids, including peripheral circulation. They show remarkable stability despite multiple freeze-thaw cycles or extreme pH (Chen et al., 2008) and are routinely measured via next generation sequencing (NGS) or qPCR (Farr et al., 2015a). Despite this, there are many infectious diseases for which miRNA biomarkers would be inappropriate, ineﬀectual, or highly diﬃcult to implement in clinical practice. For example, short- term, self-limiting infections that require minimal intervention (apart from alleviating symptoms), such as the common cold or gastroenteritis, would not beneﬁt from the development of these biomarkers. Others, particularly those with signiﬁcant impact, 1http://www.mirbase.org/ THE POTENTIAL FOR miRNAs TO IMPROVE DISEASE OUTCOMES In 2002, less than a decade after the discovery of miRNAs (Lee et al., 1993), the application of miRNAs as disease biomarkers was ﬁrst explored. Calin et al. (2002) showed that miRNA expression patterns were altered in patients suﬀering June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org 2 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. always fatal (WHO, 2019). Therefore, early intervention is key to survival. currently sub-par diagnostics, or long subclinical phases would beneﬁt greatly by the application of validated miRNA diagnostics. Therefore, it is important to understand the disease of interest and how miRNA biomarkers would be best applied before embarking on miRNA biomarker discovery. Post-mortem brain smears are the current ‘gold standard’ diagnostic test for rabies infection, as the virus’ ability to evade the immune system and reside within neurons make standard molecular diagnostics diﬃcult. Changes in miRNA proﬁles from rabies infected brain tissue have been documented and oﬀer a promising insight into the miRNA response to lyssavirus infection (Zhao et al., 2012; Qi et al., 2014). However, there has only been one study examining the release of miRNAs into peripheral circulation due to lyssavirus infection. This study, completed in a mouse model, found seven miRNAs (miR-100, -187, -322, -706, -466, and -98) that were altered in the serum of infected mice compared to uninfected controls (Han et al., 2011). Low numbers of animals (only 3 control animals) and a heterogeneity in viral RNA and protein expression (measured via immunoﬂuorescence, ELISA and nested RT-PCR) means that this work needs to be further validated. This hypothesis would also require testing in a model system that better replicates miRNA regulation in humans in order to translate miRNA proﬁling into a diagnostic tool for at-risk individuals. Advantages of miRNA Biomarkers I: Early Detection The early detection of infectious disease is often critical to improve patient prognosis and limit the spread of disease. Treatment options often become increasingly limited as a disease proceeds (Wagstaﬀet al., 1994; McLean et al., 2015) (Figure 1). Rabies is a classic example of a disease where early intervention is crucial; it has an extremely long asymptomatic period of 2– 3 months on average and once symptoms appear it is almost p g g Human immunodeﬁciency virus (HIV) is another well-known virus that would beneﬁt from early detection. Infection with HIV causes systemic T-cell destruction and a corresponding reduction in cell mediated immunity (acquired immunodeﬁciency syndrome, AIDS) (Moir et al., 2011). It also results in damage to many organs through endothelial cell dysfunction and immune activation. As a result, there are numerous diseases associated with HIV infection, including viral and bacterial infections, cancers, encephalitis, gut disease, and cardiac damage (Lucas and Nelson, 2015). While the mortality rate from HIV-AIDS is decreasing, AIDS-related disease is still the leading cause of death in persons with HIV (Smith et al., 2014). The wide, and often devastating, impact of HIV infection has prompted numerous studies identifying miRNA biomarkers of both HIV infection and AIDS-related diseases. Serum miR-21, -122, and -223 were found to distinguish HIV-positive from HIV-negative groups with a moderate (0.773, 0.726, and 0.804, respectively) receiver operating characteristic (ROC) area under the curve (AUC) (Thapa et al., 2014). A ROC AUC gives a succinct measure of the overall diagnostic accuracy of a test, with values typically ranging from 0.5 (no discrimination between groups) to 1 (perfect discrimination). For an in-depth review of ROC curves in evaluating diagnostic test accuracy, please see Hajian-Tilaki (2013). MicroRNA-3162- 3p abundance in plasma from HIV patients was shown to diﬀerentiate new (<1 year post-infection) from old (>1 year) infections (Huang et al., 2018). Interestingly, a study by Yahyaei et al. (2016) found that miR-223 and -29a were elevated in individuals that were repeatedly exposed to HIV but did not contract a productive infection. These miRNAs may be purely markers of viral exposure or may play a key role in HIV resistance. Recently, a panel of four miRNAs (miR-16-5p, - 20b-5p, -195-5p, and -223-3p) was developed and tested for HIV diagnostic utility (Biswas et al., 2019). Blind testing was able to identify all positive samples and healthy controls with 100% sensitivity and speciﬁcity. Advantages of miRNA Biomarkers I: Early Detection miRNAs (miR-16-5p, -206, -181c-3p, and let-7g-3p) were able to detect the eclipse phase of infection (the period of time between exposure and reliable diagnostic detection) with 100% sensitivity, 95.8% speciﬁcity, and a ROC AUC of 0.999. This highlights the use of miRNAs as disease biomarkers during the infectious period where conventional markers (antibodies, viral RNA/protein) are consistently undetectable (Biswas et al., 2019). Early therapeutic intervention in HIV infection has been shown to lead to long- term control of the infection with very low levels of persistent viremia (Nishimura et al., 2017). Recently, VSV has also been found to encode multiple small non-coding RNAs (Markus et al., 2017), however, these have not yet been found within the extracellular milieu. Clinically, VSV causes varicella (chickenpox) during the initial infection, but then becomes latent within dorsal root ganglion and cranial nerves and can reactivate to cause herpes zoster (shingles) (Gershon et al., 2015). A study Qi et al. (2014) found that several host miRNAs were altered in the serum of unvaccinated children with varicella compared to healthy controls. Five of these molecules, miR-197, -629, -363, -132, and -122, were found to diﬀerentiate VSV-infected individuals from controls, with a ROC AUC of 0.872. This combination of miRNAs could also distinguish the VSV-infected patients from others infected with similar pathogens, including pertussis (whooping cough), measles, and enteroviruses (Qi et al., 2014). Another study in 2016 found a further six miRNAs (miR-190b, -571, -1276, - 1303, -943, and -661) that were diﬀerentially expressed in the serum of patients with reactivated herpes zoster (Li et al., 2016). Combined, these miRNAs could distinguish patients with herpes zoster from healthy controls and individuals infected with HSV, with a ROC AUC of 0.939 (Li et al., 2016). ( ) Early detection can aid in quarantine, surveillance and biocontainment, factors that are especially relevant for diseases capable of being transmitted from animals to humans. Hendra virus (HeV) is a zoonotic paramyxovirus that can cause severe and often fatal infections in humans. The natural reservoir host for HeV is pteropid fruit bats, however, in 1994 it spilled over to horses and then humans (all with close contact to the infected horses) (Murray et al., 1995). Numerous outbreaks of HeV have occurred in Australia, mainly in horses, sparking concerns for human health. Stewart et al. Advantages of miRNA Biomarkers I: Early Detection A diﬀerent combination of four FIGURE 1 \| MicroRNA signatures during infection. Infections typically progress through three stages: (1) incubation (green), where the pathogen is present and replicating but the patient is asymptomatic, (2) prodromal (blue), where the pathogen continues to replicate (white line) and causes non-speciﬁc symptoms due to immune activation, and (3) clinical illness (orange), where the symptoms of disease are the most severe. As the infection progresses through these stages treatment options often become more limited (red dashed line). Released miRNAs may reﬂect the stage of disease and provide important clinical information, particularly when the causative pathogen is difﬁcult to detect. FIGURE 1 \| MicroRNA signatures during infection. Infections typically progress through three stages: (1) incubation (green), where the pathogen is present and replicating but the patient is asymptomatic, (2) prodromal (blue), where the pathogen continues to replicate (white line) and causes non-speciﬁc symptoms due to immune activation, and (3) clinical illness (orange), where the symptoms of disease are the most severe. As the infection progresses through these stages treatment options often become more limited (red dashed line). Released miRNAs may reﬂect the stage of disease and provide important clinical information, particularly when the causative pathogen is difﬁcult to detect. FIGURE 1 \| MicroRNA signatures during infection. Infections typically progress through three stages: (1) incubation (green), where the pathogen is present and replicating but the patient is asymptomatic, (2) prodromal (blue), where the pathogen continues to replicate (white line) and causes non-speciﬁc symptoms due to immune activation, and (3) clinical illness (orange), where the symptoms of disease are the most severe. As the infection progresses through these stages treatment options often become more limited (red dashed line). Released miRNAs may reﬂect the stage of disease and provide important clinical information, particularly when the causative pathogen is difﬁcult to detect. June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org 3 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. miRNAs in 2006 by Cui et al. (2006). These miRNAs were found to be highly expressed during active infection (miR-H1) (Cui et al., 2006) and latency (miR-H2-6) (Umbach et al., 2008), and are actively packaged into exosomes and exported to uninfected cells (Kalamvoki et al., 2014). Despite no study outlining the use of these as diagnostic markers of HSV infection, these molecules have potential as biomarkers due to the speciﬁcity of these virally encoded miRNAs. Advantages of miRNA Biomarkers II: Improved Pathogen Identiﬁcation p g When symptoms of infectious diseases ﬁrst appear, they are often non-speciﬁc (fever, malaise, headache, and lethargy) and provide little to no information about the causative agent. Consequently, clinicians may misdiagnose patients, which may have severe downstream consequences. One such example of this is viral encephalitis, where symptoms progress from an inﬂuenza-like illness to severe neurological abnormalities, including convulsion, speech loss, confusion, and coma. There are numerous neurotropic viruses that can cause viral encephalitis, ranging from common well-known agents, including herpes simplex virus (HSV), varicella-zoster virus (VSV), and enteroviruses, to deadly zoonotic pathogens such as Japanese encephalitis virus (JEV), lyssavirus (rabies), Nipah virus (NiV), and Hendra virus (HeV). Although best known for being the causative agent of cold sores and genital warts, HSV is also the most common cause of viral encephalitis (Rabinstein, 2017). Serotype HSV-1, is one of the most intensively studied viruses, and is often used as the representative member of the alpha-herpesvirus subfamily. It was this intense scrutiny that led to the discovery of HSV-encoded Advantages of miRNA Biomarkers I: Early Detection (2013) identiﬁed elevated levels of miR-146a in the blood of experimentally challenged horses and ferrets (a model for human disease). This elevation was found to occur days before the detection of the viral genome. A subsequent study took an unbiased NGS-based approach to proﬁle miRNAs within the blood of infected horses and found that 37 miRNAs were signiﬁcantly altered in the blood of infected horses relative to healthy controls (Cowled et al., 2017). This study also examined the potential use of ratios of miRNAs, rather than individual abundances, to distinguish infected from uninfected animals. By doing so, inter-replicate variability was minimized, oﬀering an alternative and possibly more robust method of identifying diagnostic miRNA signatures. Similarly, serum miRNA proﬁling of children with hand, foot and mouth disease (HFMD), caused by enteroviral infection, identiﬁed six miRNAs (miR-148a, -143, -324-3p, -628-3p, - 140-5p, and -362-3p) that were able to discriminate between infected and healthy controls with a combined ROC AUC of 0.989, a sensitivity of 97.1% and a speciﬁcity of 92.7% (Cui et al., 2011). These miRNAs also displayed a unique proﬁle between enterovirus and other microbial infections, including tuberculosis, pertussis, measles, mumps, and varicella. Interestingly, miR-140-5p was undetectable in healthy controls but was easily detectable in the serum of patients infected with any of the six pathogens investigated (Cui et al., 2011). Furthermore, ﬁve miRNAs (miR-148a, -143, -324-3p, -545, and -140-5p) were signiﬁcantly altered between the two major causative agents of HFMD, coxsackievirus-16 and enterovirus- 71, and demonstrated a moderate ability to distinguish between the two enteroviruses (combined ROC AUC of 0.761) (Cui et al., 2011). These results strengthen the hypothesis that miRNA biomarkers cannot only identify infected from uninfected individuals, but also distinguish between diﬀerent causative agents. Further details on the potential of miRNAs in infectious disease diagnostics can be found in Ojha et al. (2019). Frontiers in Microbiology \| www.frontiersin.org Advantages of miRNA Biomarkers III: Detection of Latent Infections Latency periods present signiﬁcant hurdles in the current medical landscape. During these periods, the pathogen is June 2020 \| Volume 11 \| Article 1197 4 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. present but cannot be detected via routine diagnostic methods. While the patient appears healthy, the pathogen can reactivate at any time, causing clinical disease or shedding infectious material. Tuberculosis (TB) is the leading infectious cause of death worldwide but healthcare eﬀorts are often hampered by the large number of individuals with latent infections (WHO, 2018b). It is estimated that 23% of the global population have a latent TB infection, and 5–10% of these will develop active infection and clinical disease (WHO, 2018b). As there are currently no diagnostic platforms for latent TB, it is recommended to provide treatment to priority groups, including people living with HIV and children under 5 years old living with someone with conﬁrmed TB (household contacts) (WHO, 2018b). medicine, this approach ﬁrst gained traction in oncology (Liu et al., 2014) but is now spreading to other areas, including infectious diseases. Malaria is one such disease that results in a spectrum of severity, from asymptomatic to fatal infection. Recently, Burel et al. (2017) utilized a controlled human infection model to investigate the early molecular responses to malaria infection. They found that infected individuals could be stratiﬁed into two groups based on the changes in their circulating miRNA proﬁle (low and high miR responders). Those patients in the high miR group were found to have increased CD4+ T-cell activation, a more robust antibody response, and reduced parasite burden (Burel et al., 2017). Importantly, the authors deﬁned three miRNAs (miR-15a-3p, -30c-5p, and -30e-5p) that could distinguish these high and low-miR responders within days post- infection (Burel et al., 2017). These changes provide important information about the individual’s response to infection, helping to identify high-risk patients and guide treatment decisions. This divergent immune reaction may also explain the mixed success in malaria vaccine development (Rts, 2015). Several studies have identiﬁed circulating miRNAs in active pulmonary TB (Fu et al., 2011; Zhang et al., 2013; Cui et al., 2017; Barry et al., 2018; Pedersen et al., 2019). Recently, Lyu et al. (2019) examined exosomal miRNAs from the serum of patients with active and latent TB infections, as well as healthy controls. Advantages of miRNA Biomarkers III: Detection of Latent Infections They found miRNA expression patterns unique to each group, including four miRNAs (let-7e-5p, let-7d-5p, miR-450a- 5p, and miR-140-5p) that were elevated in latent TB infection when compared to both active infection and healthy controls (Lyu et al., 2019). Although their utility as diagnostic markers was not fully explored, they highlight the potential of miRNAs to identify latent TB infection. Circulating miRNAs have also been found to predict whether adults exposed to TB via infected household contacts would progress to active infection within the next 6 months (Duﬀy et al., 2018). These prognostic markers could be used to identify people at higher risk and better target the use of prophylactic treatments. At the opposite end of the TB infection timeline, researchers found four serum miRNAs that were able to identify patients who were cured of TB infection with an accuracy of 83.96% (Wang et al., 2018). Being able to discriminate between resolved and latent infections would provide clinicians with essential information to guide decisions for treatment and management of TB. COVID-19, another pulmonary disease where latency of up to 2 weeks and asymptomatic infections are a recognized problem (Jiang et al., 2020), is another disease where a more eﬀective and accurate diagnostic is needed. Patients can also demonstrate a wide range of responses to therapeutic intervention, which can impact a clinician’s ability to provide an accurate prognosis. For example, 30–50% of individuals infected with hepatitis C virus (HCV) will not respond eﬀectively to antiviral treatment (Cavalcante and Lyra, 2015). A liver-enriched miRNA, miR-122, has been shown to predict the eﬃcacy of HCV therapy better than traditional circulating biomarkers, including alanine aminotransferase (ALT), albumin, and HCV RNA (Fan et al., 2017). Furthermore, circulating miR-122 was found to decrease alongside detectable HCV RNA during antiviral therapy, reﬂecting successful HCV treatment (Koberle et al., 2013). These results support the use of this miRNA to monitor the progression of HCV infection, predict treatment eﬃcacy, and aid in prognostic accuracy. Whilst much of this manuscript details the use of miRNA biomarkers in a diagnostic sense, the studies mentioned above highlight the potential of miRNAs to measure treatment eﬃcacy, predict disease outcome and host responses, and guide optimal therapeutic selections as companion diagnostics. Frontiers in Microbiology \| www.frontiersin.org Current miRNA Detection Platforms Conventional approaches to the detection of miRNAs include northern blotting, microarrays, qPCR, and next generation sequencing (NGS). Contemporary miRNA biomarker studies tend to utilize qPCR and NGS, with northern blotting and microarrays falling out of favor due to limitations, including low sensitivity or speciﬁcity, and higher total RNA input requirements (often several micrograms for northern blotting). Recently, researchers have utilized Mass Spectrometry (MS) for the detection of miRs (Kullolli et al., 2014). Several reviews address qPCR and NGS for miRNA detection (Hardikar et al., 2014; Vincent et al., 2017). This application of advanced bioinformatic analysis tools gives researchers more robust methods of identifying and evaluating predictive miRNAs. Feature selection algorithms (Saeys et al., 2007; Wang et al., 2016) allow computers to trawl through datasets highlighting the most predictive miRNAs, and have become more integral as researchers generate larger, more complex datasets. The introduction of machine learning into biological sciences has meant that researchers can create computational models that evaluate the predictive power of miRNA candidates on blinded samples. This approach can identify complex patterns and strengthen diagnostic classiﬁcation. Machine learning has already been used to identify miRNA biomarkers for Ebola virus and Mycobacterium tuberculosis (Duy et al., 2016; Duﬀy et al., 2018) and will likely become more widespread. For this to occur, more investment needs to be made in advanced data analytics for biological sciences. In particular, microRNA biomarker research should move beyond simple diﬀerential expression analysis to capture the full spectrum of permutations and subtleties in expression of these molecules. Two factors that will greatly enhance successful outcomes from the use of computational analysis are: (1) an immediate focus on higher replicate numbers, and (2) proper use of holdout sets (validation samples), independent of samples used for initial biomarker discovery (training sets). These multivariate approaches (including the To quantitate via qPCR, miRNAs are reverse-transcribed to cDNA and then ampliﬁed using deﬁned primers. A sequence- speciﬁc probe, which contains a ﬂuorophore and quencher, binds to the cDNA, and during ampliﬁcation is cleaved by the endonuclease activity of the DNA polymerase. This releases the ﬂuorophore and the resulting ﬂuorescence is measured (Wong et al., 2015). It is a highly sensitive, highly speciﬁc gold standard against which to evaluate other detection systems. Despite its utility, qPCR suﬀers from several limitations, namely it is relatively expensive, and requires a priori deﬁned primer and probe sequences. Advantages of miRNA Biomarkers IV: Personalized Medicine The development of host miRNAs as infectious disease biomarkers will require the successful navigation of several hurdles. Principle among these is the perceived lack of speciﬁcity among miRNAs responding to diverse infections. For example, miR-146a expression is known to ﬂuctuate in response to hepatitis B virus (Wang et al., 2019), HCV (Abdel Motaleb et al., 2017), schistosomiasis (Cai et al., 2018), malaria (Cohen et al., 2018), JEV (Baluni et al., 2018), HeV (Stewart et al., 2013), and even prion disease (Bellingham et al., 2012). This miRNA has also been identiﬁed in non-infectious diseases, such as diabetes (Farr et al., 2015b) and heart disease (Quan et al., 2018). On its own, miR-146a has minimal disease speciﬁcity; indeed, it may be only indicative of a general immune response related to activation of the NF-κB transcription factor (Testa et al., 2017). However, Disease severity and the eﬃcacy of anti-infective therapies can vary markedly between patients. Personalized medicine (also known as precision medicine or theranostics) recognizes that a ‘one size ﬁts all’ approach is not always eﬀective and attempts to tailor the treatment to achieve the best clinical outcomes. This healthcare concept is underpinned by biomarkers to provide pertinent information for timely and eﬀective treatment decisions (Figure 2). Biomarkers in the ﬁeld of personalized medicine often refer to an individual’s genotype, such as the BRCA mutation in ovarian cancer (Marchetti et al., 2018), however, this idea is being extended to other diagnostic or prognostic markers, such as miRNAs. As with many aspects of June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org 5 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. this does not mean it should be disregarded, as the immune response may be an important marker of disease. Instead, miR- 146a should be measured in conjunction with additional disease- speciﬁc miRNAs to ensure diagnostic accuracy. use of ratios) aid in the identiﬁcation of composite miRNA signatures that can mitigate the lack of speciﬁcity seen in some individual markers. Finally, despite advances in the rapidly advancing ﬁeld of miRNA detection (Cheng et al., 2018; Kalogianni et al., 2018; Kilic et al., 2018), there are currently no industry standards for the detection and quantiﬁcation of miRNAs for clinical diagnosis of disease. Advantages of miRNA Biomarkers IV: Personalized Medicine This handicap is due to the nature of miRNAs as diagnostic targets; they require isolation from biological ﬂuids prior to detection; tend to exhibit subtle nucleotide diﬀerences leading to complications in probe speciﬁcity; and need to be protected from RNAse degradation during handling and processing (De Guire et al., 2013). In the future, emerging technologies for sample preparation and the detection of miRNAs for the diagnosis of infectious diseases should assist to improve current use of miRNAs as diagnostic markers. Generalized miRNA responses to infection has important implications when trying to use these biomarkers to diagnose infections in the presence of co-morbidities or co-infections that may confound the results. Although there are studies that explore miRNA signatures in known co-infections, such as HIV/HCV (Anadol et al., 2015), this data is limited and is not able to take into account unknown confounding factors. Increasing biological replicates for miRNA biomarker discovery studies, as well as careful validation studies, will help to mitigate this issue and result in a more robust diagnostic signature. Many studies identify potential miRNA biomarkers as those whose abundance changes in bioﬂuids, utilizing a statistical cut- oﬀto justify their selections. This approach, while eﬀective in identifying large scale changes, does not automatically select for the most diagnostically relevant miRNAs. Instead, the degree of variation or the relationship of one miRNA to another may provide more eﬀective diﬀerentiation between infected and non- infected individuals. Cowled et al. (2017) found that using ratios of two miRNAs ( miR_A miR_B ) improved the adjusted P-values by normalizing for individual heterogeneity. Notably, these ratios were not arbitrarily selected; every possible combination of miRNAs was tested to identify those ratios that had the most statistically signiﬁcant diﬀerence between groups. The use of miRNA ratios will have limited impact on their use in a clinical setting compared to the absolute or relative levels of a single marker. Indeed, they would be measured in concert (using a multiplex approach), the ratio calculated, and result presented based on whether they cross a pre-deﬁned threshold. Frontiers in Microbiology \| www.frontiersin.org Current miRNA Detection Platforms It is also relatively low-throughput, although this can be partially addressed through robotic handling systems, multiplexing and qPCR arrays (Wong et al., 2015). Next generation sequencing also requires reverse- transcription and ampliﬁcation but does so without using primers or probes speciﬁc to known miRNAs. It yields millions of short read sequences that can then be either analyzed de novo or mapped to a reference sequence such as a genome or miRNA sequence database (miRbase) (Kozomara et al., 2019). This technique allows quantitation of all miRNAs present in a sample, even novel sequences. Numerous NGS platforms are available with various proprietary chemistries but all are bulky, expensive (although prices are continuing to fall), time-consuming, and require complex sample preparation, instrument operation, and June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 6 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. FIGURE 2 \| MicroRNA biomarkers in personalized medicine. Many infections result in a spectrum of disease severity, including asymptomatic (black), mild (red), and severe (blue) disease. Circulating miRNA signatures may differentiate between these groups and their ultimate disease outcome. This information would allow clinicians to tailor their approach and provide optimal therapeutic interventions. i d i b ti t 37◦C f 60 i d dditi l i t data analysis procedures. Despite this, it is the technique of choice for initial discovery of miRNA biomarkers and is typically followed by development of qPCR assays for further validation. New and Emerging miRNA Detection Platforms Emerging technologies for the detection of miRNA biomarkers in infectious diseases aim to improve upon what is currently available, focusing on one or more of the following characteristics: portable, reliable, robust, rapid, speciﬁc, sensitive, low-cost, and user-friendly. Transitioning from costly and time-consuming lab-based methodologies to point-of-care (PoC) technologies is particularly important for the diagnosis and management of infectious disease outbreaks, where accurate and timely identiﬁcation of infected individuals in resource- limited environments is critical for both patient care and disease containment. As the current in-lab miRNA detection technologies continue to improve, the next question is how to translate this technology into PoC devices, and how to improve the current protocols in such a way that they are easily translatable. The predominant barrier to the development of PoC devices for miR detection is the low concentration of miRs in most bioﬂuids. Whilst the concentration of individual miRs vary signiﬁcantly in diﬀerent bioﬂuids, in healthy individuals, miRs in plasma are generally present in the fM range (9,000–134,000 copies/µL) (Mitchell et al., 2008). To overcome this, most examples of miRNA detection rely upon complex miRNA extraction protocols (such as multiple centrifugation or pipetting steps) or highly complex and bulky detection instrumentation (such as high-throughput NGS platforms or MS-based techniques) that are impractical for use in a resource limited PoC environment. To counteract this, many researchers have begun to develop new technologies that focus on miRNA extraction and detection in portable devices. An overview of these technologies can be found in Figure 3, while Table 1 compares their attributes. Whilst most of these examples focus on the diagnosis of cancer in humans, all of the technologies could be easily adapted to the diagnosis of infectious disease by modifying the target sequences of interest. Lateral ﬂow devices (strip tests) are useful tools for robust detection of biomarkers at the PoC due to their low-cost, simplicity, portability and speciﬁcity. In light of this, numerous groups have developed strip-based biosensors for miRNA detection (Hou et al., 2012; Gao et al., 2014; Deng et al., 2017a). Given most medical diagnoses are made after testing a panel of two or more biomarkers, Zheng et al. (2018) developed a multiplexed lateral ﬂow assay for the simultaneous detection of miR-21, miR-155 and miR-210 in human serum samples at concentrations as low as 68, 7, and 17 pM, respectively, within 10 min (Figure 3A). FIGURE 2 \| MicroRNA biomarkers in personalized medicine. Frontiers in Microbiology \| www.frontiersin.org New and Emerging miRNA Detection Platforms Lateral ﬂow test for visual detection of multiple MicroRNAs, Pages 320–326, Copyright (2018), with permission from Elsevier. (B) Electrochemical biosensor. Reprinted with permission from Labib et al. (2013a). Four-Way Junction Formation Promoting Ultrasensitive Electrochemical Detection of MicroRNA. Analytical Chemistry 85(20), 9,422–9,427. doi: 10.1021/ac402416z. Copyright (2013) American Chemical Society. (C) µPAD for miRNA extraction and quantitation. Reprinted with permission from Deng et al. (2017b). Paperﬂuidic Chip Device for Small RNA Extraction, Ampliﬁcation, and Multiplexed Analysis. ACS applied materials interfaces 9(47), 41,151–41,158. doi: 10.1021/acsami.7b12637. Copyright (2017) American Chemical Society. (D) Colorimetric miRNA sensing strategy. Reprinted with permission from Feng et al. (2017). Detection of microRNA: a point-of-care testing method based on a pH-responsive and highly efﬁcient isothermal ampliﬁcation. Analytical chemistry 89(12), 6,631–6,636. doi: 10.1021/acs.analchem.7b00850. Copyright (2017) American Chemical Society. Fully integrated patterned paper-based microﬂuidic devices (µPADs) have been developed featuring simple, user-friendly, robust and low-cost solutions for determination of a range of biomarkers using a variety of fabrication and detection strategies (Dungchai et al., 2009; Delaney et al., 2011; Dungchai et al., 2011). µPADs are particularly promising for the diagnosis of infectious diseases in resource limited settings as they are single-use, low- cost and can be readily disposed of by incineration; this negates the need for extensive sterilization procedures thus reducing the risks of accidental infection or contamination. In spite of the application of µPADs for a variety of sensing applications, they are inherently limited in ﬂuorescence-based sensing applications due to high background ﬂuorescence and light scattering from the paper substrate (Pelton, 2009; Nery and Kubota, 2013). To overcome the challenges of conducting ﬂuorescence assays on paper, Liang et al. (2017) grew a ﬂower- like silver (FLS) layer on the paper substrate which decreased background ﬂuorescence and produced ‘hot-spots’ where surface enhanced ﬂuorescence of carbon nanodot labeled probes was observed. A detection limit of 30 and 60 aM was observed for miR-210 and miR-21, respectively, using the FLS µPAD. The device was also used for a qualitative visual pre-screening of analyte concentration due to a color change after the addition of hydrogen peroxide that caused oxidation of clear Ce3+ labels to orange Ce4+ (Das et al., 2007; Sardesai et al., 2013; Liang et al., 2017). A variety of µPADs have been developed for PoC miRNA detection (Deng et al., 2017b) (Figure 3C). Deng et al. New and Emerging miRNA Detection Platforms Many infections result in a spectrum of disease severity, including asymptomatic (black), mild (red), and severe (blue) disease. Circulating miRNA signatures may differentiate between these groups and their ultimate disease outcome. This information would allow clinicians to tailor their approach and provide optimal therapeutic interventions. FIGURE 2 \| MicroRNA biomarkers in personalized medicine. Many infections result in a spectrum of disease severity, including asymptomatic (black), mild (red), and severe (blue) disease. Circulating miRNA signatures may differentiate between these groups and their ultimate disease outcome. This information would allow clinicians to tailor their approach and provide optimal therapeutic interventions. Deng et al. (2017a) sought to overcome the limited sensitivity of previous lateral ﬂow based biosensors by incorporating a target recycling ampliﬁcation strategy, whereby two sequence speciﬁc hairpins are used to amplify the signal without the need for added enzymes. The improved sensing mechanism required incubation at 37◦C for 60 min and additional equipment including a handheld UV-light and mobile phone/digital camera but boasted a remarkable LOD of 200 attomoles from 20 µL of sample within 80 min. June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org 7 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. FIGURE 3 \| Representative approaches to the development of PoC miRNA sensing devices. (A) Lateral ﬂow test device. Reprinted from Sensors and Actuators B: Chemical, volume 264, Zheng et al. (2018). Lateral ﬂow test for visual detection of multiple MicroRNAs, Pages 320–326, Copyright (2018), with permission from Elsevier. (B) Electrochemical biosensor. Reprinted with permission from Labib et al. (2013a). Four-Way Junction Formation Promoting Ultrasensitive Electrochemical Detection of MicroRNA. Analytical Chemistry 85(20), 9,422–9,427. doi: 10.1021/ac402416z. Copyright (2013) American Chemical Society. (C) µPAD for miRNA extraction and quantitation. Reprinted with permission from Deng et al. (2017b). Paperﬂuidic Chip Device for Small RNA Extraction, Ampliﬁcation, and Multiplexed Analysis. ACS applied materials interfaces 9(47), 41,151–41,158. doi: 10.1021/acsami.7b12637. Copyright (2017) American Chemical Society. (D) Colorimetric miRNA sensing strategy. Reprinted with permission from Feng et al. (2017). Detection of microRNA: a point-of-care testing method based on a pH-responsive and highly efﬁcient isothermal ampliﬁcation. Analytical chemistry 89(12), 6,631–6,636. doi: 10.1021/acs.analchem.7b00850. Copyright (2017) American Chemical Society. FIGURE 3 \| Representative approaches to the development of PoC miRNA sensing devices. (A) Lateral ﬂow test device. Reprinted from Sensors and Actuators B: Chemical, volume 264, Zheng et al. (2018). Frontiers in Microbiology \| www.frontiersin.org New and Emerging miRNA Detection Platforms (2017b) inspired by previous examples of paper-based nucleic acid extraction (Connelly et al., 2015; Rodriguez et al., 2015), developed two paper based devices – one for miRNA extraction and ampliﬁcation and a second for detection of the miRNA – which could be easily interfaced using magnetic sheets. The extraction and detection procedures required only a heating block for isothermal ampliﬁcation, UV-light for quantum dot excitation and digital camera/mobile phone for ﬂuorescence detection (Deng et al., 2017b). The µPAD exhibited a LOD of 3 × 106 copies for two miRNAs (miR-21 and miR-155) from tumor cell lysate (Deng et al., 2017b). The simplicity, speed (90 min time-to-result) and low-cost of this paper-based device make it an ideal candidate for the diagnosis of infectious disease. Visually identiﬁable color changes provide the simplest and lowest-cost detection strategy available. Inspired by the simplicity of the color-coded pH test strip Feng et al. (2017) exploited the production of hydrogen ions (H+) as a by-product of netlike rolling circle ampliﬁcation (NRCA) to develop a pH dependent visual miRNA sensing methodology (Figure 3D). The group employed three pH indicators – cresol red, neutral red and m-cresol purple – and observed an LOD of 100 femtomolar (fM) for miR-21 by visual observation of the color change June 2020 \| Volume 11 \| Article 1197 Frontiers in Microbiology \| www.frontiersin.org 8 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. TABLE 1 \| Selected examples of emerging miRNA detection platforms. New and Emerging miRNA Detection Platforms References Detection Sample LOD Multiplexed Ampliﬁcation Lateral ﬂow Gao et al., 2014 Visual/photographic Cell lysate 60 pM No No Deng et al., 2017a Visual/cell phone/digital camera Cell lysate 200 attomoles (10 pM)a No Target–recycled non-enzymatic ampliﬁcation Zheng et al., 2018 Visual/photographic Human serum 7–85 pM (quantitative) 10–50 pM (visual) 3 No Electrochemical Cardoso et al., 2016 Electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) Human serum 5.7 aM No No Labib et al., 2013a SWV Human serum 2 aM No No Labib et al., 2013b SWV and EIS Human serum 5 aM No No Gai et al., 2018 Open circuit voltage Human serum 2.7 aM No No Cheng et al., 2015 SWV Human serum 0.76 aM No No µPAD Deng et al., 2017b Visual/cell phone/digital camera Cell lysate 3 × 106 copies (996 fM)b 2 Hairpin probe-exponential ampliﬁcation reaction Liang et al., 2017 Visual/ﬂuorescence spectrometer Cell lysate 30–60 aM 2 No Colorimetric Feng et al., 2017 Visual/UV-Vis Cell lysate 100 fM (Visual) 9.3 fM (UV-Vis) No Netlike rolling circle ampliﬁcation Digital Microﬂuidic Giuffrida et al., 2015 Fluorescence microscope Cell lysate 3.3 attomoles (165 pM) No Molecular-beacon (MB)- assisted isothermal circular-strand-displacement polymerization Shamsi et al., 2016 Electrochemiluminescence photomultiplier tube Cell lysate 1.5 femtomoles (1.1 nM) No No aLOD concentration calculated from 20 µL sample volume from Deng et al., 2017a. bLOC concentration calculated from 5 µL sample volume from Deng et al. (2017b). TABLE 1 \| Selected examples of emerging miRNA detection platforms. Multiplexed Ampliﬁcation further detail. Electrochemical detection strategies are readily adaptable to PoC applications due to the availability of low- cost portable potentiostats (Rowe et al., 2011; Doeven et al., 2015; Ainla et al., 2018) used in combination with low-cost commercially available screen printed electrodes or in-house produced alternatives (Dungchai et al., 2009, 2011). with the naked eye using a commercially available pH test strip or color indicator solution Where required, the samples could subsequently be analyzed via UV-Visible spectroscopy for quantitative sample analysis. Whilst the majority of PoC sensing applications have focused on the development of colorimetric or ﬂuorescence read-out strategies, these methodologies suﬀer from several drawbacks including high background signals which limit sensitivity. Numerous researchers have sought to overcome this drawback by employing less conventional detection strategies with interesting results. Cardoso et al. Frontiers in Microbiology \| www.frontiersin.org New and Emerging miRNA Detection Platforms (2016) employed electrochemical impedance spectroscopy for the determination of miR-155 in human serum at gold electrodes functionalized with a capture probe and obtained a detection limit of 5.7 aM from human serum samples. By developing a four-way junction electrochemical sensor, Labib et al. (2013a) were able to detect as little as 2 aM of miR- 122 in human serum samples without PCR ampliﬁcation. In a subsequent study, the researchers developed a three-mode duplexed sensor for the detection of two miRNAs – miR-32 and miR-122 – in human serum with a detection limit of 5 aM from 30 µL of sample (Labib et al., 2013b). Gai et al. (2018) further simpliﬁed conventional electrochemical sensing techniques by developing a two-electrode, self-powered biofuel- based sensor for the detection of miRNA at 2.7 aM. In an interesting approach combining electrochemical sensing with cadmium modiﬁed titanium phosphate nanospheres, Cheng et al. (2015) developed a miRNA-21 sensor with a detection limit of 0.76 aM that could be applied for the direct analysis of human serum samples (Figure 3B). Digital microﬂuidic (DMF) devices employ software-based electronic control of liquids and oﬀer numerous advantages over conventional microﬂuidic systems as they eliminate the need for tubing and pumps whilst maximizing automation and reducing sample volumes (Choi et al., 2012; Giuﬀrida et al., 2015). Shamsi et al. (2016) employed electrochemiluminescence in a DMF platform for the detection of miR-143 to an LOD of 1.5 femtomoles within 40 min. A number of research groups have successfully developed electrochemical biosensors for the sensitive and selective detection of miRNAs (Figure 3B). Herein, we will focus on representative examples which are particularly promising for PoC applications; a recent review by Gillespie et al. (2019) systematically addresses electrochemical microRNA sensing in June 2020 \| Volume 11 \| Article 1197 9 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. FIGURE 4 \| Metrics for diagnostic test validation. (A) Example confusion matrix from which the common metrics (accuracy, sensitivity, and speciﬁcity) can be calculated. (B) ROC graph showing a perfect ROC curve (orange line), and example ROC curve (blue line), and the diagonal indicating a random classiﬁcation model (random guess and black dotted line). VALIDATION AND TRANSLATION OF miRNA DIAGNOSTICS Once a set of miRNAs have been identiﬁed as potential disease biomarkers, their use in a diagnostic assay needs to be adequately assessed. Accuracy is an oft-cited metric but may not always be appropriate (for example, if you expect that 99% of the samples you test are going to be negative, then a test that simply labels all samples as negative will be 99% accurate). There are a multitude of other metrics available, including sensitivity (proportion of infected samples who tested positive), and speciﬁcity (proportion of non-infected samples who tested negative), all of which are simply diﬀerent ways of interpreting a confusion matrix (predicted positive or negative classiﬁcation vs. the true classiﬁcation, see Figure 4A). Another important performance metric, which has been referenced in some studies, is the Receiver Operating Characteristic (ROC) curve. A ROC curve is a graphical representation of how the true positive rate (TPR) and false positive rate (FPR) change as the classiﬁcation threshold is varied between the infected and non-infected groups. The area under the curve (AUC) is also often calculated, giving a succinct measure of the overall performance of the test. If a diagnostic test has zero predictive power, the TPR and FPR will be equal, resulting in a diagonal line and an AUC of 0.5 (Figure 4B, Random guess). Alternatively, if it correctly identiﬁes all samples 100% of the time, then the ROC curve, the TPR is equal to 1 and the FPR is equal to 0, resulting in an AUC of 1.0 (Figure 4B, Perfect ROC curve). In reality, a diagnostic test usually falls somewhere between these two possibilities (Figure 4B, Example ROC curve). A ROC curve can also help choose the classiﬁcation threshold. There is a ﬁne balance between the TPR and FPR and the threshold that is chosen is dependent on the ultimate purpose of the test. For example, if a patient is exposed to rabies, it is more acceptable to be falsely identiﬁed as infected, and therefore treated, than falsely identiﬁed as not infected, sent home, and ultimately succumb to infection. Therefore, a miRNA diagnostic test for rabies infection would heavily favor the detection of true positives (sensitivity) even if that meant it detected a larger number of false positives. It is imperative that all potential diagnostics be evaluated in the context of their use. FIGURE 4 \| Metrics for diagnostic test validation. VALIDATION AND TRANSLATION OF miRNA DIAGNOSTICS (A) Example confusion matrix from which the common metrics (accuracy, sensitivity, and speciﬁcity) can be calculated. (B) ROC graph showing a perfect ROC curve (orange line), and example ROC curve (blue line), and the diagonal indicating a random classiﬁcation model (random guess and black dotted line). (1) a prototype evaluation (phase I), (2) an evaluation under ideal conditions (phase II), or (3) an evaluation under real- life conditions (phase III). At a minimum, such evaluations should provide information not only on a test’s diagnostic accuracy but also on its repeatability, reproducibility, and ease of use. Clinical trials require biomarkers to be assessed against a gold standard or reference method, often selected in consultation with regulators during trial design. Clearly, undertaking clinical trials requires signiﬁcant ﬁnancial resources to enroll patients, disease experts and clinicians, process large sample numbers and collect clinical data. For some diseases, particularly emerging infectious diseases, this cost may be viewed as prohibitive given the market opportunity and expected return on investment. Gaining access to appropriate samples or clinical expertise also may present a challenge. The cost of clinical trials will also be signiﬁcantly impacted by scope. For instance, some would argue that in order to demonstrate that a biomarker-based diagnostic test improves patient outcomes over an existing or predicate test, a randomized controlled trial should be conducted. Here, randomized comparisons are made between two diagnostic interventions (one reference and one experimental) with identical therapeutic interventions based on the results of the competing diagnostic tests. Study outcomes Moreover, when translating a miRNA biomarker signature to a diagnostic device, it is pertinent to have a clear understanding of how the end consumer will use the test, and how to maximize its utility. Many current diagnostic tests, such as serology, need to be sent to a diagnostic laboratory to be analyzed. For infections that are time-critical this is a costly delay, hence the time it takes to generate a result is an important consideration. Some diagnostic platforms may be used for initial screening or used in rural or remote areas where access to a laboratory is limited. PoC devices may alleviate these constraints as samples do not need to be transported to a diagnostic laboratory. In other circumstances a test undertaken in an accredited diagnostic laboratory may be more desirable. Regardless of which format the test is ultimately conﬁgured, care needs to be taken to ensure it is ﬁt-for-purpose. Frontiers in Microbiology \| www.frontiersin.org FUNDING LT was supported by the CSIRO Early Research Career (CERC) Postdoctoral Fellowship. EK was supported by the National Health and Medical Research Council (NHMRC) of Australia (grant ID GNT1161573). CC was supported by Meat & Livestock Australia (MLA), CSIRO Probing Biosystems Future Science Platform, and CSIRO SpaceTech Future Science Platform. CS and AB were supported by CSIRO Health and Biosecurity. MD was supported by the CSIRO Australian Animal Health Laboratory. RF was supported by the CSIRO Probing Biosystems Future Science Platform funding scheme. CONCLUSION For many infectious diseases, the current diagnostic technologies are inadequate. Many rely on the presence of symptoms or pathogen-speciﬁc antibodies or cannot be utilized until the pathogen has replicated to a detectable titer. New biomarkers are sorely needed to underpin the next generation of diagnostic platforms. MicroRNAs have substantial potential as biomarkers of infection, as evident in the growing body of research data. These molecules are altered in bioﬂuids due to infections with bacteria, parasites, viruses, and even prions. Their use as diagnostic or prognostic markers is not without its challenges, however, with robust analytical and validation methods, their use may alleviate the shortcomings of current tests, leading to improved patient outcomes. In conjunction with the identiﬁcation of suﬃciently predictive miRNAs, the exciting progress in miRNA sensing technologies and the emergence of recent, commercially available miRNA-based diagnostics, means that miRNAs will inevitably be employed as powerful new tools in diagnostic, prognostic and even therapeutic strategies for infectious diseases. AUTHOR CONTRIBUTIONS All authors agreed on the ﬁnal draft of the manuscript prior to submission. LT, EK, and RF wrote the ﬁrst draft and revised the manuscript. CC, CS, MD, and AB reviewed and edited the manuscript. While the market for miRNA biomarkers is still in its infancy, some products (primarily in oncology) are currently available to clinicians, while others are entering pre-clinical and clinical trials (Bonneau et al., 2019). Interpace Diagnostics is one notable example, with their product ThyraMIR R⃝(a 10 miRNA panel) distinguishing between benign and malignant thyroid cancer. This test is combined with an oncogene NGS panel (ThyGeNEXT R⃝) to stratify patients on whether they require surgery and if so, the extent of the surgical intervention. This approach, ﬁrst detailed in 2015 (Labourier et al., 2015), has since been reﬁned and demonstrates signiﬁcant clinical utility in stratifying patients based on their risk of malignancy after 9 months (Sistrunk et al., 2020). Interpace Diagnostics has also been successful in obtaining coverage for these tests by some of the largest health insurance providers in the United States, a electrochemical detection with smartphones. Anal. Chem. 90, 6240–6246. doi: 10.1021/acs.analchem.8b00850 Ali Ahmed, E., Abd El-Basit, S. A., Mohamed, M. A., and Swellam, M. (2020). Clinical role of MiRNA 29a and MiRNA 335 on breast cancer management: their relevance to MMP2 protein level. Arch. Physiol. Biochem. doi: 10.1080/ 13813455.2020.1749085 [Epub ahead of print]. Anadol, E., Schierwagen, R., Elﬁmova, N., Tack, K., Schwarze-Zander, C., Eischeid, H., et al. (2015). Circulating microRNAs as a marker for liver injury in VALIDATION AND TRANSLATION OF miRNA DIAGNOSTICS A key step toward ﬁnal test adoption is evaluation in clinical trials to deﬁne test performance. Tests may be scrutinized at: June 2020 \| Volume 11 \| Article 1197 10 MicroRNA Biomarkers of Infectious Diseases Tribolet et al. are clinically important consequences of diagnostic accuracy. While considered the gold standard, randomized controlled trials are rarely cited due to their signiﬁcant costs and a lack of regulatory pressure. signiﬁcant step in clinical adoption. Case studies such as this can provide invaluable insight into the journey of miRNA biomarkers from discovery to clinical application. In addition to clinical trials, navigation through the regulatory approval process is a key step toward adoption of biomarker- based diagnostics. The requirements for licensing diagnostic tests are complex and diﬀers considerably from country to country, with many lacking regulatory procedures to assess the safety, quality or eﬀectiveness of in vitro diagnostic tests (IVDs). In the United States, the Food and Drug Administration (FDA) regulates IVDs with the intended use of the device determining how much data, type of data, and approval route to use. Within Europe the European Medicine Association (EMA) fulﬁlls this role. Whether or not the biomarker test is substantially equivalent to an existing test, and the context in which a test will be used (critical versus low or moderate risk) are factors in the level of regulation and data required. Additionally, the sample type and route of collection will impact the ease of use and clinical uptake of an IVD. Less invasive samples, such as saliva or sputum are often preferred over blood or sera. 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https://openalex.org/W3169981303	https://www.scielo.br/j/rmat/a/NL6XcSRS8kMPxZv4gKjLzGd/?lang=pt&format=pdf	Portuguese	null	Estudo do comportamento no estado fresco e endurecido de argamassa com escória de aciaria BSSF	Matéria	2,021	cc-by	9,898	Study of the behavior in the fresh and hardened state of mortar with BSSFsteel slag Felipe Alves Amancio 1, Alisson Rodrigues de Oliveira Dias 1, Douglas Alexandre Lima 2, Antônio Eduardo Bezerra Cabral 1 1 Programa de Pós-Graduação em Engenharia Civil – Estruturas e Construção Civil, Universidade Federal do Ceará, PEC/UFC, Acesso Público, bloco 733, Campus do Pici, CEP: 60455–900, Fortaleza, CE, Brasil 2 Departamento de Estruturas e Construção Civil, Universidade Federal do Ceará, Acesso Público, bloco 733, Campus do Pici, CEP: 60455–900, Fortaleza, CE, Brasil e-mail: douglas.edif@gmail.com, felipeaamancio@hotmail.com, rodrigues alisson@live.com, eduardo.cabral@ufc.br. Programa de Pós-Graduação em Engenharia Civil – Estruturas e Construção Civil, Universidade Federal do Ceará, p Departamento de Estruturas e Construção Civil, Universidade Federal do Ceará, Acesso Público, bloco 733, Campus d ci, CEP: 60455–900, Fortaleza, CE, Brasil e-mail: douglas.edif@gmail.com, felipeaamancio@hotmail.com, rodrigues_alisson@live.com, eduardo.cabral@ufc RESUMO Em 2017, a produção de aço bruto no Brasil foi de 34,4 milhões de toneladas, o que representa um cresci- mento de 9,8% em comparação com 2016. Apesar da importância econômica para a economia do país, a in- dústria siderúrgica representa um grande problema ambiental, em virtude da quantidade de escórias geradas no processo de produção do aço. Neste sentido, o presente trabalho teve por objetivo investigar as proprieda- des das argamassas de revestimento produzidas com escória de aciaria Baosteel Slag Short Flow (BSSF) em substituição à areia natural. O traço investigado foi de 1:1:6 (cimento: cal: agregado), com a substituição em volume nos teores de 0%, 20%, 40%, 60% e 80%. Os ensaios realizados foram: índice de consistência, den- sidade, teor de ar incorporado, squeeze-flow, resistências à compressão axial e à tração na flexão, módulo de elasticidade dinâmico, absorção por capilaridade, índice de vazios, e resistência potencial de aderência à tra- ção. Constatou-se que para um índice de consistência de 260 ± 5 mm, as argamassas com escória de aciaria BSSF apresentaram uma maior relação a/c, além de maior densidade de massa aparente nos estados fresco e endurecido, módulo de elasticidade dinâmico, coeficiente de capilaridade e índice de vazios. A argamassa de referência apresentou ainda retenção de água e resistência potencial de aderência superiores aos das argamas- sas com escória de aciaria BSSF, enquanto que os valores de resistência à compressão e as propriedades físi- cas foram similares. Cabe destacar ainda o aumento significativo no módulo de elasticidade dinâmico nas argamassas com escória de aciaria, em virtude da elevada massa especifica da escória, o que podem prejudi- car a durabilidade do revestimento, em virtude da redução da capacidade de absorver deformações. Palavras-chave: Escória de aciaria BSSF. Resistência potencial de aderência a tração. Durabilidade. Arga- ç p ç Palavras-chave: Escória de aciaria BSSF. Resistência potencial de aderência a tração. Durabilidade. Arga- massa de revestimento. Autor Responsável: Felipe Alves Amancio 1. INTRODUÇÃO Ç A produção mundial de aço, em 2017 alcançou novo recorde, ao totalizar 1,689 bilhões de toneladas [1]. No Brasil, está produção foi na ordem de 34,4 milhões de toneladas, ocupando a 9º posição na produção mundi- al. A indústria metalúrgica, apesar do seu papel de extrema importância na economia, produz grandes quanti- dades de subprodutos, entre eles, as escórias, que representam um problema para a fábrica e para o meio am- biente [2]. Dentre os subprodutos, a escória de aciaria é responsável por 28% dessa geração, ou seja, 5,5 mi- lhões de toneladas/ano. A principal destinação dessas escórias tem sido para venda à indústria cimenteira (37%) e reutilização interna (26%), onde tem sido utilizada para composição de bases e sub-bases de pavi- mentos e nivelamento de terrenos e aterros [3] (IABr, 2016). Devido a essa problemática, a Baosteel, uma empresa siderúrgica chinesa, com sede em Xangai, de- senvolveu um novo tratamento para as escórias de aciaria. No processo de granulação da BSSF, a escória líquida produzida é transferida para um tambor de granulação, onde ela é tratada com água injetada com um tempo de permanência entre três e cinco minutos, o que reduz representativamente a quantidade de cal livre que não reagiu [4]. Durante a geração da escória de aciaria Baosteel Slag Short Flow (BSSF), seu resfriamento se dá atra- vés do uso de jatos de água, juntamente com forças mecânicas para trituração, formando um material de ca- racterística vítrea, granulado e com baixas quantidades de cal livre [4, 5]. Com esse processo produz-se uma escória livre de expansão [5, 6]. Como resultado desse processo tem-se a obtenção da escória de aciaria do tipo BSSF, objeto de estudo deste trabalho. No Ocidente, a Companhia Siderúrgica do Pecém – CSP, localizada no Estado do Ceará - Brasil, é a única empresa siderúrgica que incluiu esse processo na sua linha de produção do aço até o presente momento. Com isto, existem poucos trabalhos quanto a empregabilidade desta escória em argamassas de revestimento, tendo apenas publicações com a utilização de escórias de aciaria obtidas pelos métodos tradici- onais, os quais foram utilizados nas discussões dos resultados. Nesse contexto, o setor da construção civil consiste em uma das indústrias mais interessadas no apro- veitamento desse tipo de resíduo e na busca de materiais de construção de menor custo e impacto ambiental reduzido. 1. INTRODUÇÃO A busca pelo uso de materiais alternativos aumentou de forma importante com a crescente consci- entização sobre a importância das questões ambientais e a busca por novas soluções que combinam cresci- mento econômico e preservação do patrimônio natural [7, 8]. Aliado a isto, a exploração de recursos naturais, a exemplo da areia empregada na produção de arga- massas e concretos, causa inúmeros danos ao meio ambiente, como desmatamento e remoção do solo [9]. Além da possibilidade de geração de tais impactos, a disponibilidade de agregados naturais com propriedades satisfatórias para serem empregados na construção civil tem se tornado crítica em muitas áreas urbanas. Neste sentido, vem crescendo as pesquisas relacionadas ao aproveitamento de agregados alternativos em compósitos cimentícios tem crescido cada vez mais, com destaque para a areia de britagem [10, 11], bor- racha de pneu [12], resíduo de vidro [13, 14], resíduos de rochas ornamentais [15], lodo de tratamento de água [16] e de esgoto [17]. Portanto, faz-se necessário buscar o aproveitamento do subproduto da indústria siderúrgica no setor da construção civil, obtendo-se assim, redução nos custos dos produtos da construção, além de reduzir os danos ambientais gerados pelo consumo e extração exacerbados de areia na produção de argamassas e concretos. Neste sentido, o presente trabalho visa analisar as propriedades das argamassas de revestimento no estado fresco e endurecido, com a substituição em volume da areia por escória de aciaria BSSF nas proporções de 0%, 20%, 40%, 60% e 80% no traço 1:1:6 (cimento: cal: agregado). ABSTRACT In 2017, crude steel production in Brazil was 34.4 million tons, which represents a growth of 9.8% compared to 2016. Despite the economic importance for the country's economy, the steel industry represents a major environmental problem, due to the amount of slag generated in the steel production process. In this sense, the present study aimed to investigate the properties of mortars produced with Baosteel Slag Short Flow (BSSF) steel slag to replace natural sand. The mix investigated was 1:1:6 (cement:lime:aggregate), with volume sub- stitution at 0%, 20%, 40%, 60% and 80%. The tests were: consistency index, density, incorporated air con- tent, squeeze-flow, compressive and flexural strength, dynamic modulus of elasticity, capillary absorption, void index, and potential tensile bond strength. It can be seen that for a consistency index of 260 ± 5 mm, the mortars with BSSF steel slag showed a higher w/c ratio, in addition to higher density of apparent mass in the fresh and hardened state, dynamic modulus of elasticity, capillarity coefficient, void index. The reference mortar also presented water retention and potential adhesion resistance higher than the mortars with BSSF steel slag, while the values of compressive strength and physical properties were similar. It is also worth mentioning the significant increase in the dynamic elasticity modulus in mortars with BSSF steel slag, due to the high specific mass of the slag, which can harm the durability of the coating, due to the reduction of the capacity to absorb deformations. p y Keywords: BSSF steel slag. Potential tensile bond strength. Durability. coating mortar. Data de aceite: 17/11/2020 Data de aceite: 17/11/2020 Data de envio: 30/04/2020 10.1590/S1517-707620210003.13031 AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. A cal utilizada foi do tipo hidratada CH-I. O cimento foi CP V, com as propriedades físicas e químicas do descritas na Tabela 1. 2.1 Materiais A cal utilizada foi do tipo hidratada CH-I. O cimento foi CP V, com as propriedades físicas e químicas do descritas na Tabela 1. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Tabela 1: Características físicas e químicas do cimento Portland CP V Propriedade Químicas Norma Unid Resultado Especificação – Norma NBR 16697:2018 Perda ao fogo NM 18/12 % 4,39 ≤ 6,5 Óxido de magnésio – MgO NM 11-2/12 % 4,17 ≤ 6,5 Anidrido sulfúrico – SO3 NM 16/12 % 3,24 ≤ 3,5 Resíduo insolúvel NM 15/12 % 0,69 ≤ 3,5 Anidrido carbônico – CO2 11583/91 % 2,97 ≤ 5,5 Propriedades Físicas Norma Unid Resultado Especificação – Norma NBR 16697:2018 Finura (#200) NBR 11579/12 % 0,20 ≤ 6,0% Resistência à compressão (MPa) NBR 7215/1996 1 dias MPa 25,9 ≥ 14,0 MPa 3 dias MPa 36,6 ≥ 24,0 MPa 7 dias MPa 44,3 ≥ 34,0 MPa Tabela 1: Características físicas e químicas do cimento Portland CP V A escória utilizada neste estudo foi proveniente da Companhia Siderúrgica do Pecém, localizada no Estado do Ceará, Brasil. A escória foi seca em estufa a 105 ± 5°C e realizado o peneiramento, conforme es- tabelece a NBR NM 248 (ABNT, 2003) [18]. Foi realizado o ajuste da granulometria da escória de aciaria. Este procedimento ocorreu da seguinte forma, primeiramente foi realizado a granulometria da areia natural, e em seguida enquadrou-se a escória de aciaria BSSF na mesma curva, a fim de igualar a distribuição granu- lométrica da areia natural, evitando influência do tamanho dos grãos na trabalhabilidade e demais proprieda- des das argamassas, sendo investigada apenas o efeito da substituição do grão. A Figura 1 apresenta a distri- buição granulométrica dos agregados miúdos, enquanto que as características físicas são apresentadas na Ta- bela 2. Nota-se que se classificam na zona utilizável e um pequeno trecho na Zona ótima, conforme NBR 7211 (ABNT, 2009) [19]. Figura 1: Curva granulométrica dos agregados Tabela 2: Propriedades físicas dos agregados miúdos. 2.1 Materiais AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Figura 2: Identificação dos agregados miúdos a) areia natural e b) escória de aciaria BSSF Figura 2: Identificação dos agregados miúdos a) areia natural e b) escória de aciaria BSSF Em relação à caracterização química da escória de aciaria BSSF, foi realizado a fluorescência de raio- X (FRX) em um espectrômetro de raios-X Rigaku ZSX mini II, do Laboratório de Raios X da UFC. Os resul- tados estão na Tabela 3, os quais mostraram que a escória é constituída basicamente de Fe2O3 e CaO (>88%). Tabela 3: Composição química da escória de aciaria BSSF Óxidos Al2O3 SiO2 P2O5 K2O CaO TiO2 Cr2O3 MnO Fe2O3 Co2O3 Amostra (%) 0,63 5,84 1,01 0,04 33,46 0,63 0,21 5,09 52,98 0,12 No que se refere à composição mineralógica da escória de aciaria BSSF, esta foi analisada por meio de difração de raio-X, realizado no Laboratório de Raios-X do Departamento de Física da Universidade Fe- deral do Ceará – UFC. O equipamento utilizado foi o difratômetro para amostras policristalinas modelo Xpert Pro MPD Panalytical. A amostra utilizada neste ensaio foi passante na peneira 200, abertura da malha de 75µm. A amostra foi distribuída num substrato de silício monocristalino, cuja superfície de pó foi o mais plano possível. A radiação utilizada foi de cobalto (Kalfa1) monocromatizado por um conjunto formado de dois cristais de germânio e um espelho parabólico. Nessa análise foi possível identificar principalmente, a presença das fases cristalinas de wustita (FeO) e magnetita (Fe3O4) (Figura 3). Figura 3: Difratograma da escória de aciaria BSSF. Apesar do difratograma apresentar um certo halo amorfo, com os ensaios de determinação do índice de atividade pozolânica, realizados conforme a NBR 5752 (ABNT, 2014) [23], foi possível determinar um índice de atividade pozolânica de 71%, o qual, conforme a norma ABNT NBR 12653 [24] não caracteriza a escória de aciaria BSSF como material pozolânico, pois o valor obtido é inferior ao estabelecido (maior ou igual a 90%). Ademais, ainda conforme as demais exigências da NBR 12653 indicam que o material não é pozolânico. Utilizou-se um equipamento com radiação de cobalto monocromatizada. Apesar das intensidades bai- xas, por volta de 50 a 100, estas não comprometem a identificação das fases. Em adição, tem-se que as inten- sidades altas, aumentaria demasiadamente o tempo de contagem. 2.1 Materiais ENSAIO NORMA UNIDADE AREIA NATURAL ESCÓRIA Dimensão máxima NBR NM 248 (ABNT,2003) [18] mm 2,36 2,36 Módulo de finura NBR NM 248(ABNT, 2003) [18] - 2,52 2,52 Massa unitária NBR NM 45 (ABNT, 2006) [20] g/cm³ 1,41 2,08 Absorção de água NBR NM 52 (ABNT, 2009) [21] % 1,3 2,1 Massa específica NBR NM 52 (ABNT, 2009) [21] g/cm³ 2,59 3,62 Foi verificada a forma dos grãos dos agregados por meio do Aggregate Image Measurement System (AIMS), realizado no Laboratório de Mecânica dos Pavimentos – LMP da Universidade Federal do Ceará UFC. Este ensaio realiza a classificação da forma dos agregados, por meio de parâmetros de forma 2D e an gularidade para agregados miúdos; e angularidade, textura superficial, lamelaridade e esfericidade para agre gados graúdos. Foi possível verificar que a areia natural tende a apresentar partículas mais arredondadas qu a escória de aciaria BSSF, além de textura mais polida, ao passo que a escória de aciaria apresentou textur com baixa rugosidade, conforme a classificação de Al Rousan (2004) [22]. A Figura 2 apresenta a amostra d areia natural e escória de aciaria BSSF. Figura 1: Curva granulométrica dos agregados Figura 1: Curva granulométrica dos agregados Tabela 2: Propriedades físicas dos agregados miúdos. Tabela 2: Propriedades físicas dos agregados miúdos. ENSAIO NORMA UNIDADE AREIA NATURAL ESCÓRIA Dimensão máxima NBR NM 248 (ABNT,2003) [18] mm 2,36 2,36 Módulo de finura NBR NM 248(ABNT, 2003) [18] - 2,52 2,52 Massa unitária NBR NM 45 (ABNT, 2006) [20] g/cm³ 1,41 2,08 Absorção de água NBR NM 52 (ABNT, 2009) [21] % 1,3 2,1 Massa específica NBR NM 52 (ABNT, 2009) [21] g/cm³ 2,59 3,62 Foi verificada a forma dos grãos dos agregados por meio do Aggregate Image Measurement System (AIMS), realizado no Laboratório de Mecânica dos Pavimentos – LMP da Universidade Federal do Ceará – UFC. Este ensaio realiza a classificação da forma dos agregados, por meio de parâmetros de forma 2D e an- gularidade para agregados miúdos; e angularidade, textura superficial, lamelaridade e esfericidade para agre- gados graúdos. Foi possível verificar que a areia natural tende a apresentar partículas mais arredondadas que a escória de aciaria BSSF, além de textura mais polida, ao passo que a escória de aciaria apresentou textura com baixa rugosidade, conforme a classificação de Al Rousan (2004) [22]. A Figura 2 apresenta a amostra de areia natural e escória de aciaria BSSF. 2.1 Materiais Como a amostra apresentava ferro na com- posição, não é recomendado radiação de cobre, pois assim, aumenta-se muito a intensidade do background, comprometendo assim a identificação das fases Figura 3: Difratograma da escória de aciaria BSSF. Figura 3: Difratograma da escória de aciaria BSSF. Apesar do difratograma apresentar um certo halo amorfo, com os ensaios de determinação do índice de atividade pozolânica, realizados conforme a NBR 5752 (ABNT, 2014) [23], foi possível determinar um índice de atividade pozolânica de 71%, o qual, conforme a norma ABNT NBR 12653 [24] não caracteriza a escória de aciaria BSSF como material pozolânico, pois o valor obtido é inferior ao estabelecido (maior ou igual a 90%). Ademais, ainda conforme as demais exigências da NBR 12653 indicam que o material não é pozolânico. p Utilizou-se um equipamento com radiação de cobalto monocromatizada. Apesar das intensidades bai- xas, por volta de 50 a 100, estas não comprometem a identificação das fases. Em adição, tem-se que as inten- sidades altas, aumentaria demasiadamente o tempo de contagem. Como a amostra apresentava ferro na com- posição, não é recomendado radiação de cobre, pois assim, aumenta-se muito a intensidade do background, comprometendo assim a identificação das fases. Foi investigado ainda a expansibilidade da escória BSSF com base no ensaio de determinação da ex- pansibilidade do cimento pelo método de Le Chatelier, conforme NBR 11582 (ABNT, 2016) [25]. A escória AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. de aciaria BSSF não apresentou expansão a frio e em relação à expansão a quente, os valores obtidos foram na ordem de 1,16mm, encontrando-se dentro do limite máximo especificado de 5 mm pela ABNT NBR 16697 (2018). de aciaria BSSF não apresentou expansão a frio e em relação à expansão a quente, os valores obtidos foram na ordem de 1,16mm, encontrando-se dentro do limite máximo especificado de 5 mm pela ABNT NBR 16697 (2018). A amostra de escória de aciaria BSSF utilizada foi passante na peneira 200, a qual foi obtida por meio de triturador mecânico. Para esse método foi adotado um teor de substituição de cimento por escória de acia- ria BSSF de 50%, em volume. Para MEHTA, 1999 apud MASUERO, 2001 [26], esse teor é suficiente para que o fenômeno ocorra, caso o material possua características expansivas. 2.1 Materiais Destaca-se que a mesma não apre- sentou expansão a frio e em relação à expansão a quente, todos os resultados obtidos foram inferiores a 5 mm. 2.2 Métodos de pesquisa As argamassas foram preparadas de acordo com a NBR 16541 (ABNT, 2016) [27]. A substituição da areia natural por escória foi realizada em volume, por meio das massas específicas, nos teores de 0%, 20%, 40%, 60% e 80% de substituição. Os traços unitários e o consumo de materiais por m³ estão apresentados na Tabe- la 4. A substituição da areia por escória de aciaria foi dada em volume, conforme a equação 1, sendo m (a massa do material) e (massa específica do material). Essa substituição se fez necessário para que os traços tivessem a mesma quantidade de cimento por m³, e assim comparar os resultados com o traço de referência (ARG0). (1) (1) Tabela 4: Traços utilizados na pesquisa. Tabela 4: Traços utilizados na pesquisa. TRAÇO UNITÁRIO EM MASSA CONSUMO DE MATERIAL POR M³ TRAÇO TEOR (%) CIMENTO (KG) CAL (KG) AREIA (KG) ESCÓRIA (KG) CIMENTO (KG) CAL (KG) AREIA (KG) ESCÓRIA (KG) ÁGUA (KG) ARG0 0 1,0 0,4 4,70 0,00 296,8 118,7 1395,2 0,0 317,9 ARG20 20 1,0 0,4 3,8 1,3 296,0 118,4 1112,9 387,7 320,2 ARG40 40 1,0 0,4 2,8 2,6 295,1 118,1 832,3 776,2 321,7 ARG60 60 1,0 0,4 1,9 3,9 293,6 117,4 551,9 1156,7 325,6 ARG80 80 1,0 0,4 0,9 5,7 292,1 116,9 274,6 1536,7 328,4 CONSUMO DE MATERIAL POR M³ A consistência foi determinada por meio do ensaio da mesa de consistência (flow table), no qual foi obtida a quantidade de água para se obter um espalhamento de 260 ± 5 mm, conforme NBR 13276 (ABNT, 2016) [28]. Após a fixação da relação a/c, as argamassas foram produzidas para analisar as demais proprie- dades no estado fresco. A determinação da densidade foi realizada pelo método gravimétrico, conforme a NBR 13278 (ABNT, 2005) [29] logo após a mistura. O teor de ar incorporado foi calculado a partir da densidade, quantidade de água e massa específica dos materiais. A retenção de água das argamassas foi determinada conforme prescre- ve a NBR 13277 (ABNT, 2005) [30], aplicando-se uma sucção de 51 mmHg durante 15 minutos na argamas- sa. Por fim, para investigar o comportamento reológico das argamassas, foi realizado o squeeze-flow, estabe- lecido pela NBR 15839 (ABNT, 2010) [31], o qual foi utilizado uma célula de carga de 2kN, o ensaio foi realizado no intervalo de tempo de 10 minutos após a mistura, a uma velocidade de 3,0 mm/s. Sendo o en- saio finalizado quando a amostra apresentar um deslocamento máximo de 9 mm ou carga máxima de 1 kN, o que ocorrer primeiro. No ensaio do squeeze flow, foi identificado ainda as fases do comportamente. Conforme Cardoso; Pi- leggi John (2005) [32] explicam que o resultado do squeeze-flow é uma curva carga versus deslocamento, dividido em 3 estágios, conforme Figura 4 AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Figura 4: Perfil típico de carga vs. deslocamento de um ensaio de squeeze-flow. Fonte: CARDOSO et al.(2005) Figura 4: Perfil típico de carga vs. deslocamento de um ensaio de squeeze-flow. Fonte: CARDOSO et al.(2005) Conforme Cardoso et al.(2005) [32] explicam que no estágio I (pequenas deformações) o material comporta-se como um sólido, apresentando deformação elástica linear. Tabela 4: Traços utilizados na pesquisa. Uma argamassa que apresente comportamento com uma parcela significativa nesse estágio, possivelmente apresentará problemas de fissuração ainda no estado fresco devido à recuperação elástica após a retirada do esforço (após a passagem da desempenadeira). No estágio II (deslocamentos intermediários) à compressão resulta em deformações radial elongacional e de cisalhamento. O material flui por deformação plástica e/ou viscosa dependendo das suas características constitutivas. Nesta etapa, o material é capaz de sofrer grandes deformações sem aumento significativo da força necessária para o deslocamento, o que aparenta ser um comportamento apropriado para aplicação e espalhamento de argamassas [32]. Por fim, no estágio III, o material é submetido a grandes deformações. Pode ocorrer um aumento exponencial da carga necessária para o deslocamento da argamassa. Devido à aproximação das partículas dos materiais constituintes, as deformações geram forças restritivas 29 ao fluxo. Este estágio relaciona-se com os procedimentos de aplicação e acabamento da argamassa devem ser dificultados, devido às altas cargas necessárias para deformá-lo, possivelmente levando a um acabamento defeituoso [32]. Em relação aos ensaios no estado endurecido, foram todos realizados aos 28 dias. Foram moldados corpos de prova prismáticos de dimensões 40 mm x 40 mm x 160 mm para o ensaio de resistência à com- pressão e à tração, módulo de elasticidade dinâmico (considerando o coeficiente de Poisson 0,20), densidade aparente e absorção de água por capilaridade, respectivamente estabelecidos pelas NBR 13279 (ABNT, 2005) [33], NBR 15630 (ABNT, 2008) [34], NBR 13280 (ABNT, 2005) [35], e NBR 15259 (ABNT, 2005) [36], sendo 3 corpos de prova para cada ensaio. Para o ensaio de módulo de elasticidade dinâmico (Ed), foram realizadas 3 determinações em corpos de provas distintos. Primeiramente, determinou-se a velocidade de propagação ultrassônica (VPU) por meio do equipamento Pundit Lab+, com frequência dos transdutores na ordem de 54 Hz, conforme Equação 2, sendo L a distância entre os pontos de acoplamento dos transdutores (comprimentos do corpo de prova, em mm) e t – o tempo registrado pelo mostrador digital, em µs. Em seguida. O módulo de elasticidade dinâmico foi determinado conforme Equação 3. 3.1 Estado fresco A Tabela 5, apresenta a caracterização das argamassas quanto as propriedades no estado fresco. A Tabela 5, apresenta a caracterização das argamassas quanto as propriedades no estado fresc Tabela 5: Propriedades no estado Fresco. Teor de água (%) Relação a/c Índice de consistência (mm) Densidade de massa (kg/m³) Teor de ar incorporado (%) Retenção de água (%) ARG0 14,9 1,071 257 2048 4 91 ARG20 14,3 1,082 261 2142 4 84 ARG40 13,7 1,090 263 2220 5 81 ARG60 13,3 1,109 263 2323 5 81 ARG80 12,9 1,124 259 2452 4 83 As argamassas com escória de aciaria BSSF apresentaram relação a/c superior à da argamassa convencional, produzida apenas com areia natural, para obter o espalhamento fixado em 260 ± 5 mm, conforme NBR 13276 (ABNT, 2005) [28]. Isto se deve a maior absorção de água da escória (Tabela 2), em comparação a areia natural, bem como a forma dos grãos (conforme análise dos grãos realizado pelo AIMS). Estudo realizado por LE; SHEEN; BUI (2017) [39] com escória de aciaria diferente da BSSF, constataram que o aumento do teor de substituição do agregado natural por escória ocasiona uma menor fluidez nas argamassas no estado plástico, o que pode vir a interferir na determinação da relação a/c pelo ensaio flow table. Os autores alegam que esse fato é causado pela textura da superfície áspera e pela forma angular da escória de aciaria, se comparada com a forma cúbica do agregado natural. Em relação à densidade das argamassas no estado fresco, houve crescimento representativo com o aumento do teor de substituição de areia por escória, atingindo um aumento de quase 20% em entre os traços ARG0 e ARG80. O aumento se deve as características dos materiais, tendo em vista que a massa específica da escória é maior que a da areia [40-42]. Quanto ao teor de ar, nota-se que esta propriedade não foi afetada pela substituição da areia natural pela escória de aciaria. Cabe citar que os valores são baixos, na ordem de 4% a 5% devido à não utilização de aditivos. Para argamassas de revestimento produzidas sem aditivos, sejam argamassas simples ou mistas, o teor de ar geralmente encontra-se em torno de 2 a 5 % do volume total [43]. Sendo: Sendo: ρ – densidade de massa aparente no estado endurecido da argamasssa (kg/m³) ρ – densidade de massa aparente no estado endurecido da argamasssa (kg/m³) ρ – densidade de massa aparente no estado endurecido da argamasssa (kg/m ρ p v – velocidade de propagação ultrassônica (mm /µs) v – velocidade de propagação ultrassônica (mm /µs) µ - coeficiente de poisson, sendo usualmente 0,20 nesta equação. Foram moldados ainda corpos de prova cilíndricos de 5cm de diâmetro por 10 cm de altura para os ensaios de índice de vazios, absorção de água e massa específica, conforme NBR 9778 (ABNT, 2005) [37]. Por fim, foram confeccionados painéis de bloco cerâmico com ranhuras ao ar livre, os quais foram umedeci- dos e logo em seguida foi realizado o preparo da base com aplicação manual de chapisco com consistência fluida no traço em volume de 1:3 (cimento CP V e areia grossa seca, com módulo de finura 3,02), no intuito de melhorar a aderência do revestimento. Após 7 dias de cura do chapisco, foram revestidos com os traços em estudos nas dimensões 50 cm x 50 cm x 2,5 cm, executados de forma manual, e com o mesmo profissio- nal que executou o chapisco, levando aproximadamente 1h e 30 min para executar os painéis. Após 28 dias foram realizados os ensaios de aderência, conforme preconiza a NBR 13528 (ABNT, 2010) [38]. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Por fim, para a análise e tratamento dos dados se deu por meio da Análise de Variância (ANOVA) e Teste de Tukey, por meio do software Statistic 7.0. Inicialmente, determinou-se os resíduos padronizados e eliminação dos dados espúrios, sendo considerados dados espúrios aqueles com valor excedente a um valor de referência de ±1,96 nos resíduos padronizados, a fim de que se tivesse um nível de confiança de 95% ou nível de significância de 0,05. 3.1 Estado fresco Este valor corresponde ao ar aprisionado, cujas bolhas de ar são irregulares e não estáveis, resultante do aprisionamento durante a mistura, enquanto que o ar incorporado é composto por bolhas de ar estáveis com aspecto de esferas microscópicas, resultante do uso de aditivos [43]. Em relação a retenção de água, nota-se que houve uma redução desta propriedade com o aumento do teor de substituição. Isto se deve possivelmente em virtude da maior relação a/c das argamassas com escória, pois conforme [44], a argamassa se torna menos retentora de água quando a proporção de água:aglomerante é aumentada. A Figura 5 apresenta os resultados obtidos pela ensaio do squeeze-flow. Constata-se que para o teor de água definido conforme o índice de consistência fixado em 260 ± 5mm, conforme a NBR 13276 (ABNT, 2016) [28], para a carga máxima do ensaio de 1kN, o deslocamento foi abaixo de 2 mm para as argamassas. Isto demonstra a alta consistência das argamassas, pouco adequadas para aplicação. A utilização de uma maior quantidade de água ou ainda o uso de aditivos auxiliaria na lubrificação, e consequentemente reduzindo as cargas de compressão. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Figura 5: Squeeze-flow das argamassas sem (referência) e com escória BSSF (3 mm/s). Figura 5: Squeeze-flow das argamassas sem (referência) e com escória BSSF (3 mm/s). Destaca-se ainda que a argamassa de referência, que apresentou uma menor relação a/c, foi a que demonstrou o melhor comportamento entre as argamassas. O traço ARG80 apesar da maior quantidade de escória de aciaria BSSF, era esperado um menor deslocamento máximo, entretanto, possivelmente em virtude da maior relação a/c, pode ter ocasionado esse aumento no deslocamento máximo, tendo em vista que as me- didas de índice de consistência por si só, não são suficientes para determinar o comportamento do material (CARDOSO et al, 2009) [45]. 3.1 Estado fresco Cabe destacar ainda que valores baixos de deslocamentos máximos encontrados no ensaio do squeeze flow podem estar relacionados com a etapa de mistura, pois ao investigar diversos métodos de mistura, inclu- indo o preconizado pela NBR 13276:20016, França et al., (2013) [46] constataram que o referido procedi- mento de mistura, o mesmo adotado nesta pesquisa, atingiu menores valores de deslocamento, se comparados com os demais procedimentos, o que demonstra que esse procedimento não foi capaz de dispersar as partícu- las aglomeradas com eficiência, resultando em unidades móveis (aglomerados) maiores, o que dificulta o fluxo da argamassa. Em adição, tem-se que todas as argamassas não apresentaram o estágio II (saltando do estágio I para o estágio III). As curvas mostram uma mudança de um estágio elástico diretamente para o strain hardening, fase III, o qual a carga aumenta consideravelmente, com pequeno acréscimo de deformação. O segundo estágio, ausente nas argamassas, relaciona-se com à deformação plástica do material, onde a argamassa é capaz de sofrer deformações sem aumento expressivo da força, o que torna um comportamento apropriado para aplicação e espalhamento da argamassa [32]. O crescimento exponencial das cargas caracteriza o enrijecimento por deformação, causado por altos níveis de atrito entre os agregados. Devido aos baixos teores de ar na argamassa, os agregados ficam muito próximos dificultando o fluxo do material. Por outro lado, o aumentando do teor de ar nas argamassas ocasiona um aumento no teor de pasta, mantendo os agregados distantes e lubrificados [47]. Com isto, as argamassas com elevado teor de ar apresentam comportamento predominantemente plástico, para os níveis de deformação impostos. As bolhas de ar aumentam o volume ocupado pela pasta e reduzem sua resistência, promovendo facilidade do fluxo, bem como do escorregamento dos grãos [32]. p g g [ ] No mesmo sentido, o estudo realizado por Amancio (2019) [48], tambem foi constatado a redução do deslocamento máximo pelo squeeze-flow nas argamassas com escória de aciaria, o qual foi justificado pelo baixo teor de ar das argamassas, ausencia de aditivo, método de mistura e dimensão máxima caracteristica do agregado (2,36mm). 3.2 Estado endurecido A Tabela 6 apresenta a caracterização das argamassas quanto às propriedades mecânicas no estado ecido. A Tabela 6 apresenta a caracterização das argamassas quanto às propriedades mecânicas no estado endurecido. Tabela 6: Propriedades mecânicas no estado Endurecido. Traço Resistência à compressão (MPa) Resistência à tração na flexão (MPa) Densidade de massa (kg/m³) Módulo dinâmico (GPa) Média (MPa) DP. CV (%) Média (MPa) DP. CV (%) Média (MPa) DP. CV (%) Média (MPa) DP. CV (%) ARG0 14,28 0,82 5,74 4,75 1,18 24,84 2,05 0,02 0,98 16,70 0,29 1,74 ARG20 15,88 0,89 5,61 4,67 0,25 5,35 2,11 0,03 1,42 22,50 1,21 5,38 ARG40 14,01 1,47 10,49 2,92 0,26 8,90 2,21 0,03 1,36 23,20 1,08 4,66 ARG60 13,19 1,38 10,46 4,20 0,31 7,38 2,21 0,02 0,91 24,10 2,22 9,21 ARG80 13,81 1,44 10,43 4,67 0,25 5,35 2,30 0,05 2,17 20,30 1,42 7,00 Tabela 6: Propriedades mecânicas no estado Endurecido AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. A Figura 6 apresenta os valores de resistências médias à compressão e tração na flexão das argamassas. Quanto aos resultados de resistência à tração na flexão, com exceção do traço ARG40, os demais traços não existem diferenças significativas conforme o teste de Tukey de comparação de médias, apesar dos valores da relação a/c serem superiores aos do traço ARG0. Entretanto, cabe destacar que o resultado da resistencia à tração na flexão para o traço ARG40 não foi esperado, tendo em vista que os a mistura da argamassa, bem como os procedimentos de moldagem foram realizados no mesmo dia dos demais, bem como pelo mesmo operador. Figura 6: Resistência à compressão média (a) e tração na flexão (b) das argamassas com e sem escória de aciaria BSSF. a) b) Figura 6: Resistência à compressão média (a) e tração na flexão (b) das argamassas com e sem escória de aciaria BSSF. Já quanto a resistência à compressão, tem-se destaque para o traço ARG20, o que houve um aumento da resistência se comparado com o traço de referência, e ambos os traços apresentaram um baixo desvio padrão, menor que 0,90, conforme Tabela 6. Isso é justificado pela forma multi-angular do agregado de escória, levando a um melhor atrito entre partículas e matriz [38]. 3.2 Estado endurecido revista Matéria, v.26, n.3, 2021. Figura 7: Resistência potencial de aderência à tração das argamassas. Figura 7: Resistência potencial de aderência à tração das argamassas. Em relação ao fator a/c, o aumento do consumo de água reduz as propriedades mecânicas das argamassas, incluindo a resistência potencial de aderência a tração, fato também constatado por Silva; Campiteli (2008) [51] que constataram que o aumento da relação a/c das argamassas gera uma redução da resistência de aderência da argamassa. Quanto a retenção de água, as argamassas com escória de aciaria apresentaram menores valores de retenção de água (Tabela 5), o que afeta negativamente a aderência das argamassas, ao passo que a rápida perda de água da argamassa causa um comprometimento na sua aderência, resistência mecânica, capacidade de absorver deformações e consequentemente provoca perda da durabilidade e estanqueidade [52]. No mais, como as argamassas com escória de aciaria BSSF atingiram menores valores de deslocamento máximo na carga máxima do ensaio (1 kN), isto pode influenciar negativamente na resistência potencial de aderência das argamassas. Silva et al. (2005) [53] explica que as argamassas trabalham em função da sua capacidade de fluir e de se deformar, quando submetida a uma determinada tensão de cisalhamento, apresentando um contato mais extenso com o substrato, otimizando o mecanismo de aderência. Quanto ao módulo de elasticidade dinâmico (Tabela 6), as argamassas com escória tiveram valores superiores aos da argamassa de referência. Isto se deve possivelmente devido a massa específica da escória (3,86 g/cm³) ser superior ao da areia natural (2,59 g/cm³), com isto, tem-se um aumento da Velocidade de propagação de pulso ultrassônico, e consequentemente, o aumento do módulo de elasticidade dinâmico. CARRIJO [54], em estudo experimental constatou que concretos com agregados mais densos tendem a gerar concretos com maiores valores de módulo de elasticidade. Mesmo fato constatado por AMANCIO et al. [55] ao investigar diversos traços de argamassas com escória de aciaria BSSF. Nota-se que para o traço ARG80, o valor de módulo de elasticidade dinâmico reduz, ocorrendo uma inversão dos demais valores. Isso se deve possivelmente em virtude do referido traço apresentar a maior relação a/c dos traços investigados, ao passo que conforme aumenta-se o consumo de água, aumenta-se a porosidade do material, e consequentemente, reduz-se a velocidade de propagação de pulso ultrassônico e o módulo de elasticidade dinâmico. 3.2 Estado endurecido Para os demais teores de substituição de areia por escória, houve redução da resistência mecânica, pelo aumento mais elevado da relação a/c, fato também constatado de perda de resistência por Campos et al (2018) [39] utilizando escória de aciaria BSSF e Lacerda (2015) [49] ao utilizar escória de aciaria de forno elétrico a arco (FEA). Entretanto, conforme o teste de comparação de médias de Tukey, foi possível constatar que com exceção do traço ARG20, que apresentou um aumento significativo na resistência à compressão, para os demais traços não existem diferenças significativas. Em adição ressalta-se que ocorre uma sobreposição de efeitos na resistência à compressão das argamassas, geradas pela relação a/c e pela massa unitária dos agregados. Por um lado, os traços com maiores teores de escória de aciaria BSSF, apresentam maiores valores de relação a/c, o que ocasiona uma redução na resistência. Por outro, o aumento do teor de substituição da areia natural por escória de aciaria, ocasiona um aumento da massa unitária da mistura referente à parcela do agregado, pois conforme Tabela 2, a massa unitária da escória de aciaria BSSF (2,08 g/cm³) é aproximadamente 48% superior à da areia natural (1,48 g/cm³). Neste sentido, Carasek et al (2018) [50] explica que as resistências das argamassas (à compressão e à tração na flexão) e a massa unitária dos agregados podem ser correlacionadas por regressão linear crescente (R>0,8), ou seja, com o aumento da massa unitária ocasiona um aumento da resistência. Os resultados de resistência potencial de aderência das argamassas são apresentados na Figura 7. Observou-se que as argamassas com maiores teores de escória, apresentaram valores inferiores ao da argamassa de referência. Entretanto, apesar do valor quantitativo da argamassa de referência ser superior as demais argamassas com escória, foi verificado por meio da ANOVA que a substituição da areia natural por escória não influencia significativamente nas propriedades de aderência das argamassas. O mesmo fato constatado por Santamaría-Vicario et al., 2015; Campos et al, 2018 [40, 41]. Isto pode ser justificado por inúmeros fatores, como a maior relação a/c das argamassas com escória de aciaria BSSF, menores valores de retenção de água, e baixo deslocamento obtido pelo ensaio do squeeze-flow. Informa-se ainda que o resulta- do do traço ARG40 foi inesperado, em virtude de ambos os traços de argamassas terem sidos realizados nas mesmas condições climáticas e pelo mesmo operador. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. 3.2 Estado endurecido Os elevados valores de módulo de elasticidade dinâmico (Ed) de todas as argamassas pode ser justificado pelo baixo teor de ar das argamassas. As bolhas de ar aumentaram a capacidade de deformação da argamassa e reduziram sua rigidez [56]. Ao passo que conforme a Tabela 5, os valores de teor de ar ficaram compreendidos entre 1 e 5%. Para argamassas de revestimento produzidas sem aditivos, seja argamassas simples ou mistas, o teor de ar geralmente encontra-se em torno de 2 a 5 % do volume total [43]o que vem de acordo com os resultados obtidos. Os elevados valores de módulo de elasticidades podem afetar negativamente a durabilidade do revestimento, tendo em vista que quanto menor o valor do módulo, maior é a capacidade da argamassa absorver deformações [57, 58]. Com isto, os revestimentos com valores de módulo de elasticidade mais elevados são mais susceptíveis a sofrerem fissuração, o que afeta diretamente a a estanqueidade e consequentemente a vida útil do revestimento. Conforme as recomendações do Centre Scientifique et Techinique de la Construction (CSTB) [59], o grau de fissurabilidade se classifica em Baixa Fissurabilidade (Ed ≤ 7,0 GPa), Média Fissurabilidade (7,0 GPa ≤ Ed ≤ 12,0 GPa) e Alta Fissurabilidade (Ed ≥ 12,0 GPa). Em consoante, a referida norma ainda traz a correlação entre o módulo de elasticidade dinâmico (Ed) e a resistência à tração na flexão (ft) e o potencial de fissurabilidade, sendo Ed/ft ≤ 25, 25< Ed/ft<35 e Ed/ft > 35, respectivamente para baixa, média e alta fissurabilidade, com isto, todas as argamassas têm alta fissurabilidade. A Tabela 7 apresenta a caracterização das argamassas quanto às propriedades físicas no estado endurecido. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. Tabela 7: Propriedades físicas do concreto no estado Endurecido Traço Índice de vazios (%) Absorção de água total (%) Coeficiente de capilaridade (g/dm².min0,5) Média DP. CV Média DP. CV Média DP. CV ARG0 25,30 2,73 10,79 14,00 1,40 10,00 7,00 1,29 18,43 ARG20 25,40 0,37 1,46 13,00 0,24 1,85 7,00 0,46 6,57 ARG40 25,90 0,35 1,35 13,00 0,24 1,85 10,00 0,31 3,10 ARG60 26,00 0,30 1,34 12,00 0,19 1,58 8,00 1,75 21,88 ARG80 26,70 0,33 1,15 12,00 0,13 6,50 10,00 0,59 5,90 O índice de vazios e o coeficiente de capilaridade das argamassas, foram maiores nas argamassas com escória de aciaria, possivelmente devido à maior relação a/c. 4. CONCLUSÕES As argamassas com escória de aciaria BSSF são menos trabalháveis e demandam uma maior relação a/c para obter o mesmo espalhamento. Quanto ao comportamento reológico, as argamassas em estudo apresentaram baixo deslocamento, além de não apresentarem o estágio II na curva do squeeze-flow, sendo o traço de refe- rência a que apresentou o melhor resultado. Em relação a massa específica no estado fresco e endurecido das argamassas, verificou-se um incre- mento com o aumento o teor de escória, devido a maior massa específica do agregado. A retenção de água foi maior na argamassa de referência, sem escória de aciaria, possivelmente em virtude na menor relação a/c, o que melhora suas propriedades no estado endurecido. A argamassa de referência apresentou menor valor de módulo de elasticidade, coeficiente de capilaridade e índice de vazios, além de maior valor de resistência de aderência. As argamassas com escória de aciaria BSSF apresentaram elevados valores de módulo de elasticidade dinâmica. Com isto, estas argamassas tendem a ser mais propensas a fissuração, em virtude da menor capacidade de sofrer deformação, sendo viável investigar o comportamento dessas argamassas com aditivos, como por exemplo, incorporador de ar. Nesta pesquisa, para os materiais e metodologias adotadas, tem-se destaque para o teor de substituição de 20%, o qual apresentou valores de resistência mecânica superiores ao da argamassa de referência. Nos ensaios de índice de vazios, capilaridade e absorção de água, o teor de 20% apresentou desempenho igual ou melhor que o traço de referência, favorecendo assim a durabilidade do revestimento. Em adição, destaca-se que com exceção da retenção de água, que foi inferior para o teor de 20 % de substituição, se comparado com o traço de referência, as demais propriedades se enquadram na mesma classe proposta pela NBR 13281 (ABNT, 2005). 3.2 Estado endurecido O valor de vazios reflete os valores de resistência mecânica, no qual maiores índices de vazios tendem a gerar argamassas com menores valores de resistência à compressão. p Por fim, a Tabela 8 apresenta a classificação das argamassas conforme a NBR 13281 (ABNT, 2005) [60]. Constata-se que apesar da substituição da areia por escória nas argamassas alterar as propriedades no estado fresco e endurecido, com exceção da propriedade de retenção de água, todas as propriedades se enquadraram na mesma classe. Tabela 8: Classificação das argamassas conforme NBR 13281:2005 Traço Resistência à compressão Densidade aparente endurecido Resistência à tração na flexão Coeficiente de capilaridade Densidade aparente fresco Retenção de água Resistência de aderência Classes ARG0 P6 M6 R6 C5 D6 U5 A3 ARG20 P6 M6 R6 C5 D6 U3 A3 ARG40 P6 M6 R5 C5 D6 U3 A3 ARG60 P6 M6 R6 C5 D6 U3 A3 ARG80 P6 M6 R6 C5 D6 U3 A3 Tabela 8: Classificação das argamassas conforme NBR 13281:2005 Os autores agradecem ao Programa de Pós-Graduação em Engenharia Civil: Estruturas e Construção Civil (PEC) e ao Laboratório de Materiais e Construção Civil (LMCC) da Universidade Federal do Ceará – UFC. 6. BIBLIOGRAFIA 2020, v.25, n.1 [14] RIBEIRO, U.G., SANTOS, D.F. Physical-mechanical potential properties of wastes from glass lapping to produce mortar as partial replacement of the conventional aggregate. Rev. IBRACON Estrut. Mater. [online]. 2020, v.13, n.1 5] SOUZA, N.S.L., et al. 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NBR NM 45: Agregados – Determinação da massa unitária e do volume de vazios. Rio de Janeiro, 2006. [21] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 52. Agregado miúdo - Determinação da massa específica e massa específica aparente. Rio de Janeiro, 2009 ç p p p [22] AL ROUSAN, T.M. (2004) Characterization of Aggregate Shape Properties Using a Computer Automated System. 2004. 211 f. Tese (Doutorado em Engenharia Civil), Office of Graduate Studies, Texas A&M University, Texas, 2004. [23] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 5752: Materiais pozolânicos — Determinação do índice de desempenho com cimento Portland aos 28 dias Rio de Janeiro 2014 [22] AL ROUSAN, T.M. (2004) Characterization of Aggregate Shape Properties Using a Computer Automated System. 2004. 211 f. Tese (Doutorado em Engenharia Civil), Office of Graduate Studies, Texas A&M University, Texas, 2004. y, , [23] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. 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Mortars Made with Fine Granulate from Shredded Tires. Journal of Materials in Civil Engineering. V. 25(4), pp. 519-529, 2013 [9] CHAVES, M.A., SANTOS, B.E. Uso de imagens CBERS na identificação de áreas de extração de areia na Região Metropolitana de Salvador – BA XIII Simpósio Brasileiro de Sensoriamento Remoto, Florianópolis, Brasil, 21 a 26 abril de 2007. INPE. [10] TOKARSKI, R.B., MATOSKI, A., CECHIN, L., et al., Comportamento das argamassas de revestimento no estado fresco, compostas com areia de britagem de rocha calcária e areia natural. Matéria (Rio J.) [online]. 2018, v.23, n.3 [11] PIMENTEL, L.L., PISSOLATO JUNIOR, O., JACINTHO, A.E.P.G.A., et al., Argamassa com areia proveniente da britagem de resíduo de construção civil – Avaliação de características físicas e mecânicas. Matéria (Rio J.) 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NBR 15630: Argamassa para assentamento e revestimento de paredes e tetos - Determinação do módulo de elasticidade dinâmico através da propagação de onda ultrassônica. Rio de Janeiro, 2009. , [35] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13280: Argamassa para assentamento e revestimento de paredes e tetos – determinação da densidade de massa aparente no estado endurecido. Rio de Janeiro, 2005. 6. BIBLIOGRAFIA Dissertação (Mestrado em Engenharia Civil) Universidade de Brasilia Faculdade de Tecnologia Brasilia DF Brasil steelmaking slag masonry mortars. Materials and design, v. 97, pp. 307-315, 2016. [43] DO Ó, S.W. Análise da retenção de água em argamassas de revestimento aditivadas. Dissertação (Mestrado em Engenharia Civil). Universidade de Brasilia. Faculdade de Tecnologia. Brasilia, DF, Brasil, 2004. [44] INCE, C., CARTER, A., WILSON, M.A., et al., Factors affecting the water retaining characteristics of limeand cement mortars in the freshly-mixed state. Materials and Structures, v. 44, pp. 509–516, 2011. [45] CARDOSO, F.A., JOHN, V.M., PILEGGI, R.G. Rheological behavior of mortars under different squeezing rates. Cement and concrete research, v. 39, n. 9, pp. 748-753, 2009. [46] FRANÇA, M.S., CARDOSO, F.A., PILEGGI, R.G. Influence of laboratory mixing procedure on the properties of mortars. Ambiente construído, Porto Alegre, v. 13, n. 2, pp. 111-124, 2013. [47] CARDOSO, F.A., CAMPORA, F.L., PILEGGI, R.G., et al., Caracterização reológica de argamassas do mercado por squeeze-flow. VII Simpósio Brasileiro de Tecnologia de Argamassas. Anais... Pernambuco, Recife, 2007. [48] AMANCIO, F.A. Estudo de propriedades no estado fresco de argamassas de revestimento com escória de aciaria BSSF. Dissertação (mestrado). Programa de Pós graduação em Engenharia Civil: Estruturas e Construção Civil. Universidade Federal do Ceará, Fortaleza, 2019, 85p. AMANCIO, F.A.; DIAS, A.R.O.; LIMA, D.A., et al.,. revista Matéria, v.26, n.3, 2021. 9] LACERDA, C. Traços de argamassa utilizando escória de aciaria elétrica. Dissertação (Mestrado) niFOA / Mestrado Profissional em Materiais, Volta Redonda: UniFOA, 2015. 70 p. [49] LACERDA, C. Traços de argamassa utilizando escória de aciaria elétrica. Dissertação (Mestrado) – UniFOA / Mestrado Profissional em Materiais, Volta Redonda: UniFOA, 2015. 70 p. [50] CARASEK, H., GIRARDI, A.C.C., ARAÚJO, R.C., et al., Estudo e avaliação de agregados reciclados de resíduo de construção e demolição para argamassas de assentamento e de revestimento. Cerâmica, v. 64, pp. 288-300, 2018. p [50] CARASEK, H., GIRARDI, A.C.C., ARAÚJO, R.C., et al., Estudo e avaliação de agregados reciclados de resíduo de construção e demolição para argamassas de assentamento e de revestimento. Cerâmica, v. 64, pp. 288-300, 2018. [51] SILVA, N.G., CAMPITELI, V.C. Correlação entre módulo de elasticidade dinâmico e resistências mecânicas de argamassas de cimento, cal e areia. Ambiente Construído, Porto Alegre, v. 8, n. 4, pp. 21-35, out./dez. 2008. [52] BAÍA, L.L.M., SABBATINI, F.H. Projeto e execução de revestimento de argamassa. 4. ed. São Paulo, SP. O Nome da Rosa 2008, 88p. 6. BIBLIOGRAFIA In: BT/PCC/359 – Boletim Técnico da Escola Politécnica da USP, Departamento de Engenharia de Construção, São Paulo, 2004 [59] CSTC – CENTRE SCIENTIFIQUE ET TECHNIQUE DE LA CONSTRUCTION. Hydrofuges de surface: choix et mise em oeuvre. Bruxelles, 1982. 24 p. (Note D`Information Technique – NIT n. 140) Ã É [58] NAKAKURA, E.H., CINCOTTO, M.A. Análise dos requisitos de classificação de argamassas de assentamento e revestimento. In: BT/PCC/359 – Boletim Técnico da Escola Politécnica da USP, Departamento de Engenharia de Construção, São Paulo, 2004 [59] CSTC – CENTRE SCIENTIFIQUE ET TECHNIQUE DE LA CONSTRUCTION. Hydrofuges de surface: choix et mise em oeuvre. Bruxelles, 1982. 24 p. (Note D`Information Technique – NIT n. 140) [60] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. 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https://openalex.org/W2239585800	https://www.revistas.usp.br/clinics/article/download/109998/108611	English	null	Is Doppler ultrasound useful for evaluating gestational trophoblastic disease?	Clinics	2,015	cc-by	5,362	Is Doppler ultrasound useful for evaluating gestational trophoblastic disease? Lawrence H. Lin,* Lisandra S. Bernardes, Eliane A. Hase, Koji Fushida, Rossana P.V. Francisco Faculdade de Medicina da Universidade de Sa˜o Paulo, Departamento de Obstetrı´cia e Ginecologia, Centro de Doenc¸as Trofobla´sticas, Sa˜o Paulo/SP, Brazi Doppler ultrasound is a non-invasive method for evaluating vascularization and is widely used in clinical practice. Gestational trophoblastic neoplasia includes a group of highly vascularized malignancies derived from placental cells. This review summarizes data found in the literature regarding the applications of Doppler ultrasound in managing patients with gestational trophoblastic neoplasia. The PubMed/Medline, Web of Science, Cochrane and LILACS databases were searched for articles published in English until 2014 using the following keywords: ‘‘Gestational trophoblastic disease AND Ultrasonography, Doppler.’’ Twenty-eight articles met the inclusion criteria and were separated into the 4 following groups according to the aim of the study. (1) Doppler ultrasound does not seem to be capable of differentiating partial from complete moles, but it might be useful when evaluating pregnancies in which a complete mole coexists with a normal fetus. (2) There is controversy in the role of uterine artery Doppler velocimetry in the prediction of development of gestational trophoblastic neoplasia. (3) Doppler ultrasound is a useful tool in the diagnosis of gestational trophoblastic neoplasia because abnormal myometrial vascularization and lower uterine artery Doppler indices seem to be correlated with invasive disease. (4) Lower uterine artery Doppler indices in the diagnosis of gestational trophoblastic neoplasia are associated with methotrexate resistance and might play a role in prognosis. CONCLUSION: Several studies support the importance of Doppler ultrasound in the management of patients with gestational trophoblastic neoplasia, particularly the role of Doppler velocimetry in the prediction of trophoblastic neoplasia and the chemoresistance of trophoblastic tumors. Doppler ﬁndings should be used as ancillary tools, along with human chorionic gonadotropin assessment, in the diagnosis of gestational trophoblastic neoplasia. KEYWORDS: Gestational Trophoblastic Disease; Trophoblastic Neoplasms; Hydatidiform Mole; Ultrasonography, Doppler; Neovascularization, Pathologic. des LS, Hase EA, Fushida K, Francisco RP. Is Doppler ultrasound useful for evaluating gestational trophoblastic disease? C 10-815 Received for publication on June 26, 2015; First review completed on August 10, 2015; Accepted for publication on September 14, 2015 E il l li @h f b Received for publication on June 26, 2015; First review completed on August 10, 2015; Accepted for publication on September 14, 2015 E-mail: l.lin@hc.fm.usp.br Corresponding author E-mail: l.lin@hc.fm.usp.br Corresponding author REVIEW REVIEW No potential conﬂict of interest was reported. DOI: 10.6061/clinics/2015(12)08 Doppler ultrasound in the prediction of GTN There are few predictors of the clinical course of hydatidi- form moles. Advanced maternal age, higher human chorionic gonadotropin (hCG) levels, larger uterine size, complete moles, large theca lutein cysts and clinical complications have been associated with malignant post-molar disease. Few studies have evaluated the role of Doppler ultrasound in the prediction of GTN development. ’ RESULTS AND DISCUSSION The initial search identified 103 articles in PubMed, 35 in Web of Science, and no articles in the Cochrane and LILACS databases. Twenty-eight articles were selected for this review according to the inclusion and exclusion criteria (Figure 1). The articles were divided into 4 groups according to the aim of the study. ’ METHODS The PubMed/Medline, Web of Science, Cochrane and LILACS databases were searched for articles published in English until 2014 using the following Medical Subject Heading (MeSH) keywords: ‘‘Gestational trophoblastic dis- ease AND Ultrasonography, Doppler.’’ Studies evaluating the role of Doppler ultrasound in the diagnosis, prognosis and follow-up of patients with gestational trophoblastic disease were included. Case reports, reviews, letters to the editor and articles not in English were excluded. All abstracts were analyzed and included according to the current study criteria. The selected articles’ bibliographies were also evaluated and the relevant references were included. Trophoblastic cells at the implantation site of each type of pregnancy seem to differ according to the level of invasiveness observed in the first trimester of molar pregnancies, particularly in complete moles (7), which might explain the difference in Doppler velocimetry of the UA. In clinical practice, the values of the UA Doppler indices do not seem to help differentiate between partial and complete moles, except when complete moles coexist with a normal fetus. Doppler ultrasound in trophoblastic disease Lin LH et al. CLINICS 2015;70(12):810-815 moles (less than 5%). In most cases, ultrasonography can identify the type of mole. However, in certain situations (such as a complete mole with a coexisting normal fetus), ancillary methods such as Doppler ultrasound are useful. arteriogram in women with GTN and this approach is currently widely used and substituted arteriography in the evaluation of such patients (1,3). Below, we summarize the main purposes of Doppler sonography in the management of patients with gestational trophoblastic disease. In a retrospective study, a higher RI of the uterine arteries (UA) was reported in normal pregnancies (n=13; mean RI=0.66±0.05) compared with molar pregnancies, but there was no significant difference between the UA RI of partial moles (n=33; mean RI=0.56±0.04) and complete moles (n=106; mean RI=0.55±0.06) (4). Jauniaux et al. (1996) described a series of 70 cases of triploidy after the first trimester and demonstrated that at least half of the cases with a UA evaluation (n=41) presented with an abnormally high RI (5). In a series of cases presented by Jauniaux & Nicolaides, the UA PI after 16 weeks was below the fifth percentile in all three complete moles that coexisted with a normal fetus, in contrast with a uterine artery PI within normal ranges in six cases of partial moles (6). ’ INTRODUCTION tumors. These tumors are highly vascularized and, in the past, were often evaluated by arteriography (1). Gestational trophoblastic disease includes a spectrum of conditions characterized by abnormal trophoblast proliferation and invasion. It primarily comprises five diseases: hydatidiform mole, invasive mole, choriocarcinoma, placental site trophoblas- tic tumor and epithelioid trophoblastic tumor. Hydatidiform mole is the most common form, with an estimated incidence of 1 case in 1,000 pregnancies. Although it is generally a benign condition, it can evolve into an invasive form in some patients. The remaining diseases are all malignant forms termed gesta- tional trophoblastic neoplasia (GTN) or gestational trophoblastic y g p y Doppler ultrasonography is a noninvasive method cur- rently used to reliably evaluate and measure blood flow. Doppler ultrasound includes color, power and spectral modalities. Color flow imaging evaluates the direction of blood flow and is based on a frequency change. In contrast, power Doppler is based on amplitude changes and is thus more sensitive to slow velocity flows but does not provide direction information. Spectral Doppler provides a functional assessment of the circulation by calculating Doppler veloci- metry indices based on the waveforms of blood flow. The most commonly used Doppler indices are the ratio of peak systolic to end diastolic velocity (S/D), resistance index (RI; calculated as [peak systolic velocity - end diastolic velocity / peak systolic velocity]) and pulsatility index (PI, calculated as [peak systolic velocity - end diastolic velocity / mean velocity]) (2). Copyright & 2015 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. y It has been shown that Doppler ultrasonography presents almost the same findings (tumor size and localization) as a pelvic DOI: 10.6061/clinics/2015(12)08 810 Doppler ultrasound in trophoblastic disease Lin LH et al. CLINICS 2015;70(12):810-815 CLINICS 2015;70(12):810-815 Lower UA Doppler indices before molar evacuation were associated with the development of GTN in 5 patients (mean S/D=5.10; mean RI=0.80; mean PI=1.82) compared with 16 patients who exhibited spontaneous remission (mean S/D=2.27; mean RI=0.55; mean PI=0.86) (8). Similarly, Gungor et al. (1998) reported a lower UA RI before molar evacuation in 12 patients who developed persistent malignant disease (mean RI=0.29) compared to 20 patients with spontaneous remission (mean RI=0.46) (9). In contrast, the study by Chan et al. (1996) showed no difference in the UA RI in 11 patients with spontaneous remission compared with 21 patients with post- molar disease (10). A preliminary study that included 8 patients reported that a lower UA PI (o1.5) after 2 weeks of evacuation was only observed in the 2 patients with GTN prior to the hCG increase (11). there is an increase or plateau in hCG titers when evaluat- ing a primary GTN site. Using Doppler ultrasonography to detect post-molar disease was found to be more efficient, primarily in microscopic disease compared with standard ultrasound scans, in a series of patients with persistent trophoblastic disease who underwent dilation and curettage (14). Emoto & Sadamori (15) described the presence of intratumoral blood flow by color Doppler ultrasonography in all forms of trophoblastic tumors (5 invasive moles, 1 choriocarcinoma and 1 placental site trophoblastic tumor), whereas this observation was not made in 15 hydatidiform moles. Moreover, after contrast enhancement using a Levovist microbubble contrast agent, there was a significant increase in flow in the small invasive moles (o2 cm) (15). Doppler ultrasound seems to play a role in predicting GTN following uterine evacuation. In a retrospective cohort, the presence of Doppler ultrasound abnormalities, such as nodules or hypervascularization in the myometrium or endometrium, after 3 weeks of molar evacuation was highly correlated with the development of GTN in 14 patients compared to 175 patients who exhibited spontaneous remission (12). Zanetta et al. (13) evaluated the efficiency of uterine morphology and Doppler ultrasound in the diag- nosis of post-molar disease. The Doppler results were considered abnormal when at least three vessels in the myometrium or residual intrauterine tissue had a PI below 1.0. In 16 patients with spontaneous regression of hCG, the PI values showed progressive increases, and the vascularization was reduced during follow-up. CLINICS 2015;70(12):810-815 Normal Doppler findings had a negative predictive value of 100% for persistent disease, and normalization of the findings occurred up to 8 weeks before hCG became undetectable. In contrast, an abnormal Doppler at the 6th week post-evacuation showed a positive predictive value of 67% for local persistence. In a follow-up of 9 patients with GTN, only one patient presented with normal ultra- sound findings and elevated hCG, which was related to extra-uterine metastasis (13). A large retrospective study of 355 patients with tropho- blastic disease reported a higher UA RI in normal pregnan- cies (n=13; mean RI=0.66), followed by partial (n=33; RI=0.56) and complete hydatidiform moles (n=106; mean RI=0.55), compared to GTN pregnancies (mean RI=0.28 and RI=0.25 for 184 presumed invasive moles and 32 chor- iocarcinomas, respectively) (4). In other studies, it was shown that patients with trophoblastic tumors had a lower resistance on UA Doppler than did those with a hydatidi- form mole or women who were post-abortion, non- pregnant or in the first trimester of pregnancy (Taylor et al. (16); Long et al. 1990; Tepper et al. (18); Chan et al. (10); Hsieh et al. 1994) (Table 2). There is also evidence of a strong correlation between UA Doppler indices and hCG regression patterns in hydatidiform moles and post-molar tumors, with a concurrent increase in the Doppler indices and a decrease in hCG after uterine evacuation (8,10,13,18,20,21). The blood flow area ratio (BFAR) is a way to quantify blood flow in an area of interest using appropriate software based on 2D power Doppler sonography. The BFAR in 19 patients with invasive trophoblastic disease was signifi- cantly higher than in 25 women with a normal pregnancy or 25 non-pregnant individuals. It was also demonstrated that the BFAR gradually decreased during and after chemother- apy, but it took 3 to 6 months after chemotherapy to normalize in value (22). Although most studies have noted that a lower resistance in the UA is associated with the development of trophoblastic tumors (Table 1), data in the literature remain controversial. New studies with larger samples are thus needed to better elucidate the role of Doppler ultrasound in predicting post-molar disease. Hsieh et al. (23) described three different vascular patterns for Doppler ultrasound in 28 trophoblastic tumors: diffuse, lacunar and compact. CLINICS 2015;70(12):810-815 These patterns showed different UA RI (more compact and fewer lacunar type), hCG levels (more compact and fewer diffuse type) and the need for more than 5 chemotherapy cycles (more compact and fewer diffuse type). Histopathological ana- lysis was available in 11 cases; of these, 7 invasive moles Doppler evaluation for the differentiation of partial and complete moles Differentiating between partial and complete moles carries prognostic significance because of the higher rate of post-molar disease in complete moles (15-20%) compared with partial Figure 1 - Flow chart describing the mechanism of search. Figure 1 - Flow chart describing the mechanism of search. 811 Doppler ultrasound in trophoblastic disease Lin LH et al. Doppler ultrasound in trophoblastic disease Lin LH et al. Post-molar follow-up and diagnosis of trophoblastic neoplasia ( ) Doppler ultrasound seems to be a useful adjuvant tool in post-molar follow-up and GTN diagnosis because it has been well-demonstrated in the literature that abnormal Doppler findings evaluated using different methods (such as abnormal myometrial vascularization and lower UA Doppler indices) are correlated with invasive disease. Increased vascularization of the myometrium, higher UA peak systolic velocity, and lower UA RI and PI were all associated with GTN, although the Doppler indices varied between studies (Table 2). Additional useful information for follow-up includes an inverse correlation between UA Doppler indices and hCG regression. However, some caution should be taken when analyzing abnormal Doppler images alone after uterine evacuation or che- motherapy because of the development and persistence of arteriovenous communications in the myometrium; in these cases, the hCG level is normal (13). Deeper myometrial invasion and lower RI from myome- trial vascularization were correlated with methotrexate resistance in 37 patients with GTN. Thirty patients showed remission with a single chemotherapy agent, whereas 7 patients required an actinomycin D combination chemo- therapy or hysterectomy. A cuff-off value of 4 cm or higher for myometrial invasion (sensibility 100% and specificity 75%) and a myometrial vascularization RIp0.28 (sensibility 87.5 and specificity 83.3) were proposed to predict single- agent chemotherapy resistance (27). Several studies have evaluated the prediction of methotrex- ate resistance with UA PI prior to chemotherapy. Long et al. (17) prospectively evaluated the UA PI of 40 patients with GTN and proposed a PI cut-off value of p1.1 for inclusion in the prognostic scoring system of trophoblastic tumors due to the elevated likelihood of these patients developing chemore- sistance (28). In a retrospective study of 164 patients, the UA PI was an independent factor for methotrexate resistance. A PIp1.0 detected resistance with a sensitivity of 68% and specificity of 62%, with an adjusted odds ratio of 2.27 for methotrexate resistance. Furthermore, 72.2% of the patients with a Charing Cross Hospital prognostic score between 6 and 8 and a PIp1 developed resistance. In contrast, only 20% of the patients with a PI41 developed resistance (29). Similarly, the UA PI was identified as an important factor associated with methotrexate resistance in 73 patients with a GTN FIGO score of 5-6. A chemoresistance risk of 67% was calculated for patients with UA PIp1, compared to 42% when the UA PI was 41 (p=0.036) (30). Post-molar follow-up and diagnosis of trophoblastic neoplasia Study GTN HM Pregnant Non-pregnant Conclusion n UA RI n UA RI n UA RI n UA RI Tepper et al., 1994 3 0.410 ± 0.04 - - 20 0.494 ± 0.06 - - Lower UA RI in GTN compared with ﬁrst-trimester pregnant woman Chan et al., 1996 32 0.68 ± 0.16 - - 18 0.8 ± 0.09 23 0.9 ± 0.08 Lower UA RI in GTN compared with non-pregnant and ﬁrst-trimester pregnant woman Hsieh et al., 1994 23 0.56 ± 0.19 15 0.75 ± 0.06 - - 55 0.80 ± 0.05 Higher peak systolic velocity and lower UA RI in GTN compared with non-pregnant and post-evacuation uneventful moles Zhou et al., 2005 32 (CC) 184 (IM) 0.25± 0.05 (CC) 0.28 ± 0.06 IM) 106 (CHM) 33 (PHM) 0.55 ± 0.06 (CHM) 0.56 ± 0.04 (PHM) 13 0.66 ± 0.05 - - Lower UA RI in GTN compared with hydatidiform moles and ﬁrst-trimester pregnant woman Study GTN HM Pregnant Non-pregnant Conclusion n UA RI n UA RI n UA RI n UA RI Tepper et al., 1994 3 0.410 ± 0.04 - - 20 0.494 ± 0.06 - - Lower UA RI in GTN compared with ﬁrst-trimester pregnant woman Chan et al., 1996 32 0.68 ± 0.16 - - 18 0.8 ± 0.09 23 0.9 ± 0.08 Lower UA RI in GTN compared with non-pregnant and ﬁrst-trimester pregnant woman Hsieh et al., 1994 23 0.56 ± 0.19 15 0.75 ± 0.06 - - 55 0.80 ± 0.05 Higher peak systolic velocity and lower UA RI in GTN compared with non-pregnant and post-evacuation uneventful moles Zhou et al., 2005 32 (CC) 184 (IM) 0.25± 0.05 (CC) 0.28 ± 0.06 IM) 106 (CHM) 33 (PHM) 0.55 ± 0.06 (CHM) 0.56 ± 0.04 (PHM) 13 0.66 ± 0.05 - - Lower UA RI in GTN compared with hydatidiform moles and ﬁrst-trimester pregnant woman were the lacunar type, and 4 choriocarcinomas were the compact type, which indicated that on some level, Doppler ultrasound is capable of differentiating trophoblastic tumors (23). addition, there was an evident decrease in the peak systolic velocity after the first three chemotherapy cycles in the group that required fewer courses of treatment (54.2 to 23.6) compared to the other group (60.1 to 60) (23). These findings indicate a greater change in uterine hemodynamics in patients with a better response to chemotherapy, which could help in the decision to change chemotherapy. Post-molar follow-up and diagnosis of trophoblastic neoplasia Another study that assessed the UA PI and methotrexate resistance in 239 patients with a FIGO score of 0-6 showed a lower UA PI in patients with chemoresistance and found that a UA PIp1 served as a risk factor, independent of the FIGO scoring system for methotrexate resistance. Patients with a FIGO score of 6 and UA PIp1 showed 100% resistance to methotrexate compared with 20% of the patients who had a UA PI41. The authors suggest that these patients might benefit from upstaging and combination therapy (31). Post-molar follow-up and diagnosis of trophoblastic neoplasia Following uterine molar evacuation, patients are fol- lowed closely with weekly clinical evaluations and hCG monitoring. An ultrasound is only generally needed when Table 1 - Studies that evaluated the correlation between UA Doppler indices and the development of post-molar trophoblastic tumors before uterine evacuation. n: number of patients; S/D: systolic/diastolic velocity; PI: pulsatility index; RI: resistance index. Study Patients with spontaneous remission Patients with persistent disease Conclusion n S/D PI RI n S/D PI RI Yalcin et al., 2001 16 5.1 1.82 0.8 5 2.27 0.86 0.55 Lower uterine artery indices (S/D, PI, RI) before uterine evacuation were associated with persistent disease (po0.01). Gungor et al., 1998 20 - - 0.46 12 - - 0.29 Lower uterine artery RI before uterine evacuation was associated with persistent disease (po0.001). Chan et al., 1996 11 - - 0.76 21 - - 0.69 No association between uterine artery RI before uterine evacuation and persistent disease. 812 Table 2 - Studies that evaluated the UA RI in gestational trophoblastic disease, normal pregnancy and non-pregnant women. GTN: gestational trophoblastic neoplasia; HM: hydatidiform mole; n: number of patients; UA RI: uterine artery resistance index; CC: choriocarcinoma; IM: invasive mole; CHM: complete hydatidiform mole; PHM: partial hydatidiform mole. CLINICS 2015;70(12):810-815 Doppler ultrasound in trophoblastic disease Lin LH et al. CLINICS 2015;70(12):810-815 Table 2 - Studies that evaluated the UA RI in gestational trophoblastic disease, normal pregnancy and non-pregnant women. GTN: gestational trophoblastic neoplasia; HM: hydatidiform mole; n: number of patients; UA RI: uterine artery resistance index; CC: choriocarcinoma; IM: invasive mole; CHM: complete hydatidiform mole; PHM: partial hydatidiform mole. Table 2 - Studies that evaluated the UA RI in gestational trophoblastic disease, normal pregnancy and non-pregnant women. GTN: gestational trophoblastic neoplasia; HM: hydatidiform mole; n: number of patients; UA RI: uterine artery resistance index; CC: choriocarcinoma; IM: invasive mole; CHM: complete hydatidiform mole; PHM: partial hydatidiform mole. Prediction of resistance to chemotherapy Several studies have demonstrated a rough correlation between uterine Doppler vascularization and the response to chemotherapy (3,24). A pilot study assessed ultrasound and Doppler signs of response (decreases in tumor size, echogenicity and abnormal myometrial Doppler signal) in seven patients with chemoresistance to methotrexate sus- pected by an hCG plateau. Three of these patients had ultrasound findings that indicated a response and achieved remission after methotrexate. This study suggests that an ultrasound can differentiate patients with a delayed res- ponse to methotrexate from those who require second-line treatment (25). An increase in the UA Doppler indices of patients responding to chemotherapy was observed in parallel with a decrease in hCG levels (18). Although the initial UA S/D ratio was not significantly different between the 16 patients with chemoresistant (2.69±1.8) and chemosensitive disease (2.72±1.31), after the completion of chemotherapy, the UA S/D ratio increased only in patients with remitted disease (6.23±2.38). However, this was not observed in patients with non-remitted disease (3.08±1.54) (26). Hsieh et al. (19) described a higher mean UA RI in 10 patients requiring fewer than 5 cycles of chemotherapy (mean RI=0.71±0.09) compared with 13 patients who required longer chemotherapy (mean RI=0.47±0.14). In p g g py Numerous studies support the utilization of Doppler ultrasound as a tool to predict chemotherapy resistance (Table 3). In particular, qualitative Doppler assessment might help in managing patients with a plateaued hCG. More 813 Table 3 - Studies that evaluated the role of Doppler ultrasound to predict chemotherapy resistance. GTN: gestational trophoblastic neoplasia; n: number of patients; UA S/D: uterine artery systolic/diastolic velocity; UA PI: uterine artery pulsatility index; UA RI: uterine artery resistance index. Doppler ultrasound in trophoblastic disease Lin LH et al. CLINICS 2015;70(12):810-815 CLINICS 2015;70(12):810-815 Table 3 - Studies that evaluated the role of Doppler ultrasound to predict chemotherapy resistance. GTN: gestational trophoblastic neoplasia; n: number of patients; UA S/D: uterine artery systolic/diastolic velocity; UA PI: uterine artery pulsatility index; UA RI: uterine artery resistance index. Table 3 - Studies that evaluated the role of Doppler ultrasound to predict chemotherapy resistance. GTN: gestational trophoblastic neoplasia; n: number of patients; UA S/D: uterine artery systolic/diastolic velocity; UA PI: uterine artery pulsatility index; UA RI: uterine artery resistance index Study Chemo-resistant GTN Chemo-sensitive GTN Conclusion Park et al., 1994 5 11 UA S/D before treatment was not signiﬁcantly different in patients with chemoresistant and sensitive disease. Prediction of resistance to chemotherapy After completion of chemotherapy, UA S/D increased in patients with remitted disease (2.72±1.31 to 6.23±2.38), whereas in patients with resistant GTN, UA S/D did not change (2.69±1.8 to 3.08±1.54) Oguz et al., 2004 7 30 A cuff-off value of 4 cm or higher of myometrial invasion and vascularization RIp0.28 were proposed to predict single-agent chemotherapy resistance Long et al., 1992 8 42 This prospective study established a cut-off value of UA PIp1.1 associated with an elevated likelihood of GTN patients developing chemoresistance Agarwal et al., 2002 47 117 This retrospective study established that a UA PIp1.0 detected resistance with a sensitivity of 68% and speciﬁcity of 62%, with an adjusted odds ratio of 2.27 for methotrexate resistance in low-risk GTN Sita-Lumsden et al., 2013 43 30 In patients with a GTN FIGO score of 5-6, the risk of chemoresistance was 67% with a UA PIp1, compared with 42% when the UA PI was 41 Agarwal et al., 2012 113 126 A lower UA PI in patients with low-risk GTN with chemoresistance and UA PIp1 was a risk factor independent of the FIGO score for methotrexate resistance. Patients with a FIGO score of 6 and UA PIp1 had 100% resistance to methotrexate Hsieh et al., 1994 13 (GTN needing X5 courses of chemotherapy) 10 (GTN needing o5 courses of chemotherapy) Higher mean UA RI in patients requiring fewer than 5 cycles of chemotherapy (0.71±0.09) compared to patients requiring longer chemotherapy (0.47±0.14). There was an evident decrease in the UA peak systolic velocity after the ﬁrst three chemotherapy cycles in the group requiring less courses of treatment (54.2 to 23.6 cm/s compared to 60.1 to 60 cm/s) Higher mean UA RI in patients requiring fewer than 5 cycles of chemotherapy (0.71±0.09) compared to patients requiring longer chemotherapy (0.47±0.14). There was an evident decrease in the UA peak systolic velocity after the ﬁrst three chemotherapy cycles in the group requiring less courses of treatment (54.2 to 23.6 cm/s compared to 60.1 to 60 cm/s) importantly, evaluating UA Doppler indices seems to be a promising marker to predict methotrexate resistance. How- ever, more studies are needed to fully understand the role of Doppler ultrasound in the prognostic risk evaluation of trophoblastic tumors. 6. Jauniaux E, Nicolaides KH. Early ultrasound diagnosis and follow-up of molar pregnancies. Ultrasound Obstet Gynecol. 1997;9(1):17-21, http://dx.doi.org/10.1046/j.1469-0705.1997.09010017.x. p g j 7. ’ AUTHOR CONTRIBUTIONS Lin LH, Bernardes LS and Hase EA performed the literature search and wrote the manuscript. Fushida K and Francisco RPV reviewed and corrected the manuscript. p g 12. Garavaglia E, Gentile C, Cavoretto P, Spagnolo D, Valsecchi L, Mangili G. Ultrasound imaging after evacuation as an adjunct to b-hCG monitoring in posthydatidiform molar gestational trophoblastic neoplasia. Am J Obs Gynecol. 2009;200(4):417.e1-417.e5, http://dx.doi.org/10.1016/j.ajog.2008.11. 032. Prediction of resistance to chemotherapy Sebire NJ, Rees H, Paradinas F, Fisher R, Foskett M, Seckl M, et al. Extravillus endovascular implantation site trophoblast invasion is abnormal in complete versus partial molar pregnancies. Placenta. 2001; 22(8-9):725-8, http://dx.doi.org/10.1053/plac.2001.0716. p Doppler ultrasound has several applications in evaluating patients with gestational trophoblastic disease. Its role in differentiating the type of molar pregnancy and predicting trophoblastic neoplasia might aid clinicians in the manage- ment and counseling of patients. However, more studies are needed to better evaluate these findings. 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https://openalex.org/W3042067970	https://pure.coventry.ac.uk/ws/files/55055728/Published.pdf	English	null	Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	27,115	DOI 10.1038/s41467-022-31652-2 ISSN 2041-1723 DOI 10.1038/s41467-022-31652-2 ISSN 2041-1723 Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression Wall, MJ, Hill, E, Huckstepp, R, Barkan, K, Deganutti, G, Leuenberger, M, Preti, B, Winfield, I, Carvalho, S, Suchankova, A, Wei, H, Safitri, D, Huang, X, Imlach, W, Mache, CL, Dean, E, Hume, C, Hayward, S, Oliver, J, Zhao, F-Y, Spanswick, D, Reynolds, CA, Lochner, M, Ladds, G & Frenguelli, BG Published PDF deposited in Coventry University’s Repository Original citation: Wall, MJ, Hill, E, Huckstepp, R, Barkan, K, Deganutti, G, Leuenberger, M, Preti, B, Winfield, I, Carvalho, S, Suchankova, A, Wei, H, Safitri, D, Huang, X, Imlach, W, Mache, CL, Dean, E, Hume, C, Hayward, S, Oliver, J, Zhao, F-Y, Spanswick, D, Reynolds, CA, Lochner, M, Ladds, G & Frenguelli, BG 2022, 'Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression', Nature Communications, vol. 13, no. 1, 4150. https://dx.doi.org/10.1038/s41467-022-31652-2 1 School of Life Sciences, University of Warwick, Gibbet Hill Rd, Coventry CV4 7AL, UK. 2 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. 3 Centre for Sport, Exercise and Life Sciences (CSELS), Faculty of Health and Life Sciences, Coventry University, Coventry CV1 2DS, UK. 4 School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK. 5 Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland. 6 NeuroSolutions Ltd, Coventry, UK. 7 Pharmacology and Clinical Pharmacy Research Group, School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia. 8 Department of Physiology, Monash Biomedicine Discovery Institute, Monash University, Innovation Walk, Clayton, VIC 3800, Australia. 9 Warwick Medical School, University of Warwick, Gibbet Hill Rd, Coventry CV4 7AL, UK. 10These authors contributed equally: Mark J. Wall, Emily Hill, Robert Huckstepp, Kerry Barkan, Giuseppe Deganutti, Michele Leuenberger, Barbara Preti, Ian Winﬁeld. ✉email: mark.wall@warwick.ac.uk; grl30@cam.ac.uk; b.g.frenguelli@warwick.ac.uk Publisher: Springer Publisher: Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.. ARTICLE Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression Mark J. Wall 1,10✉, Emily Hill 1,10, Robert Huckstepp 1,10, Kerry Barkan2,10, Giuseppe Deganutti 3,4,10, Michele Leuenberger 5,10, Barbara Preti5,10, Ian Winﬁeld2,10, Sabrina Carvalho2, Anna Suchankova2, Haifeng Wei6, Dewi Saﬁtri 2,7, Xianglin Huang2, Wendy Imlach 8, Circe La Mache 1, Eve Dean1, Cherise Hume1, Stephanie Hayward1, Jess Oliver1, Fei-Yue Zhao6, David Spanswick 6,8,9, Christopher A. Reynolds 3,4, Martin Lochner 5, Graham Ladds 2✉& Bruno G. Frenguelli 1✉ The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is ham- pered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-speciﬁc Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism. 1 School of Life Sciences, University of Warwick, Gibbet Hill Rd, Coventry CV4 7AL, UK. 2 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. 3 Centre for Sport, Exercise and Life Sciences (CSELS), Faculty of Health and Life Sciences, Coventry University, Coventry CV1 2DS, UK. 4 School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK. 5 Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland. 6 NeuroSolutions Ltd, Coventry, UK. 7 Pharmacology and Clinical Pharmacy Research Group, School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia. 8 Department of Physiology, Monash Biomedicine Discovery Institute, Monash University, Innovation Walk, Clayton, VIC 3800, Australia. 9 Warwick Medical School, University of Warwick, Gibbet Hill Rd, Coventry CV4 7AL, UK. 10These authors contributed equally: Mark J. Wall, Emily Hill, Robert Huckstepp, Kerry Barkan, Giuseppe Deganutti, Michele Leuenberger, Barbara Preti, Ian Winﬁeld. Results The novel A1R agonist BnOCPA exquisitely discriminates between native pre- and postsynaptic A1Rs in the intact mammalian CNS. BnOCPA (Fig. 1a), a molecule ﬁrst described in a patent as a potential treatment for glaucoma or ocular hypertension34, is a cyclopentyl derivative of adenosine and a highly selective and potent, full agonist at human adenosine A1Rs (hA1Rs; Fig. 1b; Supplementary Table 1)33. Our characterisation of BnOCPA, synthesised independently as part of a screen for suitable scaffolds for the generation of ﬂuorescent ligands for the A1R, began with an exploration of the binding characteristics of BnOCPA at the hA1R using classical radioligand binding (where the antagonist [3H]DPCPX was used as a tracer), and a Nano- BRET agonist binding assay (using a novel NECA-TAMRA compound, which acts as a full agonist (pEC50 –7.23 ± 0.13; See Methods). Using both assays we observed that BnOCPA was able to bind to the hA1R with an afﬁnity equal to that of the proto- typical A1R agonists CPA and NECA, and higher than that of the endogenous agonist adenosine (Fig. 1b; Supplementary Table 1). Signiﬁcantly, using NECA-TAMRA as the ﬂuorescent agonist tracer, the high-afﬁnity state of the biphasic binding proﬁle observed in the NanoBRET assay was equivalent to that reported previously for BnOCPA (3.8 nM compared to 1.7 nM34). The therapeutic limitations of promiscuous GPCR coupling might be overcome through the development of biased agonists— compounds that preferentially recruit one intracellular signalling cascade over another4,17,18. This signalling bias has most fre- quently been expressed in terms of Gα vs β-arrestin signalling19 and has been pursued at a variety of receptors20,21, for example, at the angiotensin II type 1 receptor (AT1R)22, and at neurotensin receptors in the treatment of drug addiction23. Agonist bias has been sought in the context of opioid receptors, but with some controversy24, for compounds producing analgesia with reduced respiratory depression, gastrointestinal disturbance and tolerance4. p y p These initial pharmacological studies at recombinant hA1Rs in cell lines did not reveal anything extraordinary about BnOCPA. However, when we investigated BnOCPA at native A1Rs in rat hippocampal slices, against which BnOCPA is also a potent agonist, with ~8000- and >150-fold greater efﬁcacy at rat A1Rs (rA1Rs) than at rat A2ARs (rA2ARs) and A3Rs (rA3Rs), respectively (Supplementary Table 2), we discovered properties of BnOCPA that were not consistent with those of typical A1R agonists such as adenosine, CPA and NECA. Results In accordance with the effects of standard A1R agonists, BnOCPA potently inhibited excitatory synaptic transmission in rat hippocampal slices (IC50 ~65 nM; Fig. 1c–g and Supplementary Fig. 1a–d). This effect was attributable to the activation of native presynaptic A1Rs on glutamatergic terminals9 (Fig. 1c; Supplementary Fig. 1e, f), and cannot be attributed to any action of BnOCPA at A3Rs since even a high concentration (1 µM) of the potent and selective A3R agonist 2-Cl-IB-MECA35 had no effect on synaptic transmission (Supplementary Fig. 1g, h). However, in stark contrast to adenosine and CPA, BnOCPA did not activate postsynaptic A1Rs (Fig. 1c) to induce membrane hyperpolarisation, even at concentrations 15 times the IC50 for the inhibition of synaptic transmission (Fig. 1h, i). However, while other forms of bias exist, including between individual Gα subunits17,25,26, the challenge remains in translat- ing GPCR signalling bias observed in vitro to tangible, and physiologically- and clinically-relevant, selectivity at native receptors in vivo3,4,27,28. Accordingly, while the potential to preferentially drive the G protein-coupling of A1Rs has been described in several in vitro studies29–32, to date no A1R-speciﬁc agonist has been reported that can elicit biased Gα agonism at native A1Rs in intact physiological systems, let alone the selective activation of one Gα subunit. To achieve such selectivity among Gα subunits would introduce novel therapeutic opportunities across a wide range of debilitating clinical conditions. g This peculiar and unique discrimination between pre- and postsynaptic A1Rs might possibly be explained in terms of either some hindrance in the binding of BnOCPA to A1Rs on postsynaptic neurones, or, and unprecedented for an A1R agonist, binding to the postsynaptic A1R, but without the ability to activate the receptor. To test the latter hypothesis—that BnOCPA actually bound to postsynaptic A1Rs, but without efﬁcacy—we reasoned that BnOCPA might behave in a manner analogous to a receptor antagonist in preventing or reversing activation by other A1R agonists, a property that has been predicted and observed for biased agonists at other receptors17,27. To test this, we pre-applied BnOCPA and then applied CPA (in the continued presence of BnOCPA). Remarkably, the co-application of CPA and BnOCPA resulted in a signiﬁcant reduction of the effects of CPA on membrane potential (Fig. 1i; Supplementary Fig. 2a, b). Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression ✉email: mark.wall@warwick.ac.uk; grl30@cam.ac.uk; b.g.frenguelli@warwick.ac.uk 1 NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 G protein-coupled receptors (GPCRs) are the targets of many FDA-approved drugs1,2. However, the promiscuity with which they couple to multiple G protein- and β- arrestin-activated intracellular signalling cascades leads to unwanted side effects. These side effects limit both the range of GPCRs suitable for drug-targeting, and the number of conditions for which treatments could be developed3,4. One family of GPCRs that have particularly suffered as drug targets from their pro- miscuous coupling and wide-ranging cellular actions are the four GPCRs for the purine nucleoside adenosine, despite the potential for using adenosine receptor agonists to treat many pathological conditions including cancer, and various cardiovascular, neuro- logical and inﬂammatory diseases5–7. For example, activation of the widely-distributed adenosine A1 receptor (A1R) with cur- rently available agonists elicits multiple actions in both the central nervous system (CNS) and the cardiorespiratory system. In the CNS A1Rs inhibit synaptic transmission, induce neuronal hyperpolarization, reduce seizure activity and cause sedation, while in the cardiorespiratory system A1Rs slow the heart (bra- dycardia), and contribute to reducing blood pressure (hypoten- sion), and depress respiration (dyspnoea)7–12. These multiple effects severely limit the prospects of A1R agonists as life- changing medicines, despite their potential use in a wide range of clinical conditions, such as glaucoma, type 2 diabetes mellitus, pain, epilepsy and cerebral ischaemia7,13–16, and in which there are clear unmet clinical needs that could be addressed with novel therapeutics. G m w at native receptors, highlight the physiological beneﬁts of such selectivity, and speciﬁcally speak to the possibility of unlocking the widespread clinical potential of A1R agonists. Results In addition, membrane hyperpolarisation induced by the endogenous Here we show, utilising molecular dynamics (MD) simulations, and Gαi/o subunit- and β-arrestin-speciﬁc cellular signalling assays, how one A1R-selective agonist, BnOCPA33,34, fulﬁls the criteria for a selective Gα agonist in exclusively activating Gob among the six members of the Gαi/o family of G protein subunits, and in the absence of β-arrestin recruitment. In addition, through a combination of CNS electrophysiology, physiological recordings of cardiorespiratory parameters, a sensitive assay of attention and locomotor function, and the use of a clinically-relevant model of chronic neuropathic pain, we demonstrate selective activation of native A1Rs and the delivery of potent analgesia without sedation, motor impairment or cardiorespiratory depression. Our data thus demonstrate the translation of agonist Gα selectivity in vitro to therapeutically tangible clinically-relevant observations in vivo. Such observations reveal the possibility of achieving Gα selectivity URE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 2 ARTICLE ARTICLE Fig. 1 BnOCPA is an A1R agonist that discriminates between pre- and postsynaptic A1Rs in the CNS. a Chemical structures of adenosine, CPA and BnOCPA33. bi Schematic representing assays used to characterise the afﬁnity and efﬁcacy of agonists (green arrows) to the human (h) A1R. bii agonist displacement of [3H]DPCPX, a selective antagonist for the hA1R (n = 5–10 individual repeats). biii cAMP levels measured in CHO-K1-hA1R cells following co-stimulation with 1 μM forskolin and each agonist (n = 4–10 individual repeats). biv Both CPA and BnOCPA displace the ﬂuorescent AR agonist NECA- TAMRA in a biphasic manner indicating that both agonists display high afﬁnity and low afﬁnity binding (n = 4 individual repeats). c Diagram illustrating: left, hippocampal slice preparation showing position of stimulating, patch-clamp and extracellular recording electrodes together with representative electrophysiological recordings: membrane potential (Vm), a fEPSP (ﬁeld excitatory postsynaptic potential) and seizure activity; right, pre- and postsynaptic A1Rs at hippocampal synapses and their physiological effects upon activation. d, e Increasing concentrations of CPA or BnOCPA reduced the fEPSP, an effect reversed by the A1R antagonist 8-CPT (2 µM). Inset, superimposed fEPSP averages in control (largest fEPSP) and becoming smaller with increasing concentrations of CPA or BnOCPA. Scale bars measure 0.2 mV or 0.1 mV and 5 ms for CPA and BnOCPA, respectively. f, g Concentration- response curves for the inhibition of synaptic transmission by CPA (IC50 = 11.8 ± 2.7 nM; n = 17 slices) or BnOCPA (IC50 = 65 ± 0.3 nM; n = 11 slices). h CPA hyperpolarised the membrane potential while BnOCPA had little effect. Scale bars measure 4 mV and 30 s. i Summary data for membrane potential changes. The mean hyperpolarisation produced by CPA (300 nM; 7.26 ± 0.86 mV, n = 7 cells) was signiﬁcantly different (one-way ANOVA; F(2,23) =70.46; P = 1.55 × 10−10) from that produced by BnOCPA (300 nM or 1 µM; 0.33 ± 0.14 mV, n = 10 and 5 cells, respectively; P = 8.26 × 10−11) and for CPA (300 nM) applied in the presence of BnOCPA (300 nM; 2.75 ± 0.48 mV, n = 4 cells, P = 2.89 × 10−5; See Supplementary Fig. 2a for an example trace). j In an in vitro model of seizure activity, represented as frequent spontaneous spiking from baseline, CPA (300 nM) reversibly blocked activity while BnOCPA (300 nM) had little effect. ARTICLE In contrast, equivalent concentrations of CPA completely suppressed neuronal ﬁring (Fig. 1j, k). hyperpolarisation produced by baclofen (Supplementary Fig. 2f, g), conﬁrming that the actions of BnOCPA were speciﬁc to the A1R. These observations, of a lack of effect of BnOCPA on postsynaptic membrane potential, likely explained why, in a model of seizure activity, (low Mg2+/high K+), with prominent postsynaptic depolarisation that promotes neuronal ﬁring, BnOCPA had little effect (Fig. 1j, k). In contrast, equivalent concentrations of CPA completely suppressed neuronal ﬁring (Fig. 1j, k). BnOCPA demonstrates unique Gα signalling in the selective activation of Gob. The observation that BnOCPA discriminated between pre- and postsynaptic A1Rs might be explained if these receptors were to activate different intracellular pathways to mediate their effects, and that BnOCPA was not able to activate the pathway responsible for postsynaptic membrane hyperpo- larisation. To test whether the actions of BnOCPA and the pro- totypical A1R agonists were mediated via β-arrestins (β-arrestin1 and β-arrestin2), we used a BRET assay36–40 for β-arrestin recruitment (Supplementary Fig. 3). We observed no β-arrestin recruitment at the A1R using either BnOCPA, CPA or adenosine, regardless of whether β-arrestin1 or β-arrestin2 was expressed (Supplementary Fig. 3). This was in contrast to β-arrestin2 recruitment by the A3R in response to adenosine and NECA, but not BnOCPA (Supplementary Fig. 3). Moreover, the lack of recruitment of β-arrestin1 and β-arrestin2 by the A1R was independent of any of the six G protein receptor kinase (GRK) isoforms co-expressed with β-arrestin1 and β-arrestin2; only low levels of recruitment were observed even when GRKs were highly (ﬁve-fold) overexpressed compared to the levels in the A3R assays (Supplementary Fig. 4). These observations of a lack of β-arrestin recruitment by A1Rs are consistent with those previously reported for recombinant A1Rs expressing native sequences41–45, and are likely due to the absence of serine and threonine residues in the A1R cytoplasmic tail, which makes the A1R intrinsically biased against β-arrestin signalling19,46. Accordingly, the differential actions of BnOCPA at pre- and postsynaptic A1Rs are more likely to reside in selective activation of one Gα-mediated pathway over another. The selective and unique activation of Gob among the six Gαi/ o subunits by BnOCPA could be observed in a comparison of the activation of Goa and Gob by the native and selective A1R agonists in their ability to inhibit the forskolin-stimulated accumulation of cAMP (Fig. 2f). ARTICLE Scale bars measure 0.5 mV and 200 s. k Summary data for seizure activity expressed in terms of the frequency of spontaneous spiking before, during and after CPA or BnOCPA. CPA abolished seizure activity (n = 4) whereas BnOCPA did not signiﬁcantly reduce seizure frequency (n = 6). Data represented as mean ± SEM; Two-way RM ANOVA (BnOCPA vs CPA slices): F(1, 3)=186.11, P = 8.52 × 10−4 with the following Bonferroni post hoc comparisons: BnOCPA vs Control; P = 1; CPA vs control; P = 0.010; BnOCPA vs CPA; P = 0.027. Averaged data are presented as mean ± SEM. ns, not signiﬁcant; , P < 0.05; , P < 0.02; *, P < 0.0001. Source data are provided as a Source Data ﬁle. generated a recombinant cell system (CHO-K1 cells) expressing both the hA1R and individual pertussis toxin (PTX)-insensitive variants of individual Gαi/o subunits. Against these individual Gαi/o subunits we tested adenosine, CPA, NECA, BnOCPA, and the agonist HOCPA33,47, a stereoisomer of GR7923648,49, which behaved similarly to adenosine and CPA in both inhibiting synaptic transmission and causing membrane hyperpolarisation (Supplementary Fig. 5). In cells treated with PTX to inhibit endogenous Gαi/o30,33 we observed that adenosine, CPA, NECA and HOCPA activated a range of Gαi/o subunits. Common to all of these agonists was the activation of both Gαo isoforms, Goa and Gob, with differential activation of Gi1 (HOCPA), Gi2 (NECA, CPA) and Gz (adenosine; Fig. 2a–e; Supplementary Figs. 5 and 6). Such promiscuous and biased Gα coupling has been described previously for adenosine, CPA, and NECA at recombinant A1Rs in cell lines29,50, including using novel BRET- based assays for adenosine at some Gαi/o51. These previous observations are in keeping with ours, conﬁrming the validity of the PTX-based approach. In stark contrast, BnOCPA displayed a unique and highly distinctive Gαi/o subunit activation proﬁle: BnOCPA was not able to activate Gi1, Gi2, Gi3 or Gz, and was furthermore capable of discriminating between the two Gαo isoforms via the selective activation of Gob, and not of Goa (Fig. 2a–e; Supplementary Fig. 6). hyperpolarisation produced by baclofen (Supplementary Fig. 2f, g), conﬁrming that the actions of BnOCPA were speciﬁc to the A1R. These observations, of a lack of effect of BnOCPA on postsynaptic membrane potential, likely explained why, in a model of seizure activity, (low Mg2+/high K+), with prominent postsynaptic depolarisation that promotes neuronal ﬁring, BnOCPA had little effect (Fig. 1j, k). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ne was reversed by BnOCPA (Supplementary ast, the A3R agonist 2-Cl-IB-MECA had no effect i l d did i f i h h b To test whether the inability of BnOCPA to a potential was a trivial action due to BnOCPA h l di i h i h l denosine BnOCPA bi CPA Antagonist displacement cAMP inhibion Agonist binding bii biii biv a a c d e h j g f i k denosine BnOCPA bi CPA bi a a BnOCPA bi a bi bii bi bii Antagonist displacement denosine CPA a cAMP inhibion Agonist binding biii biv cAMP inhibion To test whether the inability of BnOCPA to affect membrane potential was a trivial action due to BnOCPA blocking K+ channels mediating the postsynaptic hyperpolarisation, or in some other way non-speciﬁcally interfering with G protein signalling, we applied the GABAB receptor agonist baclofen to CA1 pyramidal neurons. BnOCPA had no effect on membrane g f i k agonist adenosine was reversed by BnOCPA (Supplementary Fig. 2c). In contrast, the A3R agonist 2-Cl-IB-MECA had no effect on membrane potential and did not interfere with the membrane hyperpolarisation caused by adenosine (Supplementary Fig. 2d, e), further reafﬁrming the actions of BnOCPA as being selectively mediated by A1Rs. denosine a c d e h j g f c c To test whether the inability of BnOCPA to affect membrane potential was a trivial action due to BnOCPA blocking K+ channels mediating the postsynaptic hyperpolarisation, or in some other way non-speciﬁcally interfering with G protein i lli li d h GABA i b l f g f i k d e h f d d g i k g i k k To test whether the inability of BnOCPA to affect membrane potential was a trivial action due to BnOCPA blocking K+ channels mediating the postsynaptic hyperpolarisation, or in some other way non-speciﬁcally interfering with G protein signalling, we applied the GABAB receptor agonist baclofen to CA1 pyramidal neurons. BnOCPA had no effect on membrane agonist adenosine was reversed by BnOCPA (Supplementary Fig. 2c). In contrast, the A3R agonist 2-Cl-IB-MECA had no effect on membrane potential and did not interfere with the membrane hyperpolarisation caused by adenosine (Supplementary Fig. 2d, e), further reafﬁrming the actions of BnOCPA as being selectively mediated by A1Rs. TURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 3 3 ARTICLE Whereas adenosine, CPA and HOCPA activated both Goa and Gob to inhibit cAMP accumulation, BnOCPA selectively activated Gob, with no discernible activation of Goa. Further quantiﬁcation of this Gα selectivity, through the application of the operational model of receptor agonism52–54 to remove potential issues of system bias, conﬁrmed selective activation of Gob by BnOCPA, with no detectable response at Goa (Fig. 2g). As further validation of the ability of BnOCPA to discriminate between the activation of Goa and Gob, we took advantage of BRET assays of GPCR activation55,56, which utilise a reduction in a Gα-Gβγ BRET signal to infer agonist-induced G protein activation, including for Goa and Gob57 (Fig. 2h; Supplementary Fig. 7a). Using the TRUPATH GPCR BRET assay55, adenosine, CPA, and HOCPA To investigate whether BnOCPA has the ability to discriminate between the various Gαi/o subunits activated by adenosine, we NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 b a d e c Acvaon of Goa vs Gob f i j Inhibion of cAMP by Goa Inhibion of cAMP by Gob Antagonism by BnOCPA Emax pEC50 % adenosine CPA NECA HOCPA BnOCPA No response Venn diagram of Gαi/o interacons pEC50 ATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ARTICL b a i Inhibion of cAMP by Goa Inhibion of cAMP by Gob Antagonism by BnOCPA a Inhibion of cAMP by Goa i Antagonism by BnOCPA i b a elicited equipotent activation of both Goa and Gob. In stark contrast to these agonists, BnOCPA was >10-fold more efﬁcacious in activating Gob than Goa, and, of all the agonists tested, BnOCPA displayed the weakest potency at Goa. While subtle differences between the Goa and Gob response exist across the two very different in vitro assays, these data nonetheless conﬁrm that BnOCPA demonstrates a previously unprecedente ability for an A1R agonist to discriminate between Gα subtype and in particular between Goa and Gob. To establish the functional implications of BnOCPA profound selectivity for Gob over Goa, we hypothesised th BnOCPA should reduce the actions of adenosine on th d e c Acvaon of Goa vs Gob f j k Emax pEC50 % Goa/b interfering pepdes adenosine CPA NECA HOCPA BnOCPA No response g Venn diagram of Gαi/o interacons Selecvity of BnOCPA for Gob vs Goa h Acvaon by BnOCPA of Gob vs Goa TRUPATH direct G protein assay pEC50 NATURE COMMUNICATIONS \| (2022)13:4150 \| https://doi org/10 1038/s41467-022-31652-2 \| www nature com/naturecommunications d c j Emax pEC50 % adenosine CPA NECA HOCPA BnOCPA No response pEC50 d j c e Acvaon of Goa vs Gob f Venn diagram of Gαi/o interacons f e Acvaon of Goa vs Gob f Venn diagram of Gαi/o interacons e Venn diagram of Gαi/o interacons k Goa/b interfering pepdes oa h Acvaon by BnOCPA of Gob vs Goa TRUPATH direct G protein assay h Acvaon by BnOCPA of Gob vs Goa TRUPATH direct G protein assay k Goa/b interfering pepdes h k g Selecvity of BnOCPA for Gob vs Goa g Goa/b interfering pepdes g Selecvity of BnOCPA for Gob vs Goa elicited equipotent activation of both Goa and Gob. In stark contrast to these agonists, BnOCPA was >10-fold more efﬁcacious in activating Gob than Goa, and, of all the agonists tested, BnOCPA displayed the weakest potency at Goa. NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 2i): BnOCPA antagonised the Goa-mediated inhibition of cAMP production by adenosine in a concentration-dependent manner. This classic attribute of an antagonist enabled a Schild analysis estimate of BnOCPA’s afﬁnity (Kd) to be 113 nM, with a pKd ~6.958, a value that was quantitatively similar to BnOCPA’s ability to bind to the hA1R (pKi ~6.6; Fig. 1b). Importantly, this observation, of the ability of BnOCPA to antagonise the actions of adenosine on cAMP inhibition (Fig. 2i), revealed no agonist action of BnOCPA at Goa at concentrations up to 100 μM (>105 greater than the IC50 against cAMP accumulation; Fig. 1b and ~104 greater than the EC50 in the TRUPATH assay; Fig. 2h), and, moreover, had parallels with the antagonising effects of BnOCPA on membrane potential in the CNS (Fig. 1h, i; Supplementary Fig. 2a, c). These data suggest that BnOCPA has the unique ability of displaying both agonist and antagonist-like properties at both recombinant and native A1Rs; properties that are expected of a truly Gα subunit-selective agonist. Thus, adenosine-mediated membrane potential hyperpolarisa- tion occurs mainly through A1R activation of Goa, in keeping with the high levels of expression of Goa vs Gob in the hippocampus59, and with the observation that the Goa-activating agonists adenosine, CPA and HOCPA (Fig. 2c–e, Supplementary Figs. 5 and 6) all induced membrane hyperpolarisation (Fig. 1h, i; Supplementary Figs. 2 and 5). Moreover, the absence of an effect of adenosine on membrane potential in Gz knockout mice70 argues against the possibility that the selective activation of Gz by adenosine observed in our PTX assays (Fig. 2c, d; Supplementary Fig. 6) contributes to membrane hyperpolarisation. The data from recombinant receptors demonstrating the inability of BnOCPA to activate Goa (Fig. 2a, c–g) thus explains why BnOCPA did not cause membrane hyperpolarisation, and indeed prevented or reversed the hyperpolarisation induced by CPA or adenosine, respectively (Fig. 1h, i; Supplementary Fig. 2a, c). y g The data from whole-cell patch-clamp recordings showed that BnOCPA did not inﬂuence neuronal membrane potential at native A1Rs (Fig. 1h, i), while experiments in recombinant hA1Rs showed that BnOCPA did not activate Goa (Fig. 2a, c–f), and indeed prevented the activation of Goa by adenosine (Fig. 2i). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Fig. 2 BnOCPA selectively activates Gob. a cAMP accumulation in PTX-pre-treated CHO-K1-hA1R cells expressing PTX-insensitive Goa following co- stimulation with 1 μM forskolin and each agonist (1 nM–1 μM; n = 6 individual repeats). b as for a but cells were transfected with PTX-insensitive Gob (n = 6 individual repeats). Stimulation of cAMP production in a reﬂects BnOCPA’s activation of endogenous, PTX-resistant Gs by the A1R (see Supplementary Figs. 5 and 6 and29,153,154). c, d Heatmaps summarising Emax (c %) and potency (d pEC50; −log [agonist concentration] required for 50% inhibition of cAMP accumulation) for individual Gα subunit and β-arrestin1 and 2 activation by selective A1R agonists for the inhibition of forskolin- stimulated cAMP production. Data taken from: adenosine, CPA, BnOCPA Fig. 1, Supplementary Figs. 3, 6; NECA, Supplementary Fig. 3, 6; HOCPA, Supplementary Fig. 5. e Venn diagram of agonist interactions with individual Gαo/i subunits. f The inhibition of cAMP accumulation via A1R:Goa or A1R:Gob by adenosine, CPA, HOCPA and BnOCPA. Each data point represents a concentration of agonist from the data in Supplementary Figs. 5 and 6. Line of unity (broken grey line) represents no bias. Data presented as mean ± SEM. g Signalling bias of A1R-selective agonists for A1R-Goa and A1R-Gob (Δ(τ/KA)) was determined relative to the natural agonist adenosine using the change in (τ/KA) ratio. The values were calculated for all compounds at each individual G protein and the data was ﬁtted globally to determine single values for τ and KA and then normalised to a reference agonist (adenosine). This approach, used by others155, precludes the provision of individual data points. Compared to adenosine, BnOCPA elicits no measurable response (NR) at Goa. h The TRUPATH assay for direct G protein activation reveals no preference between Goa and Gob by adenosine, CPA or HOCPA, but a signiﬁcant >10-fold greater activation of Gob vs Goa by BnOCPA (two-tailed unpaired Student’s t-test; P = 0.0009; see also Supplementary Fig. 7a; n = 8 individual repeats for each agonist). i Adenosine/Goa-mediated inhibition of cAMP accumulation was antagonised by BnOCPA in a concentration-dependent manner (n = 3–4 individual repeats). j Example current traces produced by adenosine (10 µM) in control conditions or in the presence of intracellular Goa interfering peptide, scrambled Goa peptide or Gob interfering peptide (all at 100 µM). Scale bars measure 25 pA and 100 s. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 k Summary data of adenosine-induced outward current experiments. The mean amplitude of the outward current induced by adenosine (40.6 ± 2.2 pA, n = 16 cells) was signiﬁcantly reduced (one-way ANOVA; F(3,37)=12.40, P = 9.22 × 10−6) to 20.9 ± 3.6 pA (n = 10 cells, P = 2.65 × 10−5) in 100 µM Goa interfering peptide. Neither the scrambled Goa peptide (Goa SCR; 43.4 ± 2.4 pA, n = 7 cells, P = 1) nor the Gob interfering peptide (39. 2 ± 2.7 pA, n = 8 cells, P = 1) reduced the amplitude of the adenosine-induced outward current compared to control, but each were signiﬁcantly different from the Goa interfering peptide (P = 8.20 × 10−5; P = 8.86 × 10−4, respectively). Averaged data are presented as mean ± SEM. , P < 0.0001 relative to other groups. Source data are provided as a Source Data ﬁle. the Goa and Gob vectors in the TRUPATH assay used in Fig. 2h (Supplementary Fig. 7b). The interfering peptides reduced the activation of their cognate G protein in a dose-dependent manner, but had no effect on the alternate Go isoform. The scrambled peptide sequence had no effect on Goa or Gob activation. inhibition of cAMP accumulation via Goa. This was indeed the case (Fig. 2i): BnOCPA antagonised the Goa-mediated inhibition of cAMP production by adenosine in a concentration-dependent manner. This classic attribute of an antagonist enabled a Schild analysis estimate of BnOCPA’s afﬁnity (Kd) to be 113 nM, with a pKd ~6.958, a value that was quantitatively similar to BnOCPA’s ability to bind to the hA1R (pKi ~6.6; Fig. 1b). Importantly, this observation, of the ability of BnOCPA to antagonise the actions of adenosine on cAMP inhibition (Fig. 2i), revealed no agonist action of BnOCPA at Goa at concentrations up to 100 μM (>105 greater than the IC50 against cAMP accumulation; Fig. 1b and ~104 greater than the EC50 in the TRUPATH assay; Fig. 2h), and, moreover, had parallels with the antagonising effects of BnOCPA on membrane potential in the CNS (Fig. 1h, i; Supplementary Fig. 2a, c). These data suggest that BnOCPA has the unique ability of displaying both agonist and antagonist-like properties at both recombinant and native A1Rs; properties that are expected of a truly Gα subunit-selective agonist. inhibition of cAMP accumulation via Goa. This was indeed the case (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 While subtle differences between the Goa and Gob response exist across the two very different in vitro assays, these data nonetheless elicited equipotent activation of both Goa and Gob. In stark contrast to these agonists, BnOCPA was >10-fold more efﬁcacious in activating Gob than Goa, and, of all the agonists tested, BnOCPA displayed the weakest potency at Goa. While subtle differences between the Goa and Gob response exist across the two very different in vitro assays, these data nonetheless conﬁrm that BnOCPA demonstrates a previously unprecedented ability for an A1R agonist to discriminate between Gα subtypes, and in particular between Goa and Gob. conﬁrm that BnOCPA demonstrates a previously unprecedented ability for an A1R agonist to discriminate between Gα subtypes, and in particular between Goa and Gob. To establish the functional implications of BnOCPA’s profound selectivity for Gob over Goa, we hypothesised that BnOCPA should reduce the actions of adenosine on the To establish the functional implications of BnOCPA’s profound selectivity for Gob over Goa, we hypothesised that BnOCPA should reduce the actions of adenosine on the 5 5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 In these simulations the G protein was omitted so that inactivation wa possible and so that the results were not G protein-dependent The BnOCPA binding Modes A–C were interchangeable during MD simulations (Table 1) but were associated with distinctly different dynamics, as monitored by changes in a structura hallmark of GPCR activation, the N7.49PXXY7.53 motif7 Adenosine (endogenous agonist) BnOCPA Mode A PSB36 (antagonist) BnOCPA Mode B HOCPA (unbiased agonist) BnOCPA Mode C a d c b f e i g j BnOCPA Mode D TM1 TM2 TM4 TM5 TM6 TM7 ECL2 ECL3 h HOCPA (unbiased agonist) b PSB36 (antagonist) BnOCPA Mode C c f PSB36 (antagonist) c HOCPA (unbiased agonist) Adenosine (endogenous agonist) a PSB36 (antagonist) Adenosine (endogenous agonist) a PSB36 (antagonist) Adenosine (endogenous agonist) a b BnOCPA Mode C f BnOCPA Mode A d f d BnOCPA Mode A e e g BnOCPA Mode D TM1 TM2 TM4 TM5 TM6 TM7 ECL2 ECL3 h i i g j TM1 TM2 TM4 TM5 TM6 TM7 ECL2 ECL3 h TM1 TM2 TM4 TM5 TM6 TM7 ECL2 ECL3 h j h Firstly, following Dror et al.76, we compared the dynamics of the BnOCPA-bound A1R with the corresponding dynamics of the receptor77,78 bound to either HOCPA (Fig. 3b), the A1R antagonist PSB36 (Fig. 3c), or the apo receptor in the absence of G protein, our hypothesis being that there may be ligand- dependent differences in the way that the intracellular region of the receptor responds in the absence of the G protein. In these simulations the G protein was omitted so that inactivation was possible and so that the results were not G protein-dependent. The BnOCPA binding Modes A–C were interchangeable during MD simulations (Table 1) but were associated with distinctly different dynamics, as monitored by changes in a structural hallmark of GPCR activation, the N7.49PXXY7.53 motif79 (Supplementary Fig. 9). Given the high ﬂexibility shown by the agonists adenosine and HOCPA, and an antagonist (PSB36) of the A1R (Fig. 3a–c). BnOCPA engaged the receptor with the same ﬁngerprint as adenosine72 (Fig. 3a) and HOCPA (Fig. 3b, Sup- plementary Movie 2). Further explorations of the BnOCPA docked state using metadynamics (MetaD) simulations73 revealed interchangeable variations on this ﬁngerprint (namely Modes A, B, and C; Fig. 3d–f; Supplementary Fig. 8) that could be dis- tinguished by the orientation of the BnOCPA-unique benzyl group. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 PSB36 (antagonist) BnOCPA Mode B HOCPA (unbiased agonist) BnOCPA Mode C c b f e i j nist) PSB36 (antagonist) BnOCPA Mode B HOCPA (unbiased agonist) BnOCPA Mode C c b f e i agonists adenosine and HOCPA, and an antagonist (PSB36) of the A1R (Fig. 3a–c). BnOCPA engaged the receptor with the same ﬁngerprint as adenosine72 (Fig. 3a) and HOCPA (Fig. 3b, Sup- plementary Movie 2). Further explorations of the BnOCPA docked state using metadynamics (MetaD) simulations73 revealed interchangeable variations on this ﬁngerprint (namely Modes A, B, and C; Fig. 3d–f; Supplementary Fig. 8) that could be dis- tinguished by the orientation of the BnOCPA-unique benzyl group. Having established the possible BnOCPA binding modes, we examined the respective contribution of the orthosteric ago- nists, the G protein α subunit α5 (C-terminal) helix (GαCT), and the Gα protein subunit74,75 to the empirically-observed G protein selectivity displayed by BnOCPA (Fig. 2a–h, Supplementary Firstly, following Dror et al.76, we compared the dynamics o the BnOCPA-bound A1R with the corresponding dynamics of the receptor77,78 bound to either HOCPA (Fig. 3b), the A1R antagonist PSB36 (Fig. 3c), or the apo receptor in the absence of G protein, our hypothesis being that there may be ligand dependent differences in the way that the intracellular region o the receptor responds in the absence of the G protein. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 We thus predicted that A1Rs in the hippocampus, where Goa is found at levels 10–15 times higher than Gob59, should act via Goa to induce membrane hyperpolarisation, and thereby providing a potential explanation for the lack of effect of BnOCPA on membrane potential. To test this prediction, we injected a series of previously-validated interfering peptides against Goa and Gob60–69 into CA1 pyramidal cells during whole-cell voltage- clamp recordings. Introduction of the Goa interfering peptide caused a signiﬁcant attenuation of the adenosine-induced outward current (Fig. 2j, k). In contrast, neither the scrambled Goa peptide, nor the Gob peptide, which reduced the modulation of Ca2+ channels by muscarinic M4 receptors in striatal cholinergic interneurons63, had any effect on outward current amplitude (Fig. 2j, k). To conﬁrm the speciﬁcity and potency of the interfering peptides used in hippocampal neurons, we transfected plasmids coding for the last 11 C-terminal amino acids of either Goa, Gob and the scrambled version of Goa, into The Gα selectivity displayed by BnOCPA is reﬂected in non- canonical binding modes and selective interaction with Gαi/o subunits. To better understand the unusual signalling properties of BnOCPA and the highly speciﬁc Gα coupling to Gob, we carried out dynamic docking simulations to study the basic orthosteric binding mode of BnOCPA in an explicit, fully ﬂexible environment using the active cryo-EM structure of the A1R (PDB code 6D9H; Supplementary Movie 1). We previously reported that modiﬁcations at position N6 of the adenine scaffold modu- lated the agonist binding path to A1R71. More precisely, N6- cyclopentyl analogues (CPA and HOCPA) markedly interact with the extracellular loop 2 (ECL2) compared to adenosine, while BnOCPA (which bears the N6-cyclopentyl-2-benzyloxy group) is most prone to engage residues of the A1R located at the top of transmembrane helix 1 (TM1) and TM7. In the present study, we compared the bound-state BnOCPA to the non-Gα selective NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 6 ARTICLE ARTICLE g p To test the prediction from the MD simulations that R2917.56 and I2928.47 were involved in A1R/Gα coupling, we performed a series of site-directed mutagenesis (to alanine) against R2917.56, I2928.47 and the adjacent hydrophilic residues Q2938.48 and K2948.49 (Fig. 4i) and compared the inhibition of forskolin- stimulated cAMP production in response to adenosine, CPA, NECA, HOCPA and BnOCPA in Flp-In-CHO cells against the wild-type (WT) hA1R (Fig. 4j). Of these residues, none of which are reported to affect binding82,83, K2948.49 had the least impact on potency; of the agonists, the mutations had minimal effects on HOCPA. In contrast A1R/Gα coupling induced by adenosine, CPA, NECA and BnOCPA was affected, but differentially so. These effects on potency (IC50 values) can be readily observed when individual mutant IC50 values are normalised to their respective WT controls (Fig. 4k), and revealed that R2917.56, I2928.47 and Q2938.48 are especially important for CPA and NECA coupling, R2917.56 for adenosine potency, and Q2938.48 for BnOCPA. These observations reinforce the MD simulations predictions related to H8 residues involved in G protein coupling of the agonist-stimulated A1R, and in particular suggest that R2917.56, I2928.47 and Q2938.48 are especially required for selective agonist coupling to Gαo/i, and may thus contribute to the Gα bias observed among these agonists (Fig. 2c, d). A more detailed analysis, involving saturation mutagenesis of these residues, is required to provide a full characterisation of their actions to direct agonist bias but is beyond the scope of this current study. µ µ Next, to simulate the agonist-driven interaction between the A1R and the G protein, the α5 (C-terminal) helix (GαCT) of the G protein (Gi2, Goa, Gob) was dynamically docked to the HOCPA- and BnOCPA-bound active A1R structure (again lacking G protein; Supplementary Movie 3). This allowed us to evaluate the effect of different GαCT on the formation of the complex with A1R to test the hypothesis that, of Goa, Gob and Gi2, only the GαCT of Gob would fully engage with the BnOCPA-bound active A1R, in line with the empirical observa- tions of G protein selectivity summarised in Fig. 2c, d. Figure 4a shows that the GαCT of Gob docked to the A1R via a metastable state (MS1) relative to the canonical state (CS1; Supplementary Movie 3), regardless of whether HOCPA or BnOCPA was bound. ARTICLE ARTICLE Fig. 3 Molecular dynamics simulations reveal that BnOCPA binding modes can uniquely drive both agonist- and antagonist-like intracellular conformations of the A1R. a Adenosine binding pose: N2546.55 (Ballesteros-Weinstein superscript enumeration) is engaged in key hydrogen bonds, while important hydrophobic contacts are shown as cyan transparent surfaces (F171ECL2 and I2747.39). b On the basis of structural similarities and the dynamic docking (Supplementary Movie 2), HOCPA was predicted to bind with a geometry analogous to adenosine; the cyclopentyl group makes further hydrophobic contacts with L2536.54, as shown by simulation. c The xanthine scaffold of the antagonist PSB36 makes hydrogen bonds with N2546.55 side chains and hydrophobic contacts with F171ECL2 and I2747.39. d BnOCPA agonist-like binding Mode A (Supplementary Movie 1): the benzyl group orients towards the ECL2 and makes hydrophobic contacts with I175ECL2 (and M1775.35) side chains. e BnOCPA antagonist-like binding Mode B: the benzyl group orients towards the top of TM5/TM6 and makes hydrophobic contacts with L2586.59 side chain. f BnOCPA agonist-like binding Mode C: the benzyl group orients towards the top of TM7 and makes hydrophobic contacts with Y2717.36 side chain. g Binding orientation of BnOCPA in antagonist-like Mode D: the benzyl group orients under ECL3 and occupies the hydrophobic pocket deﬁned by L2536.54, T2576.58, T2707.35, and L2697.34. Key hydrogen bonds with N2546.55 and T2777.42 are shown as dotted lines; main hydrophobic contacts are highlighted as cyan transparent surfaces. h Extracellular view of the A1R showing the four BnOCPA binding Modes A (cyan), B (magenta), C (green), and D (red) as randomly extracted from the MD simulations. i, j Root-mean- square deviation (RMSD) distributions considering the inactive N7.49PXXY7.53 motif on the distal part of TM7 as reference. i HOCPA (blue broken line), BnOCPA Mode A (cyan curve), BnOCPA Mode C (green curve) and the apo receptor (dark green broken line) have a common distribution centring around the active conﬁrmation of the A1R (orange broken line; Supplementary Fig. 9) leading to A1R signalling. In contrast, j PSB36 (black broken line), BnOCPA Mode B (magenta curve) and BnOCPA Mode D (red curve) RMSD values have the tendency to move closer to the inactive N7.49PXXY7.53 geometry (leftward shift of the curves towards broken grey line at x = 0) preventing A1R signalling. ARTICLE increased the complexity of the simulations by considering the Gα subunit of the Goa and Gob protein bound to the A1R:BnOCPA (Mode B or D) complex or the Gob protein bound to A1R:HOCPA (a functional system). The most visible differences between Goa (Supplementary Movie 4) and Gob (Supplementary Movie 5) comprised the formation of transient hydrogen bonds between the α4-β6 and α3-β5 loops of Goa and helix 8 (H8) of the receptor (Supplementary Table 3). Similar contacts are present in the non-canonical state of the neurotensin receptor:Gi protein complex80. Overall, Goa interacted more with TM3 and ICL2 residues (Fig. 4g, h), while TM5 and TM6, along with ICL1, were more engaged by Gob (Fig. 4g, h). Interestingly, R2917.56 and I2928.47, which are located under the N7.49PXXY7.53 motif, showed a different propensity to interact with Goa or Gob. In this scenario, it is plausible that a particular A1R conformation stabilised by BnOCPA (as suggested by the simulations in the absence of G protein, Fig. 3i, j) may favour different intermediate states during the activation process of Goa and Gob. BnOCPA benzyl group during the simulations and its lipophilic character, we hypothesised and simulated a further binding mode (namely Mode D) not explored during MD or MetaD simula- tions. This conformation involves a hydrophobic pocket under- neath ECL3 (Fig. 3g) which is responsible for the A1/A2A selectivity72. Superimposition of the four BnOCPA binding Modes A–D reveals the highly motile nature of the benzyl group of BnOCPA (Fig. 3h) under the simulated conditions. Quantiﬁcation of the N7.49PXXY7.53 dynamics revealed that HOCPA, BnOCPA Mode A, BnOCPA Mode C and the apo receptor show a similar distribution of the RMSD of the conserved N7.49PXXY7.53 motif (Fig. 3i; Supplementary Fig. 9). In contrast, the non-canonical BnOCPA binding Modes B and D were responsible for a partial transition of the N7.49PXXY7.53 backbone from the active conformation to the inactive con- formation (Supplementary Fig. 9) in a manner analogous with the antagonist PSB36 (Fig. 3j). Overall, the simulations revealed Mode D as the most stable BnOCPA pose (6.8 µs out of 9 µs simulated starting from this conﬁguration – Table 1), while Mode B accounted for 3.6 µs out of 30 µs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Having established the possible BnOCPA binding modes, we examined the respective contribution of the orthosteric ago- nists, the G protein α subunit α5 (C-terminal) helix (GαCT), and the Gα protein subunit74,75 to the empirically-observed G protein selectivity displayed by BnOCPA (Fig. 2a–h, Supplementary Fig. 6). 7 NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 es not depress heart rate, blood pressure or vidence for in vivo physiological selectivity at rate and blood pressure12. These cardiovasc through Goa, which is expressed at high lev 6D9H 6OSA 6E67 BnOCPA Gob HOCPA Gob BnOCPA Gi2 BnOCPA Goa 180° 1 S C 1 S C MS1 MS1 180° 2 S M 2 S M MS3 MS3 CS1 MS1 MS2 MS3 c b a f e d g 90° h j k i CS1 MS1 MS2 MS3 b a e d 6D9H 6OSA BnOCPA Gob HOCPA Gob 180° 1 S C 1 S C MS1 MS1 c b 6E67 BnOCPA Gi2 BnOCPA Goa 180° 2 S M 2 S M MS3 MS3 f e BnOCPA does not depress heart rate, blood pressure or respiration: evidence for in vivo physiological selectivity at native A1Rs. Given BnOCPA’s clear differential effects in a native physiological system (Fig. 1), strong Gob selectivity (Fig. 2), unique binding characteristics (Fig. 3) and selective Gob inter- action (Fig. 4), we hypothesised that these properties might cir- cumvent a key obstacle to the development of A1R agonists for therapeutic use—their powerful effects in the cardiovascular system (CVS) where their activation markedly reduces both heart rate and blood pressure12. These cardiovascular through Goa, which is expressed at high levels i particularly in the atria86, and which plays an im regulating cardiac function87. In contrast, and w differential Goa vs Gob expression in the hippo may be absent or expressed at very low levels i Given this differential expression of Goa and Gob functional effect of BnOCPA on Goa (Fig. 2a–g that BnOCPA would have minimal effects on the BnOCPA Gi2 180° 2 S M 2 S M MS3 MS3 MS3 g 90° h j k i 90° h j k i k rate and blood pressure12. These cardiovascular effects are likely through Goa, which is expressed at high levels in the heart84,85, particularly in the atria86, and which plays an important role in regulating cardiac function87. In contrast, and with parallels of differential Goa vs Gob expression in the hippocampus59, Gob may be absent or expressed at very low levels in the heart86,88. Given this differential expression of Goa and Gob, and the lack of functional effect of BnOCPA on Goa (Fig. 2a–g), we predicted that BnOCPA would have minimal effects on the CVS. ARTICLE Figure 4b, c show that the CS1 geometry corresponds to the canonical arrangement as found in the cryo-EM A1R:Gi protein complex, whereas state MS1 resembles the recently reported non- canonical state observed in the neurotensin receptor, believed to be an intermediate on the way to the canonical state80. In contrast, ﬁg. 4d–f shows that the GαCT of Goa and Gi2 docks to the A1R to form metastable states MS2 and MS3. MS2 is similar to the β2-adrenergic receptor:GsCT fusion complex81, proposed to be an intermediate on the activation pathway and a structure relevant to G protein speciﬁcity. In this case, however, it appears to be on an unproductive pathway. To test the hypothesis that the non-functional BnOC- PA:A1R:Goa complex showed anomalous dynamics, we next NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 8 ARTICLE TURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Source data are provided as a Source Data ﬁle. given the antagonism of Goa-mediated actions by BnOCPA at native and recombinant A1Rs (Fig. 1h, i, Supplementary Fig. 2a, c, Fig. 2i), we further predicted that the actions of adenosine on the CVS may be attenuated by BnOCPA. CPA, BnOCPA abolished the bradycardia induced by both agonists, indicating its ability to bind to the A1R at the dose applied (Fig. 5a, c; Fig. 6g, Supplementary Figs. 10b and 11). Volumes of saline equivalent to the drug injections had no effect on either heart rate or blood pressure and there was no waning in the effects of adenosine responses with repeated doses (Supple- mentary Fig. 10c, d). Thus, BnOCPA does not appear to act as an agonist at CVS A1Rs, but instead antagonises the bradycardic effects of A1R activation on the heart. In initial experiments, we screened BnOCPA for its effects on heart rate using an isolated frog heart preparation. In contrast to adenosine and CPA, which depress heart rate through hyperpo- larisation caused by activation of cardiac sinoatrial K+ channels89, BnOCPA had no effect on heart rate, but markedly reduced the bradycardia evoked by adenosine (Supplementary Fig. 10a). Thus, BnOCPA appears not to activate A1Rs in the heart, but instead behaves like an antagonist in preventing the actions of the endogenous agonist. These observations have parallels with BnOCPA’s inability to activate A1Rs to hyperpo- larise neurones, and indeed inhibiting or reversing the post- synaptic hyperpolarisation induced by typical A1R agonists (Fig. 1h, i; Supplementary Fig. 2a, c), and in preventing the A1R/Goa-mediated inhibition of cAMP accumulation by adeno- sine (Fig. 2i). Such antagonist-like behaviour may be explained by BnOCPA causing unique A1R conformations unlike those of conventional agonists (Fig. 3i, j), and driving non-canonical and ultimately non-productive interactions with Goa (Fig. 4). Since adverse effects on respiration (dyspnoea) limit the use of systemic A1R agonists7, we additionally examined the effects of BnOCPA on respiration. In urethane-anaesthetised, sponta- neously breathing adult rats, intravenous injection of BnOCPA had no appreciable effect on respiration (Fig. 6a–d), even if the dose of BnOCPA was doubled or trebled (Fig. 6e, f). In stark contrast the selective A1R agonist CPA caused signiﬁcant respiratory depression (Fig. 6a–d). Paralleling BnOCPA’s antag- onism of adenosine- and CPA-induced depressions of heart rate (Fig. 5a, c; Supplementary Figs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ARTICLE Fig. 4 BnOCPA selectively induces canonical activation states at A1R:Gob, but non-productive metastable states at other Gαi/o subunits. a–c Dynamic docking of the Gob-GαCT (last 27 residues) performed on the BnOCPA-A1R (black) and the HOCPA-A1R (magenta) complex, respectively. a Frequency distribution of the RMSD of the last 15 residues of Gob-GαCT (alpha carbon atoms) relative to the Gi2-GαCT conformation reported in the A1R structure PDB code 6D9H (dashed grey line indicates 3.6 Å resolution). The two most probable RMSD ranges, canonical state (CS) CS1 and metastable state (MS) MS1, can be observed. b, c Side views of representative MD frames of the most populated α5 clusters from CS1 and MS1. The last 15 residues of Gob-GαCT in CS1 of both BnOCPA and HOCPA resemble the Gi2-bound state (PDB code 6D9H; cyan). MS1 is characterised by a binding geometry similar to the non- canonical Gi intermediate state reported for the neurotensin receptor structure (PDB Code 6OSA; orange). d–f Dynamic docking of the Goa- and Gi2- GαCT (last 27 residues) performed on the BnOCPA-A1R complex. d As for a except Gob was replaced with Goa (red) and compared to Gi2 (blue), with the two most probable RMSD ranges labelled as MS2 and MS3. e, f Side views of representative MD frames of the most populated GαCT clusters from MS2 and MS3. The Goa and Gi2 last 15 residues in MS2 overlap well with the putative Gs intermediate state (PDB code 6E67; green). In MS3, the GαCT helix orients in unique conformations that differ from those previously described. g, h For each residue the interaction plotted on the backbone is the difference between the Goa and Gob occupancies in the presence of orthosteric BnOCPA (% of MD frames in which interaction occurred). BnOCPA/A1R/Goa (inactive coupling) had the tendency to interact more with ICL2, TM3 TM7, and H8 (red), while BnOCPA/A1R/Gob (active coupling) formed more contacts with TM5 and TM6 (blue). i Residues in TM7 and H8 of the hA1R predicted by MD simulations to be of importance to A1R coupling to Goa (left) and Gob (right). j, k Mutations of R2917.56, I2928.47, Q2938.48 and K2948.49 to alanine in the hA1R differentially affect agonist efﬁcacy against stimulated cAMP production. j Data points represent individual IC50 values (n = 5-13 individual experiments), with the mean represented as the horizontal bar and the box limits indicating ±1 SD. k Spider plot summarising data from j. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 10b and 11), BnOCPA reduced the depression of respiratory frequency and minute ventilation caused by CPA (Fig. 6g, Supplementary Fig. 11). These data suggest that while BnOCPA targets and clearly engages the A1Rs responsible for adenosine and CPA’s cardiorespiratory depres- sion, BnOCPA has no agonist action at these A1Rs. y p ( g ) To investigate the effects of BnOCPA in an intact mammalian system, we measured the inﬂuence of BnOCPA on heart rate and blood pressure in urethane-anaesthetised, spontaneously breath- ing adult rats. As expected, both resting heart rate and arterial blood pressure were signiﬁcantly reduced by adenosine and CPA (Fig. 5a–d). In complete contrast, BnOCPA had no effect on either heart rate (Fig. 5a, c) or blood pressure (Fig. 5b, d), even when applied at two or three times the initial dose (Supplemen- tary Fig. 11; Fig. 6e, f). These negative observations could not be explained by metabolism of BnOCPA to an inactive substance since BnOCPA is a very stable compound (half-life (t1/2) >240 min in PBS at 37 °C) with a human plasma stability of ~100% remaining after 120 mins suggesting a t1/2 > 240 min at 37 °C. In addition, the in vitro metabolic t1/2 of BnOCPA was determined as >60 mins using human liver microsomes (0.1 mg/ mL, 37 °C), and the intrinsic clearance (CLint) calculated as <115.5 μL/min/mg. This was in contrast to the reference compounds verapamil and terfenadine (0.1 μM) with t1/2 in human plasma determined as 33 and 10 min and CLint as 213.1 and 683.0 μl/min/mg, respectively (see Supporting Data File 1). Further evidence that BnOCPA was present and active during these experiments was obtained from studies analogous to those in frog heart when BnOCPA was applied together with adenosine. In the intact anaesthetised rat, when co-applied with adenosine or BnOCPA is a potent analgesic. Our observations of a lack of effect of BnOCPA on the CVS and respiration prompted an investigation into a potential application of A1R agonists that had previously been severely curtailed by adverse cardiorespiratory events7,16, namely the use of A1R agonists as analgesics. Since sedation or motor impairment can be mistaken for analgesia, we tested BnOCPA in a sensitive assay for balance and motor coordination, the rotarod, in which the ability of a rodent to remain upon a slowly accelerating rotating cylinder is a measure of alertness and motor function. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Moreover, BnOCPA does not depress heart rate, blood pressure or respiration: evidence for in vivo physiological selectivity at native A1Rs. Given BnOCPA’s clear differential effects in a native physiological system (Fig. 1), strong Gob selectivity (Fig. 2), unique binding characteristics (Fig. 3) and selective Gob inter- action (Fig. 4), we hypothesised that these properties might cir- cumvent a key obstacle to the development of A1R agonists for therapeutic use—their powerful effects in the cardiovascular system (CVS) where their activation markedly reduces both heart 9 9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Discussion sedation or locomotor impairment that could confound inter- pretations of analgesia. Biased agonists at GPCRs offer great potential for the preferential activation of desirable intracellular signalling pathways, while avoiding, or indeed blocking those pathways that lead to adverse or unwanted effects3,27. While this, and the potential to exploit previously unattractive drug targets such as the A1R, have been appreciated, translation of in vitro observations, particularly of Gα bias, to native receptors in vivo has been problematic3,4,27. Here we have shown that translation of in vitro selectivity among Gα subunits, identiﬁed using two separate assays, to an intact physiological system is possible through a benzyloxy derivative (BnOCPA) of the selective A1R agonist CPA. Moreover, this Gα selectivity has occurred in the context of the A1R, an attractive, but notoriously intractable drug target by virtue of the profound cardiorespiratory consequences of its activation by conventional A1R agonists. To assess the potential of BnOCPA as an analgesic, we used a rat model of chronic neuropathic pain (spinal nerve ligation)90 a feature of which is mechanical allodynia whereby the affected limb is rendered sensitive to previously innocuous tactile stimuli, and which models the debilitating human clinical condition of chronic pain, which affects between 20 and 50% of the population91,92, and which carries a major global burden of disability93. Both intrathecal (Fig. 7b) and intravenous (Fig. 7c) BnOCPA potently reversed mechanical allodynia in a dose-dependent manner. Thus, BnOCPA exhibits powerful analgesic properties at doses devoid of sedative or cardiorespiratory effects, and at several orders of magnitude lower than the non-opioid analgesics pregabalin and gabapentin94. To test if this analgesia was mediated by the activation of A1Rs by BnOCPA, we used the selective A1R antagonist, DPCPX. Prior administration of DPCPX prevented the reversal of mechanical allodynia by BnOCPA (Fig. 7d), conﬁrming the importance of A1Rs in mediating the analgesic actions of BnOCPA. In contrast, the rat A3R-selective antagonist MRS152395,96, which is effective in reversing analgesia caused by selective A3R agonists97–99, had no effect on the analgesia caused by BnOCPA, and indeed may have provoked a slight facilitation. This may be due to the reported g BnOCPA was ﬁrst reported as a ﬁnal compound in a patent where it was described to be selective for the A1R with respect to its binding afﬁnity, and effective in reducing elevated intraocular pressure for the potential treatment of glaucoma or ocular hypertension34. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 As a positive control for the sensitivity of the test, we showed that the ability of animals treated with morphine to remain on the rotating cylinder was strongly impaired (Fig. 7a). In contrast, the performance of ani- mals treated with BnOCPA, delivered either intravenously or intraperitoneally, was indistinguishable from vehicle-treated mice (Fig. 7a). This was true even if BnOCPA was injected intrave- nously at three times the dose (Fig. 7a), which, while having no cardiorespiratory actions on its own, prevented cardiorespiratory depression caused by adenosine and CPA (Figs. 5 and 6; Sup- plementary Figs. 10 and 11). Thus, BnOCPA does not induce NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 10 ARTICLE BnOCPA had no effect on its own on MAP (88 ± 11 vs 85 ± 13 mm Hg; P = 1) and did not prevent adenosine in lowering MAP to a value similar to adenosine on its own (51 ± 4 mm Hg; P = 1 vs adenosine; P = 0.012 vs BnOCPA alone). CPA signiﬁcantly decreased MAP (from 83 ± 8 to 51 ± 5 mm Hg; P = 0.017), a decrease that was not different to the effect of adenosine in the absence or presence of BnOCPA (P = 1 for both). ns, not signiﬁcant; , P < 0.02; *, P < 0.001; , P < 0.0001. Source data are provided as a Source Data ﬁle. elevated adenosine tone100 and activation of A3Rs101 in the neuropathic spinal cord, which may have resulted in the desensitisation of A1R-mediated responses102. These observations conﬁrm that the analgesia provoked by BnOCPA is mediated via the selective activation of A1Rs. ARTICLE CPA signiﬁcantly decreased MAP (from 83 ± 8 to 51 ± 5 mm Hg; P = 0.017), a decrease that was not different to the effect of adenosine in the absence or presence of BnOCPA (P = 1 for both). ns, not signiﬁcant; , P < 0.02; *, P < 0.001; *, P < 0.0001. Source data are provided as a Source Data ﬁle. Fig. 5 BnOCPA does not affect heart rate or blood pressure. a Examples of heart rate (HR) and b arterial blood pressure traces from a urethane- anaesthetised, spontaneously breathing rat showing the effects of adenosine (1 mg kg−1), BnOCPA (8 µg kg−1) and CPA (6 µg kg−1). Grey diamonds reﬂect saline ﬂushing of the femoral vein catheter. Insets are expanded HR and blood pressure responses to adenosine and BnOCPA (black and blue traces, respectively; boxed regions in a and b). Scale bars measure: HR, 200 BPM and 6 s; blood pressure, 40 mm Hg and 6 s. c, d Summary data for 4 rats where each rat is shown as a different symbol with the means (± SEM, light grey bars) connected. One-way RM ANOVA for: c HR, Greenhouse-Geisser corrected F(2.33, 7.00) = 68.27, c HR, Greenhouse-Geisser corrected F(2.33, 7.00) = 68.27, P = 2.07 × 10−5; d mean arterial blood pressure (MAP), Greenhouse- Geisser corrected F(1.84, 5.52) =10.51, P = 0.014; with the following Bonferroni post hoc comparisons: The resting HR of 432 ± 21 BPM was signiﬁcantly reduced to 147 ± 12 BPM (~66%, P = 2.76 × 10−11) by adenosine. BnOCPA had no effect on HR (~6%, 442 ± 20 vs 416 ± 21 BPM; P = 1) but prevented the bradycardic effects of adenosine (P = 2.71 × 10−9 vs adenosine) when co-injected (mean change 51 ± 4 BPM; ~12%; P = 0.67). CPA signiﬁcantly decreased HR (from 408 ± 17 to 207 ± 29 BPM; ~50%, P = 1.85 × 10−8), a decrease that was not different to the effect of adenosine (P = 0.12), but was signiﬁcantly different to the effect of both BnOCPA (P = 9.00 × 10−9) and adenosine in the presence of BnOCPA (P = 6.69 × 10−7). The resting MAP (86 ± 9 mm Hg) was signiﬁcantly reduced by adenosine (~47%, 46 ± 4 mm Hg; P = 0.001). ARTICLE ARTICLE d l h ld f d Fig. 5 BnOCPA does not affect heart rate or blood pressure. a Examples of heart rate (HR) and b arterial blood pressure traces from a urethane- anaesthetised, spontaneously breathing rat showing the effects of adenosine (1 mg kg−1), BnOCPA (8 µg kg−1) and CPA (6 µg kg−1). Grey diamonds reﬂect saline ﬂushing of the femoral vein catheter. Insets are expanded HR and blood pressure responses to adenosine and BnOCPA (black and blue traces, respectively; boxed regions in a and b). Scale bars measure: HR, 200 BPM and 6 s; blood pressure, 40 mm Hg and 6 s. c, d Summary data for 4 rats where each rat is shown as a different symbol with the means (± SEM, light grey bars) connected. One-way RM ANOVA for: c HR, Greenhouse-Geisser corrected F(2.33, 7.00) = 68.27, P = 2.07 × 10−5; d mean arterial blood pressure (MAP), Greenhouse- Geisser corrected F(1.84, 5.52) =10.51, P = 0.014; with the following Bonferroni post hoc comparisons: The resting HR of 432 ± 21 BPM was signiﬁcantly reduced to 147 ± 12 BPM (~66%, P = 2.76 × 10−11) by adenosine. BnOCPA had no effect on HR (~6%, 442 ± 20 vs 416 ± 21 BPM; P = 1) but prevented the bradycardic effects of adenosine (P = 2.71 × 10−9 vs adenosine) when co-injected (mean change 51 ± 4 BPM; ~12%; P = 0.67). CPA signiﬁcantly decreased HR (from 408 ± 17 to 207 ± 29 BPM; ~50%, P = 1.85 × 10−8), a decrease that was not different to the effect of adenosine (P = 0.12), but was signiﬁcantly different to the effect of both BnOCPA (P = 9.00 × 10−9) and adenosine in the presence of BnOCPA (P = 6.69 × 10−7). The resting MAP (86 ± 9 mm Hg) was signiﬁcantly reduced by adenosine (~47%, 46 ± 4 mm Hg; P = 0.001). BnOCPA had no effect on its own on MAP (88 ± 11 vs 85 ± 13 mm Hg; P = 1) and did not prevent adenosine in lowering MAP to a value similar to adenosine on its own (51 ± 4 mm Hg; P = 1 vs adenosine; P = 0.012 vs BnOCPA alone). NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunicatio Methods Approvals. All experiments involving animals were conducted with the knowledge and approval of the University of Warwick Animal Welfare and Ethical Review Board, and in accordance with the U.K. Animals (Scientiﬁc Procedures) Act (1986) and the EU Directive 2010/63/EU. In vivo cardiorespiratory studies were con- ducted under the auspices of UK PPL 70/8936 and chronic neuropathic pain studies under the auspices of P9D9428A9. Rotarod studies were approved by the Monash University Animal Ethics Committee in accordance with the Australian Code for the Care and Use of Animals for Scientiﬁc Purposes (2013) under Monash AEC protocol number 13333. g yp The unique and unprecedented Gα selectivity displayed by BnOPCA has physiological importance since it is able to inhibit excitatory synaptic transmission without causing neuronal membrane hyperpolarisation, sedation, bradycardia, hypotension or dyspnoea. BnOCPA thus overcomes cardiovascular and respiratory obstacles to the development of adenosine-based therapeutics that have plagued the ﬁeld since their ﬁrst descrip- tion nine decades ago105. As a ﬁrst, but signiﬁcant, step towards this, we demonstrate that BnOCPA has powerful analgesic properties via A1Rs in an in vivo model of chronic neuropathic pain, potentially through a mechanism that may involve a com- bination of inhibition of synaptic transmission in peripheral and spinal pain pathways, and the hyperpolarisation of Gob- containing nociceptive neurons. Chronic pain, a condition that a large proportion of the population suffers on a constant or frequent basis91,92 and associated with a major global burden of disability93 is, however, a disorder for which the current treat- ments are either severely lacking in efﬁcacy106 or, in the case of opioids, come with unacceptable harms such as adverse gastro- intestinal effects, respiratory depression, tolerance, dependence and abuse potential107. Accordingly, novel treatments for chronic pain are urgently required. Preparation of hippocampal slices. Sagittal slices of hippocampus (300–400 µm) were prepared from male Sprague–Dawley rats, at postnatal days 12–20108. Rats were kept on a 12-hour light-dark cycle with slices made 90 min after entering the light cycle. In accordance with the U.K. Animals (Scientiﬁc Procedures) Act (1986), rats were killed by cervical dislocation and then decapitated. The brain was removed, cut down the midline and the two sides of the brain stuck down to a metal base plate using cyanoacrylate glue. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ARTICLE Fig. 6 BnOCPA does not cause respiratory depression. a Examples of tracheal airﬂow, respiratory frequency (f), tidal volume (VT) and minute ventilation (VE) from a urethane-anaesthetised, spontaneously breathing rat showing the lack of effect of BnOCPA on respiration and the respiratory depression caused by CPA. BnOCPA or CPA were given intravenously at the times indicated by the vertical broken lines (BnOCPA, 8 µg kg−1, blue; CPA, 6 µg kg−1, red). Grey diamonds indicate spontaneous sighs. Scale bars measure: 180 s and: airﬂow, 0.5 mL; f, 50 breaths per minute (BrPM); VT, 0.25 mL; VE, 50 mL/min. b–d Summary data for 8 animals. Data from each rat is shown before and after the injection of BnOCPA (blue squares and broken lines) or CPA (red circles and broken lines) together with the mean value for all animals (solid lines) for f, VT and VE, respectively. One-way RM ANOVA: For: b f, Greenhouse-Geisser corrected F(1.20, 8.38) = 30.4, P = 3.48 × 10−4; c VT, F(3, 21) = 15.9, P = 1.25 × 10−5, and d VE, Greenhouse-Geisser corrected F(1.19, 8.34) =15.77, P = 0.003, with the following Bonferroni post hoc comparisons: Following BnOCPA, f (149 ± 12 BrPM), VT (1.0 ± 0.1 mL), and VE (152 ± 26 ml/min) were not altered (P = 1) compared to resting values f (149 ± 12 BPM), VT (1.0 ± 0.1 mL), and VE (153 ± 26). In contrast to CPA, which reduced f (108 ± 10 BrPM), VT (0.8 ± 0.1 mL), and VE (99 ± 19 ml/min) compared to resting values f (143 ± 11 BrPM; p = 4.05 × 10−6), VT (1.1 ± 0.1 mL; P = 2.58 × 10−5), and VE (155 ± 28; P = 5.52 × 10−5). The control resting values before administration of BnOCPA and CPA were not different to one another (P = 1). The effects of CPA were signiﬁcantly greater than BnOCPA for f (P = 4.48 × 10−7), VT (P = 1.15 × 10−4), and VE (P = 1.16 × 10−4). Horizontal signiﬁcance indicators above the data show differences between resting values and following IV administration of either BnOCPA (blue line) or CPA (red line). Vertical signiﬁcance indicators show differences between the effects of BnOCPA and CPA. e Individual data (n = 4–12 rats) for the three doses of BnOCPA (blue circles) compared to their preceding baseline (black squares). Discussion We have previously prepared BnOCPA (and HOCPA)33 for assessment as part of our synthetic campaign to develop potent and A1R-selective ﬂuorescent ligands. The N6- substituent (1R,2 R)-2-aminocyclopentan-1-ol) present in BnOCPA and HOCPA is also found in the experimental and later discontinued103 drug CVT-3619 (later named GS 9667), which 11 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 cribed as a partial, selective agonist of the A1R and duce cAMP content and lipolysis in rat adipocytes104. dentiﬁed BnOCPA as a selective Gob agonist at BnOCPA were observed at A1Rs expressed b species: amphibian, rat, and human. While Bn and induced A1R coupling to Gαi/o subunits r E NATURE COMMUNICATIONS \| https://doi.org/10.1038/s4 has been described as a partial, selective agonist of the A1R and shown to reduce cAMP content and lipolysis in rat adipocytes104. has been described as a partial, selective agonist of the A1R and shown to reduce cAMP content and lipolysis in rat adipocytes104. Having identiﬁed BnOCPA as a selective Gob agonist at recombinant A1Rs in vitro, we established that this unusual property can be translated into the selective activation of native A1Rs in both the in vitro CNS and in vivo cardiorespiratory and peripheral nervous systems. Moreover, these properties of BnOCPA were observed at A1Rs expressed by three different species: amphibian, rat, and human. While BnOCPA bound to and induced A1R coupling to Gαi/o subunits recruited by pro- totypical A1R agonists such as adenosine and CPA, BnOCPA selectively activated Gob among the six Gαi/o subunits. This likely reﬂected BnOCPA’s non-canonical binding proﬁle at the A1R, which had profound implications for the interaction with the BnOCPA were observed at A1Rs expressed by three different species: amphibian, rat, and human. While BnOCPA bound to and induced A1R coupling to Gαi/o subunits recruited by pro- totypical A1R agonists such as adenosine and CPA, BnOCPA selectively activated Gob among the six Gαi/o subunits. This likely reﬂected BnOCPA’s non-canonical binding proﬁle at the A1R, which had profound implications for the interaction with the p y p y Having identiﬁed BnOCPA as a selective Gob agonist at recombinant A1Rs in vitro, we established that this unusual property can be translated into the selective activation of native A1Rs in both the in vitro CNS and in vivo cardiorespiratory and peripheral nervous systems. Moreover, these properties of NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 The mean is shown as an open symbol. One-way ANOVA with Bonferroni corrections found no differences in: HR (p = 0.07), MAP (p = 1), Freq (P = 0.2), VT (p = 1), or VE (p = 0.9). f Average data from the animals in e showing cardiorespiratory variables as a percentage of their preceding baseline and as a function of increasing dose of BnOCPA (log10 scale). g Individual data from four rats showing the effect (difference from the previous baseline) of CPA in the absence (red squares) and presence (purple circles) of BnOCPA (8 µg kg−1). The mean is shown as an open symbol. Two-tailed paired t-tests indicated a signiﬁcant reduction in the effects of CPA by BnOCPA on: HR (CPA: 179 ± 15 bpm vs BnOCPA: 159 ± 10 bpm; p = 0.035), VE (CPA: 59 ± 9 ml/min vs BnOCPA: 21 ± 3 ml/min; p = 0.041) and Freq (CPA: 52 ± 8 breaths/min vs BnOCPA: 17 ± 3 breaths/min; p = 0.009), with no change in: MAP (p = 0.807) or VT (p = 0.609). Data are shown as mean ± SEM. Raw traces from a representative experiment can be found in Supplementary Fig. 11. Source data are provided as a Source Data ﬁle. GαCT in terms of different binding pathways and intermediate states, and in the different intra- and intermolecular hydrogen bond patterns and contacts observed in the simulations of the A1R in complex with either Goa (inactive) or Gob (active). Pre- dictions from the MD simulations suggested four hitherto uncharacterised residues as being important for the interaction between the A1R and Gαi/o. Individual mutations in three of these contacts, R2917.56, I2928.47, Q2938.48, differentially impac- ted agonist efﬁcacy, with adenosine and HOCPA being relatively unaffected compared to the stronger effects on the efﬁcacy of CPA, NECA and BnOCPA. These and other molecular differ- ences in the coupling of the A1R to Gαi/o are likely to underlie the ability of the BnOCPA-bound A1R to selectively trigger Gob activation among the six Gαi/o subtypes. one potential and wide-reaching therapeutic application. Such discoveries are of importance in both understanding GPCR- mediated signalling, and in the generation of both new research tools and therapeutics based on the untapped potential of biased, and indeed Gα-selective, agonists such as BnOCPA. NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 performed using pClamp 10 (Molecular Devices, USA). For voltage-clamp experiments, CA1 pyramidal cells were held at −60 mV. Peptides to interfere with G protein signalling were introduced via the patch pipette into the recorded cell. The cell was held for at least 10 min before adenosine (10 µM) was added to induce an outward current. performed using pClamp 10 (Molecular Devices, USA). For voltage-clamp experiments, CA1 pyramidal cells were held at −60 mV. Peptides to interfere with G protein signalling were introduced via the patch pipette into the recorded cell. The cell was held for at least 10 min before adenosine (10 µM) was added to induce an outward current. Seizure model. Seizure activity was induced in hippocampal slices using nominally Mg2+-free aCSF that contained no added Mg2+ and with the total K+ con- centration increased to 6 mM with KCl. Removal of extracellular Mg2+ facilitates depolarisation via glutamate N-methyl-D-aspartate (NMDA) receptor activation. Increasing the extracellular concentration of K+ depolarises neurons leading to ﬁring and release of glutamate to sustain activity. Both the increase in K+ con- centration and removal of Mg2+ are required to produce spontaneous activity in hippocampal slices110. Spontaneous activity was measured with an aCSF-ﬁlled microelectrode placed within stratum radiatum in area CA1. Frog heart preparation. Young adult male Xenopus leavis frogs were obtained from Portsmouth Xenopus Resource Centre. Frogs were euthanized with MS222 (0.2% at a pH of 7), decapitated and pithed. The animals were dissected to reveal the heart and the pericardium was carefully removed. Heart contractions were measured with a force transducer (AD instruments). Heart rate was acquired via a PowerLab 26 T (AD instruments) controlled by LabChart 7 (AD instruments). The heart was regularly washed with Ringer solution and drugs were applied directly to the heart. Whole-cell patch-clamp recording from hippocampal pyramidal cells. A slice was transferred to the recording chamber and perfused at 3 ml min−1 with aCSF at 32 ± 0.5 °C. Slices were visualised using IR-DIC optics with an Olympus BX151W microscope (Scientiﬁca) and a CCD camera (Hitachi). Whole-cell current- and voltage-clamp recordings were made from pyramidal cells in area CA1 of the hippocampus using patch pipettes (5–10 MΩ) manufactured from thick walled glass (Harvard Apparatus, Edenbridge UK) and containing (mM): potassium gluconate 135, NaCl 7, HEPES 10, EGTA 0.5, phosphocreatine 10, MgATP 2, NaGTP 0.3 and biocytin 1 mg ml−1 (290 mOSM, pH 7.2). Methods Slices were cut along the midline with a Microm HM 650 V microslicer in cold (2–4 °C), high Mg2+, low Ca2+ artiﬁcial cerebrospinal ﬂuid (aCSF), composed of (mM): 127 NaCl, 1.9 KCl, 8 MgCl2, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 D-glucose (pH 7.4 when bubbled with 95% O2 and 5% CO2, 300 mOSM). Slices were stored at 34 °C for 1–6 h in aCSF (1 mM MgCl2, 2 mM CaCl2) before use. Extracellular recording. A slice was transferred to the recording chamber, sub- merged in aCSF and perfused at 4–6 ml min−1 (32 ± 0.5 °C). The slice was placed on a grid allowing perfusion above and below the tissue and all tubing was gastight (to prevent loss of oxygen). An aCSF-ﬁlled glass microelectrode was placed within stratum radiatum in area CA1 and recordings were made using either a differential model 3000 ampliﬁer (AM systems, WA USA) or a DP-301 differential ampliﬁer (Warner Instruments, Hampden, CT USA). Field excitatory postsynaptic potentials (fEPSPs) were evoked with either an isolated pulse stimulator model 2100 (AM Systems, WA) or ISO-Flex (AMPI, Jerusalem, Israel). For fEPSPs a 10–20 min baseline was recorded at a stimulus intensity that gave 40-50% of the maximal response. Signals were acquired at 10 kHz, ﬁltered at 3 kHz, and digitised on line (10 kHz) with a Micro CED (Mark 2) interface controlled by Spike software (Vs 6.1, Cambridge Electronic Design, Cambridge UK) or with WinLTP109. For fEPSP slope, a 1 ms linear region after the ﬁbre volley was measured. Extracellular recordings were made independently on two electrophysiology rigs. As the data obtained from each rig was comparable, both sets of data have been pooled. We have shown that highly selective Gα agonism in vitro can be translated into selective activation of native A1Rs to mediate differential physiological effects, and have identiﬁed a novel molecule capable of doing so. We have also explored molecular mechanisms by which this could occur, and demonstrated pain as 13 e model. Seizure activity was induced in hippocampal slices using nominally free aCSF that contained no added Mg2+ and with the total K+ con- ion increased to 6 mM with KCl. Removal of extracellular Mg2+ facilitates risation via glutamate N-methyl-D-aspartate (NMDA) receptor activation. ing the extracellular concentration of K+ depolarises neurons leading to and release of glutamate to sustain activity. Methods Both the increase in K+ con- ion and removal of Mg2+ are required to produce spontaneous activity in ampal slices110. Spontaneous activity was measured with an aCSF-ﬁlled lectrode placed within stratum radiatum in area CA1. performed using pClamp 10 (Molecular Devices, USA). For volt experiments, CA1 pyramidal cells were held at −60 mV. Peptides G protein signalling were introduced via the patch pipette into t The cell was held for at least 10 min before adenosine (10 µM) wa an outward current. Frog heart preparation. Young adult male Xenopus leavis frogs from Portsmouth Xenopus Resource Centre. Frogs were euthani (0.2% at a pH of 7), decapitated and pithed. The animals were d h h d h d f ll d TICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s4146 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 ARTICLE Fig. 7 BnOCPA is a potent analgesic without causing sedation or motor impairment. a BnOCPA did not induce sedation or affect motor function when injected intraperitoneally (IP; 10 µg kg−1) or intravenously (IV; 10 or 25 µg kg−1). In contrast, morphine caused sedation and motor impairment (15 mg kg−1 subcutaneously, SC). Saline (Veh, SC) did not affect rotarod performance. Data points (mean ± SEM; n = 6 for each compound) are normalised to pre-dose performance and are offset for clarity. b, c BnOCPA alleviates mechanical allodynia in neuropathic pain when administered b via an intrathecal (IT) or c IV route. Pre-surgery (pre-surg) animals had similar sensitivity to tactile stimulation as assessed by von Frey hair stimulation. Spinal nerve ligation caused hypersensitivity to touch (mechanical allodynia) at 1 week after surgery as evidenced by the reduction in the tactile pressure necessary to elicit paw withdrawal (paw withdrawal threshold; PWT). PWT reaches a similar nadir across all groups prior to the vehicle or BnOCPA infusion (pre-dose). Administration of BnOCPA signiﬁcantly increased PWT in the limb ipsilateral to the site of injury in a dose-dependent manner (one-way ANOVA (pre- dose, 1, 2 and 4 hrs) for IT BnOCPA F(3,88) = 21.9, P = 1.10 × 10−10; for IV BnOCPA F(3,92) =18.1, P = 2.70 × 10−9). Fisher LSD post hoc comparisons showed signiﬁcant differences at: IT 1 nmol at 1 and 2 hrs, P = 0.001 and 4.16 × 10−5, respectively, and 3 nmol at 1, 2 and 4 hrs, P = 9.52 × 10−11, 1.42 × 10−11 and 1.41 × 10−8, respectively; IV 3 µg kg−1 at 1, 2 and 4 hrs, P = 0.044, 0.008 and 0.019, respectively, and 10 µg kg−1 at 1, 2 and 4 hrs, P = 1.37 × 10−8, 6.81 × 10−14 and 3.23 × 10−4, respectively. b, c n = 6 per treatment, except for 1 nmol BnOCPA, n = 5. d The analgesic effects of BnOCPA (6 µg kg−1 IV) were prevented by the A1R antagonist DPCPX (1 mg kg−1 IP), but not the A3R-selective antagonist MRS1523 (2 mg kg−1 IP). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Airﬂow measurements were used to calculate: tidal volume (VT; mL; pressure sensors were calibrated with a 3 mL syringe), and respiratory frequency (f; breaths min−1; BrPM). Minute ventilation (VE; mL min−1) was calculated as f × VT. maintained at 36.7 °C via a thermocoupled heating pad (TCAT 2-LV; Physitemp). The trachea was cannulated and the femoral artery catheterised, and both were connected to pressure transducers (Digitimer) to record respiratory airﬂow and arterial blood pressure, respectively. Blood pressure and airﬂow signals were ampliﬁed using the NeuroLog system (Digitimer) connected to a micro1401 interface and acquired on a computer using Spike2 software (v7.08a; Cambridge Electronic Design). Arterial blood pressure recordings were used to derive heart rate (HR: beats.minute−1; BPM), and to calculate mean arterial blood pressure (MAP: Diastolic pressure + 1/3[Systolic Pressure−Diastolic pressure]). Airﬂow measurements were used to calculate: tidal volume (VT; mL; pressure sensors were calibrated with a 3 mL syringe), and respiratory frequency (f; breaths min−1; BrPM). Minute ventilation (VE; mL min−1) was calculated as f × VT. Cardiovascular and respiratory parameters were allowed to stabilise before experiments began. A1R agonists were administered by intravenous (IV) injection and the changes in HR, MAP, f, VT, and VE were measured. In pilot studies, the optimal dose of adenosine was determined by increasing the dose until robust and reliable changes in HR and MAP were produced (1 mg kg−1). The dose of CPA was adjusted until equivalent effects to adenosine were produced on HR and MAP (6 µg kg−1). For BnOCPA we initially used 1 µg kg−1, but saw no agonist effect on HR and MAP. To ensure this was not a false negative we increased the dose of BnOCPA (8 µg kg−1), which still gave no agonist effect on HR and MAP. However, as BnOCPA produced an antagonistic effect when co-administered with adenosine (Fig. 5, Supplementary Fig. 10b) and CPA (Fig. 6, Supplementary Fig. 11), it must have reached A1Rs at a high enough concentration to be physiologically active. These observations conﬁrmed that the lack of agonistic effects on HR and MAP were not due to a type II error. 8 µg kg−1 BnOCPA was used for all further experiments. All injections were administered IV as a 350 µl·kg-1 bolus. Spinal nerve ligation (Chung model90). Adult male Sprague–Dawley rats, 7-8 weeks old, weighing around 250 g at the time of Chung model surgery, were purchased from Charles River UK Ltd. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 The animals were housed in groups of 4 in an air-conditioned room on a 12-hour light/dark cycle. Food and water were available ad libitum. They were allowed to acclimatise to the experimental envir- onment for three days by leaving them on a raised metal mesh for at least 40 min. The baseline paw withdrawal threshold (PWT) was examined using a series of graduated von Frey hairs (see below) for 3 consecutive days before surgery and re- assessed on the 6th to 8th day after surgery and on the 13th to 17th day after surgery before drug dosing. g g Prior to surgery each rat was anaesthetised with 3% isoﬂurane mixed with oxygen (2 L min−1) followed by an intramuscular injection of ketamine (60 mg kg−1) plus xylazine (10 mg kg−1). The back was shaved and sterilised with povidone-iodine. The animal was placed in a prone position and a para-medial incision was made on the skin covering the L4-6 level. The L5 spinal nerve was carefully isolated and tightly ligated with 6/0 silk suture. The wound was then closed in layers after a complete haemostasis. A single-dose of antibiotics (Amoxipen, 15 mg/rat, intraperitoneally, IP) was routinely given for prevention of infection after surgery. The animals were placed in a temperature-controlled recovery chamber until fully awake before being returned to their home cages. The vehicle (normal saline or DMSO) was administered via the IV route at 1 ml·kg−1 and via the intrathecal (IT) route at 10 µl for each injection. The A1R-selective antagonist DPCPX (1 mg kg−1) and the A3R-selective antagonist MRS1523 (2 mg kg−1) were delivered IP 30 mins before vehicle or BnOCPA treatment. The rats with validated neuropathic pain state were randomly divided into 11 groups: vehicle IV, BnOCPA at 1, 3, 6, 10 µg kg−1 IV; vehicle IT, BnOCPA at 0.3, 1, 3 nmol IT; 6 µg kg−1 BnOCPA IV plus 1 mg kg−1 DPCPX IP; 6 µg kg−1 BnOCPA IV plus 2 mg kg−1 MRS1523 IP groups and tested blind to treatment. In the experimental studies, rats either: In the experimental studies, rats either: 1. received an injection of adenosine. After cardiorespiratory parameters returned to baseline (5–10 min), rats were given BnOCPA. After allowing sufﬁcient time for any effect of BnOCPA to be observed, rats received adenosine with BnOCPA co-administered in a single injection. After cardiorespiratory parameters returned to baseline, rats were injected with CPA, or 2. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Post hoc LSD comparisons across all four groups and four-time points (pre-dose, 1, 2 and 4 hrs; F(15,116) = 26.8, P = 0) revealed that BnOCPA at 6 µg kg−1 (IV) elicited signiﬁcant analgesia compared to vehicle-treated animals at 1, 2, and 4 h post-dosing (P = 4.69 × 10−9, 3.50 × 10−16, 4.69 × 10−9, respectively), which persisted in the presence of the selective A3R antagonist MRS1523 over the same time period (P = 4.42 × 10−13, 3.38 × 10−14, 1.81 × 10−10, respectively). In contrast, the PWT in DPCPX-treated animals did not differ from those in the vehicle group (P = 0.872, 0.748, 0.453 at 1, 2, and 4 h, respectively). n = 11 for BnOCPA and vehicle groups; n = 6 for the DPCPX group and n = 5 for the MRS1523 group. Averaged data are presented as mean ± SEM. ns, not signiﬁcant; , P < 0.05; , P < 0.02; , P < 0.001; *, P < 0.0001. Source data are provided as a Source Data ﬁle. After allowing the animal to stabilise following surgery, BnOCPA (8 µg kg−1) was administered. After a 20 min recovery period CPA (6 µg kg−1) was administered. All injections were administered IV as a 350 µl kg-1 bolus. Changes in f, VT, and VE were measured. If the dosing occurred close to a respiratory event such as a sigh a second IV dose was administered, with 20 min recovery periods either side of the injection. Measurements for the effect of BnOCPA were time- matched to when CPA induced a change in respiration in the same preparation. As no difference was observed between the respiratory responses to BnOCPA in these rats (n = 4) and those instrumented for both cardiovascular and respiratory recordings (n = 4), the data were pooled (n = 8; Fig. 6a–d). maintained at 36.7 °C via a thermocoupled heating pad (TCAT 2-LV; Physitemp). The trachea was cannulated and the femoral artery catheterised, and both were connected to pressure transducers (Digitimer) to record respiratory airﬂow and arterial blood pressure, respectively. Blood pressure and airﬂow signals were ampliﬁed using the NeuroLog system (Digitimer) connected to a micro1401 interface and acquired on a computer using Spike2 software (v7.08a; Cambridge Electronic Design). Arterial blood pressure recordings were used to derive heart rate (HR: beats.minute−1; BPM), and to calculate mean arterial blood pressure (MAP: Diastolic pressure + 1/3[Systolic Pressure−Diastolic pressure]). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Voltage and current recordings were obtained using an Axon Multiclamp 700B ampliﬁer (Molecular Devices, USA) and digitised at 20 KHz. Data acquisition and analysis was In vivo anaesthetised rat preparation for cardiorespiratory recordings. Anaesthesia was induced in adult male Sprague–Dawley rats (230-330 g) with isoﬂuorane (2–4%; Piramal Healthcare). The femoral vein was catheterised for drug delivery. Anaesthesia was maintained with urethane (1.2–1.7 g kg−1; Sigma) in sterile saline delivered via the femoral vein catheter. Body temperature was 14 NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunicati ARTICLE Brieﬂy, in sterile tubes containing 150 mM NaCl, DNA or PEI was added (ﬁnal volume 50 μl) and allowed to incubate at room temperature for 5 min before mixing together and incubating for a further 10 min prior to adding the combined mix dropwise to the cells. 24 h post-transfection, HEK 293 T cell were harvested, resuspended in reduced serum media (MEM, NEAA; Thermo Fisher Scientiﬁc) supplemented with 1% L-glutamine (2 mM ﬁnal; Thermo Fisher Scientiﬁc), 2% FBS and 1x antibiotic- antimycotic solution and seeded (50,000 cells/well) in a poly-L-lysine-coated (MW: 150,000-300,000 Da; Sigma-Aldrich) white 96-well plate (PerkinElmer Life Sci- ences). 24 h post seeding, media was removed, cells gently washed in PBS and 90 μl of furimazine (4 μM)-containing solution added (PBS supplemented with 0.49 mM MgCl2, 0.9 mM CaCl2 and 0.1% BSA) to each well before incubating in the dark for 10 min. After incubation, 10 μl of agonist (NECA, CPA, adenosine, BnOCPA) was added (0.01 μM to 10 μM) and ﬁltered light emissions measured at 450 nm and 530 nm every minute for 1 h using a Mithras LB 940 (Berthold technology) using MikroWin 2000 software. Here, Nluc on the C-terminus of A1R or A3R acted as the BRET donor (luciferase oxidising its substrate) and YFP acted as the ﬂuorescent acceptor. Vehicle control (1% DMSO) was added to determine background emission, and data was corrected for baseline reading, vehicle and the response obtained in the absence of YFP-β-arrestin1/2, when appropriate. Rotarod test for motor function. A rotarod test was used to assess motor coor- dination following intravenous and intraperitoneal administration of BnOCPA. An accelerating rotarod (Ugo Basile) was set so speed increased from 6 to 80 rpm over 170 seconds. Male Sprague–Dawley rats (n = 24), 7 weeks of age (212-258 g) were trained on the rotarod twice daily for two days (≥2 trials per session) until per- formance times were stable. On the day of the experiment, three baseline trials were recorded. The compound was administered IP (10 µg kg−1, n = 6) or IV via tail vein injection (10 and 25 µg kg−1, n = 6 per group). The control group received subcutaneous saline and the positive control group received subcutaneous mor- phine (15 mg kg−1). Latency to fall (seconds) was measured in triplicate at 1, 2, 3 and 5 h post drug administration. Constructs, transfections and generation of stable cell lines. ARTICLE Cell lines were maintained using standard subculturing routines as guided by the European Col- lection of Cell Culture (ECACC) and checked annually for mycoplasma infection using an EZ-PCR mycoplasma test kit from Biological Industries (Kibbutz Beit- Haemek, Israel). To investigate the signalling properties of the rat A3R (rA3R) and mutants of the human A1R (hA1R), stable cell lines were generated using Flp-In- CHO cells (ThermoFisher - R75807). Untagged hA1R from sigNanoLuciferase (Nluc)-A1R in pcDNA3.1+ and untagged rA3R from sigNluc-A3R in pcDNA3.1 + (both gifted by Dr Steve Briddon (University of Nottingham)) were cloned into the pcDNA5/FRT expression vector (Thermo Fisher Scientiﬁc). Mutations within the hA1R were made using the QuikChange Lightening Site- Directed Mutagenesis Kit (Agilent Technologies) in accordance with the manu- facturer’s instructions. Constructs for generating Goa/b interfering and scrambled peptides were generated by PCR and cloned into the BamHI/HindIII site of pcDNA3.1- as described in Gilchrist et al.60,. Prior to the initiator codon a Kozak sequence was included for enhanced translation. The peptide sequences used were: for Goa MGIANNLRGCGLY, for Gob MGIAKNLRGCGLY, and for the scrambled peptide MGLNRGNAYLCIGMG was used. Constructs were sequenced to conﬁrm ﬁdelity. Flp-In-CHO cells were generated through co-transfection of the cell line with pcDNA5/FRT expression vector (Thermo Fisher Scientiﬁc) containing the WT or mutant hA1R, or rA3R, and the Flp recombinase expressing plasmid, pOG44 (Thermo Fisher Scientiﬁc), in accordance with the manufacturer’s instructions. Co-transfection of cells in a T25 ﬂask, with a total of 5 μg of adenosine receptor (AR)/pcDNA5/FRT and pOG44 (AR:pOG44 ratio of 1:9), was performed using Fugene HD (Promega), at a ratio of 3:1 (v/w) (Fugene:DNA). 24 h after transfection, cells were harvested and resuspended in growth media containing 600 μg/ml Hygromycin B (Thermo Fisher Scientiﬁc), and subsequently seeded into a fresh T25 ﬂask. Media was replaced every 2–3 days and cells stably expressing the receptor of interest were selected using 600 μg/ml Hygromycin B. To generate CHO-K1 (European Collection of Authenticated Cell Cultures (ECAC) catalogue number 85051005) cells stably expressing the rat A2AR (CHO-K1-rA2AR), CHO- K1 cells were seeded onto a 6-well plate and transfected with 1 μg rA2AR using Fugene HD (Promega) at a ratio of 3:1 (v/w) (Fugene:DNA). 48 h after transfec- tion, media was replaced with growth media containing 800 μg/ml G418 (Thermo Fisher Scientiﬁc) and changed every 2–3 days until cells were >80% conﬂuent. ARTICLE To investigate rat A1R-mediated signalling, CHO-K1 cells seeded onto a 6-well plate were transiently transfected with 1 μg rat A1R (rA1R) using Fugene HD (Promega) at a ratio of 3:1 (v/w) (Fugene:DNA), for 48 h. The plasmids encoding the rA1R and rA2AR (Nluc-A1R/pcDNA3.1(+) and Nluc-A2AR/pcDNA3.1(+), respectively) were kindly gifted by Stephen Hill and Stephen Briddon (University of Nottingham). Radioligand binding. Radioligand displacement assays were conducted using crude membrane preparations (100 μg protein per tube) acquired from homogenisation of CHO-K1-hA1R cells in ice-cold buffer (2 mM MgCl2, 20 mM HEPES, pH 7.4). The ability to displace binding of the A1R-selective antagonist radioligand, 1,3-[3H]- dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) at a concentration (1 nM) around the Kd value (1.23 nM, as determined by saturation binding experiments) by increasing concentrations of NECA, adenosine, CPA, BnOCPA or HOCPA (0.1 nM −10 μM) allowed the binding afﬁnities (Ki) to be determined. Non-speciﬁc binding was determined in the presence of 10 μM DPCPX. Membrane incubations were con- ducted in Sterilin™scintillation vials (Thermo Fisher Scientiﬁc; Wilmington, Mas- sachusetts, USA) for 60 min at room temperature. Free radioligand was separated from bound radioligand by ﬁltration through Whatman® glass microﬁber GF/B 25 mm ﬁlters (Sigma-Aldrich). Each ﬁlter was then placed in a Sterilin™scintillation vial and radioactivity determined by: addition of 4 mL of Ultima Gold XR liquid scintillant (PerkinElmer), overnight incubation at room temperature and the retained radioactivity determined using a Beckman Coulter LS 6500 Multi-purpose scintilla- tion counter (Beckman Coulter Inc.; Indiana, USA). NanoBRET ligand-binding studies. Real-time pharmacological interactions between ligands and receptors was quantitated using NanoBRET as described previously112. In brief, using N-terminally Nluc-tagged rA1R-, rA2AR- or rA3R-expressing HEK 293 cell lines, competition binding assays were conducted. In all antagonist assays CA200645, which acts as a ﬂuorescent antagonist with a slow off- rate113, was used, with the exception of the rat A3R where the ﬂuorescent compound was AV039114. The data was ﬁtted with the ‘one-site – Ki model’ derived from the Cheng and Prusoff equation, built into Prism to determine afﬁnity (pKi) values for all unlabelled agonists at all AR subtypes assayed. For the hA1R we also performed an agonist binding competition assay using NECA-TAMRA (Noel et al., unpublished). Here data was ﬁtted with the ‘two-site Ki model’, build into Prism to determine high afﬁnity and low afﬁnity values for the unlabelled agonists. ARTICLE For all ARs, ﬁltered light emission at 450 nm and >610 nm (640-685 nm band pass ﬁlter) was measured using a Mithras LB 940 and the raw BRET ratio calculated by dividing the 610 nm emission with the 450 nm emission. The Nluc acts as the BRET donor (luciferase oxidising its substrate) and CA200645/AV039/NECA-TAMRA acted as the ﬂuorescent acceptor. CA200645 was used at 25 nM, as previously reported115, AV039 was used at 100 nM112 and NECA-TAMRA at its Kd of 15.2 μM (Noel et al., unpublished). BRET was measured following the addition of the Nluc substrate, Furimazine (0.1 μM). Non-speciﬁc binding was determined using a high concentration of unlabelled antagonist, DPCPX for rA1R, ZM241385 for the rA2AR and MRS1220 for rA3R. Cell signalling assays. CHO cell lines expressing ARs of interest (including mutants of the hA1R) were routinely cultured in Ham’s F-12 nutrient mix sup- plemented with 10% foetal bovine serum (FBS), at 37 °C with 5% CO2, in a humidiﬁed atmosphere. For cAMP accumulation experiments, cells were seeded at a density of 2000 cells per well of a white 384-well optiplate and stimulated, for 30 min, with a range of agonist concentrations (100 pM – 100 μM) in the presence of 25 μM rolipram (Cayman Chemicals). For cAMP inhibition experiments, cells were co-stimulated with 1 μM forskolin and a range of agonist concentrations (1 pM–100 μM), in the presence or absence of 1 μM antagonist. cAMP levels were then determined using a LANCE® cAMP kit as described previously33,111. For determination of individual Gαi/o/z couplings, CHO-K1-hA1R cells (made in-house) were transfected with pcDNA3.1-GNAZ or, pcDNA3.1 containing pertussis toxin (PTX) insensitive Gαi/o protein mutants (C351I, C352I, C351I, C351I, C351I, for Gi1, Gi2, Gi3, Goa, Gob, respectively, obtained from cDNA Resource Centre; www.cdna.org), using 500 ng plasmid and Fugene HD at a 3:1 (v/w) (Fugene:Plasmid) ratio. Cells were then incubated for 24 h before addition of 100 ng/ml PTX, to inhibit activity of endogenous Gαi/o, and then incubated for a further 16-18 h. Transfected cells were then assayed as per cAMP inhibition experiments, but co-stimulated with agonist and 100 nM forskolin. TRUPATH G protein dissociation assay. Cells were plated in a density of 1,500,000 cells/well in a 6-well plate and grown in DMEM /F-12 GlutaMAX™ media (Thermo Fisher Scientiﬁc, UK) supplemented with 10% FBS (Sigma, UK) and 1% AA (Sigma, UK). ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 transiently co-transfected with either A1R-Nluc or A3R-Nluc, β-arrestin1/2-YFP and hGRK1-6, or pcDNA3.1 vector (total 2 μg, at a AR:β-arrestin:hGRK ratio of 1:5:4) using polyethyleneimine (PEI, 1 mg/ml, MW = 25,000 g/mol; Polysciences Inc) at a DNA:PEI ratio of 1:6 (w/v). As a negative control for the A1R, trans- fections were also set up in the absence of β-arrestin1/2-YFP. Brieﬂy, in sterile tubes containing 150 mM NaCl, DNA or PEI was added (ﬁnal volume 50 μl) and allowed to incubate at room temperature for 5 min before mixing together and incubating for a further 10 min prior to adding the combined mix dropwise to the cells. 24 h post-transfection, HEK 293 T cell were harvested, resuspended in reduced serum media (MEM, NEAA; Thermo Fisher Scientiﬁc) supplemented with 1% L-glutamine (2 mM ﬁnal; Thermo Fisher Scientiﬁc), 2% FBS and 1x antibiotic- antimycotic solution and seeded (50,000 cells/well) in a poly-L-lysine-coated (MW: 150,000-300,000 Da; Sigma-Aldrich) white 96-well plate (PerkinElmer Life Sci- ences). 24 h post seeding, media was removed, cells gently washed in PBS and 90 μl of furimazine (4 μM)-containing solution added (PBS supplemented with 0.49 mM MgCl2, 0.9 mM CaCl2 and 0.1% BSA) to each well before incubating in the dark for 10 min. After incubation, 10 μl of agonist (NECA, CPA, adenosine, BnOCPA) was added (0.01 μM to 10 μM) and ﬁltered light emissions measured at 450 nm and 530 nm every minute for 1 h using a Mithras LB 940 (Berthold technology) using MikroWin 2000 software. Here, Nluc on the C-terminus of A1R or A3R acted as the BRET donor (luciferase oxidising its substrate) and YFP acted as the ﬂuorescent acceptor. Vehicle control (1% DMSO) was added to determine background emission, and data was corrected for baseline reading, vehicle and the response obtained in the absence of YFP-β-arrestin1/2, when appropriate. transiently co-transfected with either A1R-Nluc or A3R-Nluc, β-arrestin1/2-YFP and hGRK1-6, or pcDNA3.1 vector (total 2 μg, at a AR:β-arrestin:hGRK ratio of 1:5:4) using polyethyleneimine (PEI, 1 mg/ml, MW = 25,000 g/mol; Polysciences Inc) at a DNA:PEI ratio of 1:6 (w/v). As a negative control for the A1R, trans- fections were also set up in the absence of β-arrestin1/2-YFP. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 received an injection of CPA. After cardiorespiratory parameters returned to baseline (5-10 min) rats were given 8 µg kg−1 BnOCPA. After allowing sufﬁcient time for any effect of BnOCPA to be observed, rats received CPA with 8 µg kg−1 BnOCPA co-administered in a single injection. After cardiorespiratory parameters returned to baseline, rats were injected with successive injections of 17 µg kg−1 and 25 µg kg−1 BnOCPA, with sufﬁcient time given for any effect of BnOCPA to be observed. To test for mechanical allodynia the animals were placed in individual Perspex boxes on a raised metal mesh for at least 40 min before the test. Starting from the ﬁlament of lower force, each ﬁlament was applied perpendicularly to the centre of the ventral surface of the paw until slightly bent for 6 seconds. If the animal withdrew or lifted the paw upon stimulation, then a hair with force immediately lower than that tested was used. If no response was observed, then a hair with force immediately higher was tested. The highest value was set at 15 g. The lowest amount of force required to induce reliable responses (positive in 3 out of 5 trials) was recorded as the value of PWT. On the testing day, PWT were assessed before and 1, 2 and 4 h following BnOCPA or vehicle administration. The animals were returned to their home cages to rest (about 30 min) between two neighbouring testing time points. At the end of each experiment, the animals were deeply anaesthetised with isoﬂurane and killed by decapitation. To check that the volume of solution injected with each drug did not itself induce a baroreﬂex response leading to spurious changes in cardiorespiratory responses, equivalent volumes of saline (0.9%) were injected. These had no effect on either heart rate or MAP (Supplementary Fig. 10c). To conﬁrm that repeated doses of adenosine produced the same response and that the responses did not run- down, rats were given two injections of adenosine (1 mg kg−1). There was no signiﬁcant difference in the changes in cardiovascular parameters produced by each adenosine injection (Supplementary Fig. 10d). An additional series of experiments (n = 4) were undertaken to directly compare BnOCPA and CPA on respiration. Adult male Sprague–Dawley rats (400- 500 g) were anaesthetised with urethane and instrumented as described above, with the exception that the arterial cannulation was not performed. 15 ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Frey hair) when they arrived, and those that showed this, i.e., a paw withdrawal threshold (PWT) less than 8 g, were eliminated from the study. Those rats suc- cessfully developing mechanical allodynia (with PWT lower than 4 g) were ran- domly assigned into different groups according to their PWT values of pre-surgery, 1 week post-surgery and pre-dosing to ensure a balanced distribution across groups. For the rotarod studies, rats were randomly assigned to treatment groups by animal care technicians not involved in the experimental studies. Experiments occurred over a period of time with various types of manipulations interleaved to avoid batch effects. Key observations were replicated with different batches of animals and reagents and by different experimenters. Results across different independent labs working in different experimental systems were consistent with the main themes and conclusions of the study. media was discarded and replaced with 80 μl assay buffer (1× Hank’s balanced salt solution (HBSS) with calcium, supplemented with 20 mM HEPES and 0.1% BSA at pH 7.4). The assay was started by adding 10 μl of coelenterazine 400a (Nanolight technology, USA) to a ﬁnal concentration of 5 μM. The plates were then incubated in the dark for 5 min, prior to the addition of 10 μl compounds (in a range of 0.01 nM – 1 µM). In order to investigate the effect of interfering peptides on Goa and Gob activation, cells were transfected with the TRUPATH constructs for Goa and Gob with the A1R as described above. However, the vector was replaced by either interfering or scrambled peptides, as appropriate, with increasing con- centration: 0, 100, and 400 ng and was complemented by pcDNA3.1(-) up to 400 ng. CPA 10 µl was used as the ligand in a range of 1 nM–1 µM. BRET signal was recorded for 30 min on a Mithras LB940 plate reader allowing sequential integration of signal detected from GFP2 and Rluc8. The BRET ratio corresponds to the ratio of light emission from GFP2 (515 nm) over Rluc8 (400 nm). Net BRET ratio was used to generate the concentration-response curve by taking 11-minute time-point after baseline correction. Data was analysed as change in the presence of the interfering peptides relative to control alone at 1 µM CPA. Drugs and substances. Drugs were made up as stock solutions (1-10 mM) and then diluted in aCSF or saline on the day of use. Statistics and reproducibility The area of precursor compound remaining after each of the time points relative to the amount remaining at time 0, expressed as a percentage, was calculated. Subsequently, the t1/2 is estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming ﬁrst-order kinetics. Half-life determination in human plasma. At the end of incubation at each of the time points (0, 0.5, 1, 1.5, 2 h), acetonitrile was added to the incubation mixture followed by centrifugation. Samples were analysed by HPLC-MS/MS and peak areas were recorded for each analyte. The area of precursor compound remaining after each of the time points relative to the amount remaining at time 0, expressed as a percentage, was calculated. Subsequently, the t1/2 is estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming ﬁrst-order kinetics. All in vivo cardiovascular and respiratory data were analysed using OriginPro 2018. One-way ANOVAs, with repeated measures as appropriate, and with Bonferroni correction for multiple comparisons were used. Statistical signiﬁcance for the effects of IV saline and the antagonist effect of BnOCPA on CPA were tested using two-tailed paired t-tests. Data are reported throughout as mean ± SEM and n values are reported for each experiment. For the neuropathic pain studies, one-way ANOVAs with Fisher’s Least Signiﬁcant Difference (LSD) post hoc test was used to compare drug treatment groups to the vehicle group (OriginPro 2018). The signiﬁcance level was set at P < 0.05, with actual P values reported in the ﬁgure legends and summaries, by way of abbreviations and asterisks, on the graphs: ns, not signiﬁcant; * P < 0.05; , P < 0.02; , P < 0.001; **, P < 0.0001. Intrinsic clearance by human liver microsomes. Metabolic stability, expressed as a percentage of the parent compound remaining, was calculated by comparing the peak area of the compound at the time-point (0, 15, 30, 45, 60 min) relative to that at time 0. The t1/2 was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the ﬁrst-order kinetics. The apparent intrinsic clearance (CLint, in µL/min/mg) was calculated according to the following Eq. (3): CLint ¼ 0:693 t1=2 ´ ð0:0001mg protein=μLÞ ð3Þ Reproducibility. Statistics and reproducibility At the end of the incubation at each of the time points (0, 1, 2, 3, 4 h), an equal volume of an organic mixture (acetonitrile/ methanol, 50/50 v/v) was added to the incubation mixture. Samples were analysed by HPLC-MS/MS and corresponding peak areas were recorded for each analyte. The ratio of precursor compound remaining after each time-point relative to the amount present at time 0, expressed as a percentage, is reported as chemical stability. The t1/2 was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming ﬁrst-order kinetics. Half-life determination in PBS. At the end of the incubation at each of the time points (0, 1, 2, 3, 4 h), an equal volume of an organic mixture (acetonitrile/ methanol, 50/50 v/v) was added to the incubation mixture. Samples were analysed by HPLC-MS/MS and corresponding peak areas were recorded for each analyte. The ratio of precursor compound remaining after each time-point relative to the amount present at time 0, expressed as a percentage, is reported as chemical stability. The t1/2 was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming ﬁrst-order kinetics. ð2Þ where D’ and D = EC50 values of adenosine with and without BnOCPA present, respectively, [A] = the concentration of BnOCPA, and K2 = the afﬁnity constant (KA) of the BnOCPA58. Statistical signiﬁcance relative to adenosine was calculated using a one-way ANOVA with a Dunnett’s post-test for multiple comparisons. Radioligand displacement curves were ﬁtted to the one-site competition binding equation yielding log(Ki) values. One-way ANOVA (Dunnett’s post-test) was used to determine signiﬁcance by comparing the log(Ki) value for each compound when compared to adenosine. To determine the extent of ligand-induced recruitment of β-arrestin2-YFP to either the A1R or A3R, the BRET signal was calculated by subtracting the 530 nm/450 nm emission for vehicle-treated cells from ligand- treated cells (ligand-induced ΔBRET). ΔBRET for each concentration at 5 min (maximum response) was used to produce concentration-response curves. Half-life determination in human plasma. At the end of incubation at each of the time points (0, 0.5, 1, 1.5, 2 h), acetonitrile was added to the incubation mixture followed by centrifugation. Samples were analysed by HPLC-MS/MS and peak areas were recorded for each analyte. Statistics and reproducibility Samples sizes for in vitro pharmacological or in vivo cardior- espiratory experiments were based on prior experience and were routinely in excess of 4 individual biological replicates. In many instances the pool of replicates was added to over time via interleaved experiments with other compounds and often by other investigators, ensuring the consistency and reproducibility of experimental observations. Data was only excluded when technical failures occurred. Rando- misation and blinding was not performed for in vitro or cardiorespiratory studies. In the pain and rotarod studies, the sample sizes for which (typically 6 animals) were based on prior experience, the experimenters were blinded to the reagents used. For the pain studies, all animals were pre-screened for hypersensitivity (von ð3Þ The behaviour of BnOCPA was compared to appropriate standards. Data is available in Supplemental Data File 1 ARTICLE BnOCPA34 ((2 R,3 R,4 S,5 R)−2-(6- {[(1 R,2 R)−2-benzyloxycyclopentyl]amino}−9H-purin-9-yl)−5-(hydroxymethyl)oxo- lane-3,4-diol) and HOCPA47 ((2 R,3 R,4 S,5 R)−2-(6-{[(1 R,2 R)−2-hydro- xycyclopentyl]amino}−9H-purin-9-yl)−5-(hydroxymethyl)oxolane-3,4-diol), the [(1 R,2 R)−2-hydroxycyclopentyl]amino bis-epimer of known A1R agonist GR7923649, were synthesised as described previously33 and dissolved in dimethyl-sulphoxide (DMSO, 0.01% ﬁnal concentration). Adenosine, 8-CPT (8-cyclopentyltheophylline), NECA (5′-(N-Ethylcarboxamido) adenosine), DPCPX, ZM241385, MRS1220 and CPA (N6-Cyclopentyladenosine) were purchased from Sigma-Aldrich (Poole, Dorset, UK). MRS1523 was purchased from Cayman Chemicals (Cambridge Bioscience Ltd., Cambridge UK). [3H]-DPCPX was purchased from PerkinElmer (Life and Analytical Sciences, Waltham, MA). CA200645 and peptides for interfering with G protein sig- nalling were obtained from Hello Bio (Bristol, UK) and were based on published sequences60. NECA-TAMARA was synthesised in-house (Noel et al., in preparation), while AV039, a highly potent and selective ﬂuorescent antagonist of the human A3R based on the 1,2,4-Triazolo[4,3-a]quinoxalin-1-one linked to BY630 was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). For Goa the peptide had a sequence of MGIANNLRGCGLY. The scrambled version was LNRGNAYLCIGMG. For Gob the peptide had a sequence of MGIAKNLRGCGLY. Peptides were made up as stock solutions (2 mM) and stored at −20 °C. The stock solutions were dissolved in ﬁltered intracellular solution just before use. Statistics and reproducibility Data and statistical analysis. Concentration-response curves for the effects of A1R agonists on synaptic transmission were constructed in OriginPro 2018 (OriginLab; Northampton, MA, USA) and ﬁtted with a logistic curve using the Levenberg Marquadt iteration algorithm. OriginPro 2018 was also used for statistical analysis. Statistical signiﬁcance was tested as indicated in the text using paired or unpaired two-tailed t-tests or one-way or two-way ANOVAs with repeated measures (RM) as appropriate. Bonferroni corrections for multiple comparisons were performed. All in vitro cell signalling assay data was analysed using Prism 8.4 (Graphpad software, San Diego, CA), with all concentration-response curves being ﬁtted using a 3 parameter logistic equation to calculate response range and IC50. All cAMP data was normalised to a forskolin concentration-response curve ran in parallel to each assay. Where appropriate the operational model of receptor agonism52,111 was used to obtain efﬁcacy (log τ) and equilibrium disassociation constant (log KA) values. Calculation of bias factors (Δlog(Tau/KA)) relative to adenosine was performed as described in Weston et al. (2016)111. Error for this composite measure was pro- pagated by applying the following Eq. (1). Pooled SEM ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðSEMAÞ2 þ ðSEMBÞ2 q ð1Þ ð1Þ BnOCPA Pharmacokinetics. The stability in solution and metabolism of BnOCPA (0.1 µM or 1 µM) was assessed by Euroﬁns Panlabs (Supplementary Data 1). The parameters examined were: half-life (t1/2) in PBS (1 µM BnOCPA, 37 °C, pH 7.4; Assay #600); t1/2 in human plasma (1 µM BnOCPA, 37 °C; Assay #887) and intrinsic clearance by human liver microsomes (0.1 µM BnOCPA, 0.1 mg/ml, 37 °C; Assay #607). Where, σA and σB are the standard deviations of measurement A and B with mean of xA and xB is the composite mean and n is the number of repeats. Single-dose Schild analysis was performed on data using BnOCPA as an antagonist to adenosine in the cAMP assays so enabling determination of BnOCPA’s afﬁnity constant (KA) using the following Eq. (2) Where, σA and σB are the standard deviations of measurement A and B with mean of xA and xB is the composite mean and n is the number of repeats. Single dose Schild analysis was performed on data using BnOCPA as an antagonist to adenosine in the cAMP assays so enabling determination of BnOCPA’s afﬁnity constant (KA) using the following Eq. (2) D0 D ¼ 1 þ A ½ K2 ð2Þ Half-life determination in PBS. NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications ARTICLE After being grown overnight, cells in each well were transfected using polyethylenimine 25 kDa (PEI, Polysciences Inc., Germany) at a 6:1 ratio of PEI to DNA, diluted in 150 mM NaCl. Cells were transfected with hA1R, Goa-RLuc8 or Gob-RLuc8, Gβ3, Gγ8-GFP2, and pcDNA3.1 with the ratio of 1:1:1:1:1 (400 ng per construct) in accordance with previously published methods55. Gɑ (either Goa-RLuc8, or Gob-RLuc8), Gβ3 and Gγ8-GFP2 constructs were purchased as part of the TRUPATH sensor kit from Addgene, pcDNA3.1- A1R was obtained from cDNA resource centre, and pcDNA3.1 (-) zeo was pur- chased from Invitrogen. After 24 h, cells were trypsinised and re-seeded onto poly- L-lysine (PLL)-coated white 96-well plates (Greiner, UK) at the density of 50,000 cells/well in a complete DMEM/F-12 medium. After grown overnight, the culture β-arrestin recruitment assays. HEK 293 T cells (ATCC CRL-3216) were routi- nely grown in DMEM/F-12 GlutaMAXTM (Thermo Fisher Scientiﬁc) supple- mented with 10% foetal bovine serum (FBS; F9665, Sigma-Aldrich) and 1x antibiotic-antimycotic solution (DMEM complete; Thermo Fisher Scientiﬁc). For analysis of β-arrestin recruitment following ligand stimulation at the hA1R or hA3R, HEK 293 T cells in a single well of 6-well plate (conﬂuency ≥80%) were 16 NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 a distance between the adenine and the receptor residues centres of mass <3 Å), a classic (unsupervised and without energetic bias) MD simulation was performed for at least a further 100 ns. orders of magnitudes faster than the corresponding classical (unsupervised) MD simulations139. SuMD has been successfully applied to small molecules and peptides140–146. In the present work, the distances between the centres of mass of the adenine scaffold of the A1R agonist and N2546.55, F171ECL2, T2777.42 and H2787.43 of the receptor were considered for the supervision during the MD simulations. The dynamic docking of BnOCPA was hindered by the ionic bridge formed between the E172ECL2 and K265ECL3 side chains. A metadynamics147–149 energetic bias was there- fore introduced in order to facilitate the rupture of this ionic interaction, thus favoring the formation of a bound complex. More precisely, Gaussian terms (height = 0.01 kcal mol−1 and widths = 0.1 Å) were deposited every 1 ps along the distance between the E172ECL2 carboxyl carbon and the positively charged K265ECL3 nitrogen atom using PLUMED 2.3150. A similar SuMD-metadynamics hybrid approach was previously employed to study binding/unbinding kinetics151 on the A2AR subtype. For each replica (Table 1), when the ligands reached a bound pose (i.e. a distance between the adenine and the receptor residues centres of mass <3 Å), a classic (unsupervised and without energetic bias) MD simulation was performed for at least a further 100 ns. Paramchem webserver118. Higher penalties were associated with a few BnOCPA dihedral terms, which were therefore optimised at the HF/6-31 G level of theory using both the high throughput molecular dynamics (HTMD)121 parameterise functionality and the visual molecular dynamics (VMD)122 Force Field Toolkit (ffTK)123, after fragmentation of the molecule. Short MD simulations of BnOCPA in water were performed to visually inspect the behaviour of the optimised rotatable bonds. Systems preparation for fully dynamic docking of BnOCPA and HOCPA. Coordi- nates of the A1R in the active, adenosine- and G protein-bound state were retrieved from the Protein Data Bank124,125 database (PDB ID 6D9H77). Intracellular loop 3 (ICL3) which is missing from PDB ID 6D9H was rebuilt using Modeller 9.19126,127. The G protein, with the exception of the C-terminal helix (helix 5) of the G protein alpha subunit (the key region responsible for the receptor TM6 active-like con- formation) was removed from the system as in previous work128,129. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 BnOCPA and HOCPA were placed in the extracellular bulk, in two different systems, at least 20 Å from the receptor vestibule. The resulting systems were prepared for simulations using in-house scripts able to exploit both python HTMD121 and Tool Command Language (TCL) scripts. Brieﬂy, this multistep procedure performs the preliminary hydrogen atoms addition by means of the pdb2pqr130 and propka3131 software, considering a simulated pH of 7.0 (the proposed protonation of titratable side chains was checked by visual inspection). Receptors were then embedded in a square 80 Å × 80 Å 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphocholine (POPC) bilayer (previously built by using the VMD Membrane Builder plugin 1.1, Mem- brane Plugin, Version 1.1.; http://www.ks.uiuc.edu/Research/vmd/plugins/ membrane/) through an insertion method132, considering the A1R coordinates retrieved from the OPM database133 to gain the correct orientation within the membrane. Lipids overlapping the receptor transmembrane bundle were removed and TIP3P water molecules134 were added to the simulation box (ﬁnal dimensions 80 Å × 80 Å × 125 Å) using the VMD Solvate plugin 1.5 (Solvate Plugin, Version 1.5; http://www.ks.uiuc.edu/Research/vmd/plugins/solvate/). Finally, overall charge neutrality was achieved by adding Na+/Cl−counter ions (concentration of 0.150 M) using the VMD Autoionize plugin 1.3 (Autoionize Plugin, Version 1.3; http://www.ks.uiuc.edu/Research/vmd/plugins/autoionize/). All histidine side chains were considered in the delta tautomeric state, with the exception of H251 (epsilon tautomer) and H278 (protonated). 135 BnOCPA bound state metadynamics. We decided to perform a detailed analysis of the role played by the E172ECL2–K265ECL3 ionic interaction in the dynamic docking of BnOCPA. Three 250 ns long well-tempered152 metadynamics simulations were performed using the bound state obtained from a previous dynamic docking simu- lation, which resulted in binding mode A, as a starting point. The collective variables (CVs) considered were: i) the distance between the E172ECL2 carboxyl carbon and the positively charged K265ECL3 nitrogen atom and ii) the dihedral angle formed by the 4 atoms linking the cyclopentyl ring to the phenyl moiety (which was the most ﬂexible ligand torsion during the previous SuMD simulations). Gaussian widths were set at 0.1 Å and 0.01 radians respectively, heights at 0.01 kcal/mol−1, and the deposition was performed every 1 ps (bias-factor = 5). Although complete convergence was probably not reached, three replicas (Table 1) allowed sampling of three main energetic minima on the energy surface (Supplementary Fig. 8); these correspond to the representative binding poses shown in Fig. 3d to f. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Table 1 Summary of the simulations performed. Ligand MD approach # Replicas Total simulated time♦ BnOCPA SuMD 6 1.9 μs BnOCPA SuMD-Metadynamics 5 4.3 μs HOCPA SuMD 5 3.4 μs BnOCPA (bound state after dynamic docking) Metadynamics 3 0.75 μs BnOCPA(A) Classic MD 6 9.0 μs BnOCPA(B) Classic MD 6 9.0 μs BnOCPA(C) Classic MD 3 3.0 μs BnOCPA(D) Classic MD 6 9.0 μs HOCPA Classic MD 4 8.0 μs PSB36 Classic MD 4 6.0 μs Apo A1 Classic MD 4 8.0 μs GαCT Goa (BnOCPA) SuMD + Classic MD 10 0.36 μs + 3.0 μs GαCT Gob (BnOCPA) SuMD + Classic MD 10 0.33 μs + 3.0 μs GαCT Gi2 (BnOCPA) SuMD + Classic MD 10 0.37 μs + 3.0 μs GαCT Gob (HOCPA) SuMD + Classic MD 10 0.29 μs + 3.0 μs BnOCPA(D):Gob Classic MD 4 4.0 µs BnOCPA(B):Gob Classic MD 3 3.0 µs HOCPA:Gob Classic MD 4 4.0 µs BnOCPA(D):Goa Classic MD 5 5.0 µs BnOCPA(B):Goa Classic MD 4 4.0 µs ♦For SuMD and SuMD-metadynamics simulations the time is the sum of productive SuMD time windows. (A), (B), (C) and (D) indicate the respective BnOCPA binding modes. ♦For SuMD and SuMD-metadynamics simulations the time is the sum of productive SuMD time windows. (A), (B), (C) and (D) indicate the respective BnOCPA binding modes. orders of magnitudes faster than the corresponding classical (unsupervised) MD simulations139. SuMD has been successfully applied to small molecules and peptides140–146. In the present work, the distances between the centres of mass of the adenine scaffold of the A1R agonist and N2546.55, F171ECL2, T2777.42 and H2787.43 of the receptor were considered for the supervision during the MD simulations. The dynamic docking of BnOCPA was hindered by the ionic bridge formed between the E172ECL2 and K265ECL3 side chains. A metadynamics147–149 energetic bias was there- fore introduced in order to facilitate the rupture of this ionic interaction, thus favoring the formation of a bound complex. More precisely, Gaussian terms (height = 0.01 kcal mol−1 and widths = 0.1 Å) were deposited every 1 ps along the distance between the E172ECL2 carboxyl carbon and the positively charged K265ECL3 nitrogen atom using PLUMED 2.3150. A similar SuMD-metadynamics hybrid approach was previously employed to study binding/unbinding kinetics151 on the A2AR subtype. For each replica (Table 1), when the ligands reached a bound pose (i.e. Dynamic docking of BnOCPA and HOCPA. The supervised MD (SuMD) approach is an adaptive sampling method138 for simulating binding events in a timescale one or two Molecular dynamics simulations Ligand parameterisation. The CHARMM36116,117/CGenFF118–120 force ﬁeld combination was employed in all the MD simulations performed. Initial topology and parameter ﬁles of BnOCPA, HOCPA, and PSB36 were obtained from the 17 TURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications ARTICLE References A & Varani, K. Pharmacology of adenosine receptors: the state of the art. Physiol. Rev. 98, 1591–1625 (2018). 7. Sawynok, J. Adenosine receptor targets for pain. Neuroscience 338, 1–18 (2016). 8. Headrick, J. P., Ashton, K. J., Rose’meyer, R. B. & Peart, J. N. Cardiovascular adenosine receptors: expression, actions and interactions. Pharm. Ther. 140, 92–111 (2013). 9. Dunwiddie, T. V. & Masino, S. A. The role and regulation of adenosine in the central nervous system. Annu Rev. Neurosci. 24, 31–55 (2001). 10. Vecchio, E. A. et al. New paradigms in adenosine receptor pharmacology: allostery, oligomerization and biased agonism. Br. J. Pharm. 175, 4036–4046 (2018). Analysis of the classic MD simulations. During the classic MD simulations that started from Modes A-C (Fig. 3d–g), BnOCPA had the tendency to explore the three conformations by rapidly interchanging between the three binding modes. In order to determine the effect exerted on the TM domain by each conformation, 30 μs of MD simulations (Table 1: BnOCPA mode A, BnOCPA mode B, BnOCPA mode C, BnOCPA mode D) were subjected to a geometric clustering. More pre- cisely, a simulation frame was considered in pose A if the distance between the phenyl ring of BnOCPA and the I175ECL2 alpha carbon was less than 5 Å; in pose B if the distance between the phenyl ring of BnOCPA and the L2586.59 alpha carbon was less than 6 Å, and in pose C if the distance between the phenyl ring of BnOCPA and the Y2717.36 alpha carbon was less than 6 Å. During the MD simulations started from mode D (Fig. 3g), a frame was still considered in mode D if the root mean square deviation (RMSD) of the benzyl ring to the starting equilibrated conformation was less than 3 Å. For each of the resulting four clusters, the RMSD of the GPCR conserved motif NPXXY (N7.49 PIV Y7.53 in the A1R; Supplementary Fig. 9) was computed using Plumed 2.3150 considering the inactive receptor state as reference, plotting the obtained values as frequency distributions (Fig. 3i, j). Rearrangement of the NPXXY motif, which is located at the intracellular half of TM7, is considered one of the structural hallmarks of GPCR activation79. Upon G protein binding, it moves towards the centre of the receptor TM bundle (Supplementary Fig. 9). Unlike other activation micro-switches (e.g. References A the break/ formation of the salt bridge between R3.50 and E6.30), this conformational transition is believed to occur in timescales accessible to MD simulations76. 11. Kaczynska, K. & Szereda-Przestaszewska, M. The potential role of the nodose ganglion adenosine A1 receptor in regulation of breathing in anaesthetized rats. J. Physiol. Pharm. 59, 759–770 (2008). 12. Koeppen, M., Eckle, T. & Eltzschig, H. K. Selective deletion of the A1 adenosine receptor abolishes heart-rate slowing effects of intravascular adenosine in vivo. PLoS ONE 4, e6784 (2009). 13. Varani, K., Vincenzi, F., Merighi, S., Gessi, S. & Borea, P. A. Biochemical and pharmacological role of A1 adenosine receptors and their modulation as novel therapeutic strategy. Adv. Exp. Med. Biol. 1051, 193–232 (2017). 14. Baltos, J. A. et al. Quantiﬁcation of adenosine A1 receptor biased agonism Implications for drug discovery. Biochem. Pharm. 99, 101–112 (2016). p g y 15. Weltha L., Reemmer J., Boison D. The role of adenosine in epilepsy. Brain Res. Bull. 151, 46–54 (2018). 16. Deb, P. K., Deka, S., Borah, P., Abed, S. N. & Klotz, K. N. Medicinal chemistry and therapeutic potential of agonists, antagonists and allosteric modulators of A1 adenosine receptor: current status and perspectives. Curr. Pharm. Des. 25, 2697–2715 (2019). 17. Kenakin, T. Biased receptor signaling in drug discovery. Pharm. Rev. 71, 267–315 (2019). 18. Wootten, D., Christopoulos, A., Marti-Solano, M., Babu, M. M. & Sexton, P. M. Mechanisms of signalling and biased agonism in G protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 19, 638–653 (2018). Hydrogen bonds and atomic contacts were computed using the GetContacts analysis tool (https://github.com/getcontacts/getcontacts) and expressed in terms of occupancy (the percentage of MD frames in which the interaction occurred). receptors. Nat. Rev. Mol. Cell Biol. 19, 638–653 (2018). 19. Violin, J. D. & Lefkowitz, R. J. Beta-arrestin-biased ligands at seven- transmembrane receptors. Trends Pharm. Sci. 28, 416–422 (2007). Analysis of the Goa, Gob and Gi2-GαCT classic MD simulations after SuMD. For each system, only the classic MD simulations performed after the GαCT reached the A1R intracellular binding site were considered for the analysis. 20. Smith, J. S., Lefkowitz, R. J. & Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nat. Rev. Drug Discov. 17, 243–260 (2018). g y The RMSD values to the last 15 residues of the Gi2-GαCT reported in the A1R cryo-EM PDB structure 6D9H were computed using VMD122. Received: 21 July 2020; Accepted: 23 June 2022; Dynamic docking of the Goa, Gob and Gi2-GαCT helix. A randomly extracted frame from the classic MD performed on the BnOCPA:A1R complex was prepared for three sets of simulations placing the GαCT helix 5 (last 27 residues) of the Gα proteins Goa, Gob and Gi2 in the intracellular solvent bulk side of the simulation boxes. As a further control, a frame from the classic MD performed on the unbiased ligand HOCPA:A1R complex was randomly extracted and prepared along with the Gob-GαCT. The resulting four systems were embedded in a POPC membrane and prepared as reported above. p boxes. As a further control, a frame from the classic MD performed on the unbiased ligand HOCPA:A1R complex was randomly extracted and prepared along with the Gob-GαCT. The resulting four systems were embedded in a POPC membrane and prepared as reported above. References A 1. Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829 (2017). The different structural effects putatively triggered by BnOCPA and HOCPA on the recognition mechanism of Goa, Gob and Gi2-GαCT were studied by The different structural effects putatively triggered by BnOCPA and HOCPA on the recognition mechanism of Goa, Gob and Gi2-GαCT were studied by The different structural effects putatively triggered by BnOCPA and HOCPA on the recognition mechanism of Goa, Gob and Gi2-GαCT were studied by performing 10 SuMD replicas (Table 1). During each replica (Video S3), the distance between the centroid of the GαCT residues 348-352 and the centroid of the A1R residues D422.37, I2326.33, and Q2938.48 was supervised until it reached a value lower than 8 Å. A classic MD simulation was then run for a further 300 ns. 2. Congreve, M., de Graaf, C., Swain, N. A. & Tate, C. G. Impact of GPCR structures on drug discovery. Cell 181, 81–91 (2020). structures on drug discovery. Cell 181, 81–91 (2020). 3. Kenakin, T. Is the quest for signaling bias worth the effort? Mol. Pharm. 93, 266–269 (2018). 4. Michel, M. C. & Charlton, S. J. Biased agonism in drug discovery-is it too soon to choose a path? Mol. Pharm. 93, 259–265 (2018). Classic MD simulations on the A1R:Goa and Gob complexes. The A1R cryo-EM structure (PDB ID 6D9H) was used as template for all the ﬁve systems simulated (Table 1). The endogenous agonist adenosine was removed and HOCPA and BnOCPA (modes B and D) were inserted in the orthosteric site superimposing 6D9H to the systems prepared for the classic MD simulations in the absence of G protein. ICL3 was not modelled, nor were the missing part of the G protein α subunit. As subunits β and γ were removed, the Gα NT helix was truncated to residue 27 to avoid unnatural movements (NT is constrained by Gβ in 6D9H). The Gα subunit was mutated according to the Goa and Gob primary sequences87 using in-house scripts. The resulting ﬁve systems (Table 1) were embedded in a POPC membrane and prepared as reported above. 5. Jacobson, K. A. & Muller, C. E. Medicinal chemistry of adenosine, P2Y and P2X receptors. Neuropharmacology 104, 31–49 (2016). 6. Borea, P. A., Gessi, S., Merighi, S., Vincenzi, F. Data availability The active cryo-EM structure of the A1R (PDB code 6D9H) was retrieved from https:// www.rcsb.org/structure/6d9h; the human neurotensin receptor 1 (PDB code 6OSA) from https://www.rcsb.org/structure/6OSA, and the β2 adrenergic receptor (PDB code 6E67) from https://www.rcsb.org/3d-view/6e67. Source data are provided with this paper. 26. Von Moo, E. et al. Ligand-directed bias of G protein signaling at the dopamine D2 receptor. Cell Chem. Biol. 29, 226–238.e224 (2022). p 27. Luttrell, L. M., Maudsley, S. & Gesty-Palmer, D. Translating in vitro ligand bias into in vivo efﬁcacy. Cell Signal. 41, 46–55 (2018). bias into in vivo efﬁcacy. Cell Signal. 41, 46–55 (2018). 28. Benredjem, B. et al. Exploring use of unsupervised clustering to associate signaling proﬁles of GPCR ligands to clinical response. Nat. Commun. 10, 4075 (2019). References A The MD frames associated with the peaks in the RMSD plots (states CS1, MS1, MS2 and MS3 in Fig. 4a, d) were clustered employing the VMD Clustering plugin (https://github. com/luisico/clustering) by selecting the whole GαCT helixes alpha carbon atoms and a cutoff of 3 Å. 21. Foster, D. J. & Conn, P. J. Allosteric modulation of GPCRs: new insights and potential utility for treatment of schizophrenia and other CNS disorders. Neuron 94, 431–446 (2017). 22. Wingler, L. M. et al. Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR. Science 367, 888–892 (2020). 23. Slosky L. M., et al. Beta-arrestin-biased allosteric modulator of NTSR1 selectively attenuates addictive behaviors. Cell 181, 1364–1379.e14 (2020). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 24. Kliewer, A. et al. Morphine-induced respiratory depression is independent of beta-arrestin2 signalling. Br. J. Pharm. 177, 2923–2931 (2020). 25. Yano, H. et al. Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor. Nat. Commun. 9, 486 (2018). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Received: 21 July 2020; Accepted: 23 June 2022; Received: 21 July 2020; Accepted: 23 June 2022; ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31652-2 Classic MD simulations of BnOCPA binding modes A, B, C and D. To test the hypothesis that BnOCPA and HOCPA may differently affect TM6 and/or TM7 mobility when bound to A1R (and to further sample the stability of each BnOCPA binding mode), putative binding conformations A, B and C (Fig. 3) were super- posed to the experimental A1R active state coordinates with the modelled ICL3. This should have removed any A1R structural artefacts, possibly introduced by metadynamics. As reference and control, two further systems were considered: i) the pseudo-apo A1R and ii) the selective A1R antagonist PSB3672 superposed in the same receptor active conformation (Table 1). The BnOCPA binding mode D was modelled from mode B by rotating the dihedral angle connecting the cyclopentyl ring and the N6 nitrogen atom in order to point the benzyl of the agonist toward the hydrophobic pocket underneath ECL3 (Fig. 3g) delimited by L2536.56, T2576.52, K265ECL3, T2707.35, and L2697.34. The G protein atoms were removed, and the resulting systems prepared for MD as reported above. A similar comparison was performed in a milestone study on the β2 adrenergic receptor76 which sought to describe the putative deactivation mechanism of the receptor. The MD engine ACEMD135 was employed for both the equilibration and productive simulations. Systems were equilibrated in isothermal-isobaric conditions (NPT) using the Berendsen barostat136 (target pressure 1 atm), the Langevin thermostat137 (target temperature 300 K) with a low damping factor of 1 ps−1 and with an integration time step of 2 fs. Clashes between protein and lipid atoms were reduced through 2000 conjugate-gradient minimisation steps before a 2 ns long MD simulation was run with a positional constraint of 1 kcal mol−1 Å−2 on protein and lipid phosphorus atoms. Twenty nanoseconds of MD simulation were then performed constraining only the protein atoms. Lastly, positional constraints were applied only to the protein backbone alpha carbons for a further 5 ns. Dynamic docking of BnOCPA and HOCPA. 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Highly potent and selective ﬂuorescent antagonists of the human adenosine 21 TURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications Acknowledgements We gratefully acknowledge the support of the University of Warwick (URSS Awards to S.H. and J.O.; Warwick Ventures Proof of Concept Fund awards to M.J.W. & B.G.F.), the Leverhulme Trust (RPG-2017-255, CAR and G.L. to fund K.B. and G.D.), the BBSRC (BB/M00015X/2, G.L., and BB/M01116X/1, Ph.D. studentship to E.H.), the MRC (MR/ J003964/1; I.W. and 2270402, iCASE PhD Studentship with NeuroSolutions to C.L.M.) and The Swiss National Science Foundation (PP00P2_123536 and PP00P2_146321, M.Lo). A.S. is supported by a European Scholarship from the Cambridge Trust, S.C. is funded by an AstraZeneca Ph.D. studentship and X.H. is funded by a China Scholarship Council Cambridge International Scholarship. RH is supported by an MRC Discovery Award (MC_PC_15070). C.A.R. is a Royal Society Industry Fellow. We would like to thank: Stephen Hill, Stephen Briddon, and Mark Soave (University of Nottingham) for gifting the Nluc-tagged adenosine receptor cell lines, the ﬂuorescent antagonist AV039, and technical advice; Kathleen Caron and Duncan Mackie (University of North Car- olina) for the β-arrestin1/2-YFP constructs, and Annette Gilchrist (Midwestern Uni- versity) and Heidi Hamm (Vanderbilt University) for assistance with the Gαo interfering We gratefully acknowledge the support of the University of Warwick (URSS Awards to S.H. and J.O.; Warwick Ventures Proof of Concept Fund awards to M.J.W. & B.G.F.), the Leverhulme Trust (RPG-2017-255, CAR and G.L. to fund K.B. and G.D.), the BBSRC (BB/M00015X/2, G.L., and BB/M01116X/1, Ph.D. studentship to E.H.), the MRC (MR/ J003964/1; I.W. and 2270402, iCASE PhD Studentship with NeuroSolutions to C.L.M.) and The Swiss National Science Foundation (PP00P2_123536 and PP00P2_146321, 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/. M.Lo). A.S. is supported by a European Scholarship from the Cambridge Trust, S.C. Acknowledgements is funded by an AstraZeneca Ph.D. studentship and X.H. is funded by a China Scholarship Council Cambridge International Scholarship. RH is supported by an MRC Discovery Award (MC_PC_15070). C.A.R. is a Royal Society Industry Fellow. We would like to thank: Stephen Hill, Stephen Briddon, and Mark Soave (University of Nottingham) for gifting the Nluc-tagged adenosine receptor cell lines, the ﬂuorescent antagonist AV039, and technical advice; Kathleen Caron and Duncan Mackie (University of North Car- olina) for the β-arrestin1/2-YFP constructs, and Annette Gilchrist (Midwestern Uni- versity) and Heidi Hamm (Vanderbilt University) for assistance with the Gαo interfering NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunications 22 NATURE COMMUNICATIONS \| (2022) 13:4150 \| https://doi.org/10.1038/s41467-022-31652-2 \| www.nature.com/naturecommunicatio
https://openalex.org/W3184567430	https://www.hal.inserm.fr/inserm-03312491/document	English	null	Variability of multi-omics profiles in a population-based child cohort	BMC medicine	2,021	cc-by	12,338	To cite this version: Marta Gallego-Paüls, Carles Hernández-Ferrer, Mariona Bustamante, Xavier Basagaña, Jose Barrera- Gómez, et al.. Variability of multi-omics profiles in a population-based child cohort. BMC Medicine, 2021, 19 (1), pp.166. 10.1186/s12916-021-02027-z. inserm-03312491 Variability of multi-omics profiles in a population-based child cohort Marta Gallego-Paüls, Carles Hernández-Ferrer, Mariona Bustamante, Xavier Basagaña, Jose Barrera-Gómez, Chung-Ho E Lau, Alexandros P Siskos, Marta Vives-Usano, Carlos Ruiz-Arenas, John Wright, et al. Variability of multi-omics profiles in a population-based child cohort Marta Gallego-Paüls1,2,3, Carles Hernández-Ferrer1,2,3, Mariona Bustamante1,2,3,4, Xavier Basagaña1,2,3, Jose Barrera-Gómez1,2,3, Chung-Ho E. Lau5,6, Alexandros P. Siskos7, Marta Vives-Usano1,2,3,4, Carlos Ruiz-Arenas1,2,3, John Wright8, Remy Slama9, Barbara Heude10, Maribel Casas1,2,3, Regina Grazuleviciene11, Leda Chatzi12, Eva Borràs2,4, Eduard Sabidó2,4, Ángel Carracedo13,14, Xavier Estivill4, Jose Urquiza1,2,3, Muireann Coen6,15, Hector C. Keun7, Juan R. González1,2,3, Martine Vrijheid1,2,3 and Léa Maitre1,2,3* © 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. Abstract Background: Multiple omics technologies are increasingly applied to detect early, subtle molecular responses to environmental stressors for future disease risk prevention. However, there is an urgent need for further evaluation of stability and variability of omics profiles in healthy individuals, especially during childhood. Methods: We aimed to estimate intra-, inter-individual and cohort variability of multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) measured 6 months apart in 156 healthy children from five European countries. We further performed a multi-omics network analysis to establish clusters of co-varying omics features and assessed the contribution of key variables (including biological traits and sample collection parameters) to omics variability. Results: All omics displayed a large range of intra- and inter-individual variability depending on each omics feature, although all presented a highest median intra-individual variability. DNA methylation was the most stable profile (median 37.6% inter-individual variability) while gene expression was the least stable (6.6%). Among the least stable features, we identified 1% cross-omics co-variation between CpGs and metabolites (e.g. glucose and CpGs related to obesity and type 2 diabetes). Explanatory variables, including age and body mass index (BMI), explained up to 9% of serum metabolite variability. Conclusions: Methylation and targeted serum metabolomics are the most reliable omics to implement in single time-point measurements in large cross-sectional studies. In the case of metabolomics, sample collection and individual traits (e.g. BMI) are important parameters to control for improved comparability, at the study design or analysis stage. This study will be valuable for the design and interpretation of epidemiological studies that aim to link omics signatures to disease, environmental exposures, or both. Keywords: Multi-omics, Exposome, Variability, Population study, Metabolomics, DNA methylation, Cross-omics, mRNA, miRNA, Children * Correspondence: lea.maitre@isglobal.org 1ISGlobal, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain Full list of author information is available at the end of the article HAL Id: inserm-03312491 https://inserm.hal.science/inserm-03312491v1 Submitted on 2 Aug 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Gallego-Paüls et al. BMC Medicine (2021) 19:166 https://doi.org/10.1186/s12916-021-02027-z * Correspondence: lea.maitre@isglobal.org 1ISGlobal, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain Full list of author information is available at the end of the article Background due to genetics, although some proteins are highly in- fluenced by body mass composition and acute inflam- mation [28]. g Characterizing early indicators of health and disease tra- jectories during pregnancy and childhood is at the core of the life course approach [1–4]. Early life englobes the most critical/sensitive periods for organ development, which makes it especially vulnerable to the effects of en- vironmental exposures [5, 6]. The integration of multiple omics data—such as epigenomics, transcriptomics, pro- teomics and metabolomics—is increasingly applied to detect early, subtle molecular responses to environmen- tal exposures because it employs a holistic view on all cellular processes [7–10]. However, there is an urgent need for further evaluation of stability and variability of omics profiles, between and within healthy children. Epi- demiological studies that incorporate omics profiles to monitor healthy individuals over time need to be in- formed of technical and biological variability in order to interpret changes in omics profiles, even if they are small. Omics variability may be determined by factors hindering subtle biological changes of interest, such as seasonality, individual characteristics (age and BMI), stage of life (i.e. hormones might vary between pre- puberty and adulthood stages) as well as by technical variability (due to measurement error and limited preci- sion of analytic tools), which therefore must be con- trolled at the design of the study [2, 5, 6]. To date, multi-omics platforms have mainly been used in studies with a small sample size that focused on diet- ary or physical activity interventions rather than follow- ing up healthy people from the general population [2, 29, 30]. They agreed that inter-individual variation, ra- ther than intra-individual variation, was the main ex- planatory factor for all omics measurements. However, previous multi-omics profile studies have not considered changes related to short and medium term. Especially, there is a lack of evidence regarding children from the general population, nor single or multi-omics studies, and the contribution of several factors such as age, sex and BMI. In the present study, we estimated intra- and inter- individual variability in multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) in 156 children from five European countries at two time points with a 6-month interval. We further aimed to assess interrelationships between the variability in different omics layers by performing a cross- omics correlation network analysis. © 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. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 2 of 16 Page 2 of 16 Background Finally, we aimed to decompose the variance in multi-omics profiles according to (1) inter-individual characteristics: sex, ancestry, age, maternal education, Mediterranean diet quality index (KIDMED score) and zBMI (which do not change in a 6- month period); and (2) intra-individual characteristics: hours of fasting before the visit for blood/ urine sampling, heavy exercise practice the same day or the day before sampling, having a cold at the moment of the visit, hour of sampling, day of the week and season (Fig. 1). g y Temporal variability in omics profiles has been de- scribed previously to assess the reliability of single time-point measurements in cross-sectional studies and to understand ageing and disease processes [11, 12]. In this paper, we define “intra-individual variabil- ity” as the variability estimated within individuals over time and “inter-individual variability” as the between- individuals variability. We also define the “short-term” as a time span of hours or days, the “medium-term” as a time span of months, and the “long-term” when considering years. For example, in the short term, the metabolome in urine and blood is assumed to be more dynamic than other omics as it is the down- stream result of in vivo substances and environmental factor influence [4]. On the other hand, DNA methy- lation is generally considered to be the most stable omics profile over short periods of time [13] and could provide more valuable information for environ- mental epidemiological purposes than other omics, as some methylomics signatures (e.g. smoking signatures) have been shown to persist over time even when the exposure no longer exists [2, 13–19]. Previous studies have also shown low levels of intra-individual variabil- ity in > 95% of the gene expression profile and in 25.5% of the miRNAs analysed, which are proposed as good biomarkers for many human diseases [20– 27]. Overall, the blood proteome is considered quite stable over time with strong inter-individual variability Summary of laboratory processing of omics signatures, quality control and normalization We performed in-depth omics profiling at two time points ~ 6 months apart for all 156 children. Because only 87 children had complete data of all omics analyses at both visits, we decided to analyse each omics profile independ- ently (i.e. all paired samples available for each specific omics layer). The final sample size for each omics layer was 149, 105, 100, 149, 154 and 154 children for DNA methylation, blood gene expression, miRNA expression, proteins, serum metabolites and urine metabolites, re- spectively. Details on laboratory and data processing methods are available in Additional file 1 - Supplementary Methods [39–48]. While DNA methylation, gene and miRNA expression screenings were based on genome- wide arrays, the other methods were targeted or semi- targeted. From now on, we use the term “features” to refer to the omics variables in our study: CpGs, gene and miRNA transcripts, proteins and metabolites. Because our study did not have technical replicates (biological samples systematically aliquoted in two replicates before sample preparation), which would be the ideal way to measure technical variability, potential technical variability was fil- tered out as much as possible in each omics layers, before fitting the variance partition models. Concretely, the panel study consisted of two visits (A and B) where data on exposures, individual behaviours, phenotypes and omics profiles were collected [38]. The mean difference between the two visits (A and B) was 6.11 months (standard deviation (SD): 2.18 months). In order to characterize in depth the variability of the omics measurements, the study population was further restricted to children with complete information for both visits (A and B) for at least one of the omics (N= 156). Prior to the start of data collection, national ethics committees had granted all the required permissions that allowed cohort participant recruitment and follow- up visits. Additionally, all the participants were asked to sign a HELIX specific informed consent. g p All samples were randomized in the arrays by sex and cohort, and in addition, the samples from the same indi- vidual in the microarray-based platforms were paired in the same plate/array (see Supplementary Methods). In the methylation, gene and miRNA expression data, we corrected remaining technical batch effects and blood cell composition by calculating surrogate variables (SVs) while protecting for cohort, sex and age with the SVA and SmartSVA methods [44, 49]. Study design and population y g p p The HELIX (Human Early Life Exposome) study is a collaborative project of six-population based cohorts in different European Countries: UK (BiB: Born in Brad- ford) [31], France (EDEN: Étude des Déterminants pré et postnatals du développement et de la santé de l’Én- fant) [32], Spain (INMA: Infancia y Medio Ambiente) [33], Lithuania (KANC: Kaunus cohort) [34], Norway (MoBa: Norwegian Mother and Child Cohort Study) [35] and Greece (RHEA: Mother Child Cohort study in Crete) [6, 36]. The aim of the HELIX study was to meas- ure and describe multiple environmental exposures dur- ing early life (pregnancy and childhood) in a prospective cohort and associate these exposures with molecular omics signatures and child health outcomes. From the six existing European longitudinal population-based birth cohorts studies participating in HELIX, a subcohort of 1301 mother-child pairs was se- lected to be fully characterized for a broad suite of Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 3 of 16 Gallego-Paüls et al. BMC Medicine Fig. 1 Study workflow Fig. 1 Study workflow Page 4 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 extraction, red cells and a buffy coat for DNA extraction. After processing, these samples were frozen at −80 °C under optimized and standardized procedures [6]. environmental exposures and omics data, to be clinic- ally examined, and to have biological samples col- lected [6]. From this subcohort, 156 children were selected to be part of the panel study: 28 from BiB (UK), 28 from EDEN (France), 40 from INMA Saba- dell (Spain), 30 from KANC (Lithuania) and 30 from RHEA (Greece) [37]. Children from MoBa (Norwe- gian Mother and Child Cohort Study; Norway) were not included in this panel study. The Child Panel Study had the same inclusion criteria as the HELIX subcohort: (a) age 6–11 years at the time of the visit, with a preference for ages 7–9 years if possible; (b) sufficient stored pregnancy blood and urine samples; (c) complete address history available; and (d) no ser- ious health problem. Summary of laboratory processing of omics signatures, quality control and normalization We used residuals from the correction process to analyse blood DNA methylation, gene and miRNA expression. We excluded probes for CpGs that did not reach a 62.5% interclass correlation coefficient (ICC) to minimize technical vari- ability, based on a previous analysis with technical repli- cates [46]. Gene and miRNA expression were filtered out based on call rate or other omics based on other technical parameters (see Table 1). All omics measure- ments were normalized and log2 transformed except for DNA methylation. Sample collection Biological samples were collected using the same stan- dardized protocols across all five cohorts. Urine samples were collected twice daily (first morning void and bed- time sample) in high-quality polypropylene tubes. The two urine samples were brought by the participants to the centre in cool packs and stored at −4 °C until pro- cessing. After aliquoting, the urine samples were frozen at −80 °C under optimized and standardized procedures. A pooled sample of both the morning and the night urine samples was used for the analysis when available (94.9% of individuals in the first visit and 95.5% in the second visit). In visit A, 7 children only had a morning sample available, and 1 child only had night sample. For visit B, this happened in 4 and 3 children for morning and night samples, respectively. Eighteen milliliters of blood was collected at the end of the clinical examination of the child, ensuring an approxi- mate 3-h fasting time since the last meal (visit A mean: 3: 34 h (SD: 1:11 h); visit B mean: 2:35 h (SD: 1:31 h)). Blood samples were collected using a ‘butterfly’ vacuum clip and local anaesthetic and processed into a variety of sample matrices for serum, plasma, whole blood for RNA Linear mixed effect models biological traits and sample collection parameters that were obtained through questionnaires. All omics, except the urine metabolome, were corrected for omics platform technical variables; therefore, we only included the run order as a covariate in the urine metabolome model. Time to last meal and hour of sample collection were not in- cluded in the urine metabolome model because we used pooled samples (morning and night). Variability present in the different omics layers was cal- culated with the variancePartition R package [47]. Briefly, it fits a linear mixed effect model to partition the variance attributed to multiple variables in the data. As this analytical process uses a multiple regression model, the effect of each variable is assessed while correcting for the others. Therefore, variancePartition assessed the contribution of each meta-data variable to variation in each feature. Before running the models, we ensured the absence of collinearity between the explanatory variables by obtain- ing a collinearity score: if this score were to be > 0.99, the variance partition estimates would produce mislead- ing results and overestimate the contribution of variables modelled as fixed effects [47]. No variables were elimi- nated due to collinearity. We considered two mixed effect models: (1) a model to estimate the proportion of variance attributable to intra-individual, inter-individual and cohort variability and (2) the same model adjusting for several explanatory variables (inter- and intra-individual variability-related variables, see list below) to determine the proportion of variance they accounted for. Individual IDs were entered in the models to account for inter-individual variability, whereas we took residuals as a measure of intra- individual variability. In the case of the methylome, we aimed to assess the amount of variance attributed to differences in the im- munological cell type composition. For this purpose, an extra model was performed with a dataset corrected for batch effect using the ComBat method [52] instead of using SVA method, in order to keep the effect of cell type composition. Statistical analyses All statistical analyses were performed using R version 3.6.3 [50]. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 5 of 16 Table 1 Omics data description and technical variability management Omics profile Matrix Sample size (omics available for both visits) Number of features Laboratory processing Batch correction Criteria for feature exclusion DNA methylation Blood leukocytes 149 91601 Randomized by cohort and sex, and panel samples paired in plate and array Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 98% call rate and < 62.5% ICC Gene expression Whole blood 105 45438 Randomized by cohort and sex, and panel samples paired in plate. Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 25% call rate miRNA expression Whole blood 100 453 Randomized by cohort and sex, and panel samples paired in plate and array. Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 25% call rate Proteins Plasma 149 36 Randomized by cohort Overall protein average minus plate specific protein average subtracted for each individual and each protein < 30% measurements in the linear range (LIN) Serum metabolites Serum 154 177 Fully randomized - > 30% CV and > 30% BLD + zeros Urine metabolites Urine 154 44 Fully randomized - > 30% CV Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below limit of detection Table 1 Omics data description and technical variability management Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below li i f d i Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below limit of detection DNA methylation A total of 156 children from five cohorts across Europe (BIB in the UK, EDEN in France, KANC in Lithuania, RHEA in Greece and INMA in Spain) were followed up for this study, with demographic data detailed in Table 2. Time span between both visits (A and B) was 6.1 months (2.2 SD). Children were on average 7.8 (1.7 SD) years old in visit A. Most of the participants of the study (71.2%) were in the healthy BMI range and remained in the same category from visit A to visit B. Samples were collected on average 3:36 h (1:12 SD) after the last meal during visit A, and after 2:36 h (1:30 SD) during visit B. The hour of sam- ple collection at both visits was almost the same, being 16: 54 h (2:54 SD) and 16:18 h (3:06 SD) for visits A and B, re- spectively. Table 2 shows the description of other explana- tory variables measured in the study. Variation in blood DNA methylation was mainly due to intra-individual variability with a median of 62.2% across all CpG sites and a median inter-individual variability of 37.6% (Q1: 4.1%; Q3: 65.8%). We note however a large heterogeneity between the CpGs with the lower and upper quartiles ranging from 33.9 to 95.6% for intra- individual variability and from 4.1 to 65.8%, for inter. This heterogeneity was partially expected due to the intra-experimental variation in Illumina BeadChip data. Indeed, intra-individual variability of each CpG site in our study was associated to previously reported ICCs, described in the same tissue and the same array, but in adults [46] (Additional file 1 - Figure S1). CpG sites with the highest inter-individual variability were enriched for CpG shores, whereas CpG sites with high intra-individual variability were enriched in open sea areas. We further aimed to evaluate the importance of blood cellular composition in blood DNA methylation variability. For this, we applied the same model for vari- ance partition but without residualizing the effect of blood cell proportions (Additional file 1 - Figure S2). Linear mixed effect models The data matrix contained 139 samples × 13,167 features (13,103 CpGs, 9 proteins, 44 serum me- tabolites and 11 urine metabolites). We computed GGMs using the ggm.estimator.pcor function from the R package “GeneNet” [48]. This function estimates pair- wise partial correlation coefficients conditioned against all remaining variables, allowing to filter out indirect as- sociations that may appear in omics data [48, 54]. We considered significant partial correlations between fea- tures those with p values below the false discovery rate (FDR) threshold of 0.05 (p < 1.28 × 10−7). To construct and visualize the resulting GGM networks, we used Cytoscape 3.8.2 [59]. Edges connecting the nodes repre- sent significant partial correlations and they were weighted using partial correlation coefficients (PCCs). The opacity of each node is based on its connectivity de- gree (number of edges connecting a particular feature). omics features presented little variability due to cohort (median variability of features ranging from 0% for the methylome to 15.7% for the proteome). Linear mixed effect models The following explanatory variables were added to the model as a measure of (1) inter-individual variation: sex, ancestry of the child, age, maternal education—as a gen- eral measure of socio-economic status—KIDMED score as a measure of healthy diet pattern [51] and zBMI, as we did not observe significant changes in the 6-month period; and of (2) intra-individual variation: time to last meal (hours of fasting), heavy exercise practice the same day or the day before the sample collection, having a cold at the moment of sampling, hour of sample collection, run order for the urine metabolome model, day of the week and sea- son at which the samples were collected. These were Additionally for the methylome, we checked whether CpGs located in the 4th quartile for intra- and inter- individual variability were enriched for CpG island rela- tive position (island, shore, shelf, open sea) and for over- lap with CpGs associated with exposures/traits in the EWAS Atlas [53]. Gaussian graphical modelling (GGM)—network analysis A GGM was used to assess direct associations between changes in omics features and elucidate biologically Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 6 of 16 Table 2 Population description (N=156) Start of the study Sex Male 89 Female 67 Ancestry European ancestry 145 Pakistani 10 Other 1 Cohort BIB, UK 28 EDEN, France 28 KANC, Lithuania 30 RHEA, Greece 30 INMA, Spain 40 Age (years); mean (SD) Total 7.8 (1.7) BIB 6.7 (0.2) EDEN 10.8 (0.5) KANC 6.7 (0.5) RHEA 6.3 (0.12) INMA 8.6 (0.5) zBMI; mean (SD) 0.4 (1.2) zBMI categories Thinness (zBMI < −2) 1 Healthy (−2 ≤zBMI < 1) 111 Overweight (1 ≤zBMI ≤2) 27 Obese (zBMI > 2) 17 Table 2 Population description (N=156) relevant associations [54–58]. GGMs were built on the delta matrix calculated as the change in omics features between visits (i.e. the change in feature X for a child be- tween time points is correlated with the change in Y, where the correlation is again calculated across all chil- dren). The omics features included: CpG sites with 100% intra-individual variability and proteins, serum and urine metabolites in the highest quartile for intra-individual variability. Gene expression and miRNAs were excluded because the number of participants with complete data including these layers was significantly lower (n=87) and would penalize the identification of biologically relevant associations. Serum and urine metabolites On average, for all the omics, intra-individual variabil- ity was negligibly affected by the inclusion of the ex- planatory variables. However, these variables explained a large percentage of variance in some particular features (Fig. 5) as described further below. Considering all the omics features as a whole, we identified that age, zBMI and hour of sample collection had a major effect on fea- ture variability. The serum metabolome presented, in average, the high- est inter-individual variability (median: 43.4%; Q1: 31.1%; Q3: 53.7%), and the lowest intra-individual vari- ability (median: 50.7%; Q1: 41.3%; Q3: 60.3%). Urine me- tabolites also presented a relatively high median inter- individual variability (median: 28.82%; Q1: 16%; Q3: 40.2%), compared to other omics. Proteins Proteins presented large heterogeneity as well (median cohort variability median: 15.7%; Q1: 4.5%; Q3: 35.3%; median intra-individual variability: 60%; Q1: 48.8%; Q3: 66.3%;). For instance, the variability of C-reactive protein (CRP) was largely explained by intra-individual variabil- ity (87.2%), while the variability of the epidermal growth factor protein (EGF) was attributed to cohort by 55%. Variance partition analysis shows large heterogeneity between and within omics A large heterogeneity in terms of the proportion of vari- ance explained by cohort, inter- and intra-individual variability between omics layers and within the same omics layer was found (Fig. 2 and Table S1). Overall, Page 7 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Page 8 of 16 Page 8 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 When these were added as explanatory variables, differ- ences in immune cell type composition explained a me- dian of 14.0% of the intra-individual variance (Q1: 6.0%; Q3: 34.8%). model (P=9) did not show any significant partial correlation to other features. Among the connected components com- posed by features from different omics layers, all consisted of one serum or urine metabolite directly correlated to a group of CpGs, indicating CpG-metabolite change dependencies. These metabolites included trimethylamine oxide (TMAO), carnitine C3-DC (C4-OH), PC ae C38:1, glucose and citrate (Fig. 4A–E, respectively). CpGs cg16076587 and cg08510264 present in the same connected component as glucose are an- notated to INPP5A (Inositol polyphosphate-5-phosphatase A) and IRS2 (Insulin receptor substrate 2) genes, respectively. Gene expression Intra-individual variability explained the majority of the variance in most of the transcript clusters—groups of probes that define the expression of a gene (median: 93%; Q1: 78%; Q3: 100%). Biological traits can help to interpret some of the omics variability, especially in the serum metabolome MiRNAs presented a variance partition pattern similar to the gene expression, although not as pronounced. Intra-individual variability was predominant for most of the miRNAs (median: 65.2%; Q1: 51.8%; Q1: 81.4%). y y We further aimed to evaluate the association of omics variability with several anthropometric and dietary traits. Overall, the inclusion of explanatory variables accounted for up to 9% of the serum metabolite inter-individual variability (change from a median of 43% to 34% with additional explanatory variables) and up to 3–4% (me- dian change) of the intra-individual variability in the gene expression, miRNA and proteins (Additional file 1 - Table S1). Percentage of omics features explained by each explanatory variable per omics dataset, considering three different thresholds: ≥1%, ≥2% and ≥5% of vari- ance explained are also provided (Additional file 1 – Table S2). Gaussian graphical model networks identify few CpG- metabolite change dependencies Generally, DNA methylation was poorly explained by the explanatory variables: only 3% of the CpGs had more than 2% of the variance explained by individual traits; the KIDMED score explained the most variability (≥2% of the variance in 9.6% of CpGs). Our multi-omics study design also allowed us to analyse co- dependencies in the variability of biological features across the different molecular layers. After applying GGM on the delta matrix (e.g. correlations on the change in omics fea- tures between visits, see the “Methods” section [54–58]), we found 70 connected components and a total of 755 nodes and 1781 undirected edges (FDR < 0.1, Fig. 3). Edges were weighted using partial correlation coefficients [PCCs ranged from 0.003 to 0.007, p values ranged from 2.22 × 10−16 to 1.28 × 10−7]. The largest connected component contained 409 nodes mainly formed by CpG sites (99%), plus 69 smaller connected components that contained from 26 to 2 nodes. Most connected components (88.5%) were formed by features from the same omics layer, including three formed exclusively by serum metabolites as follow: (1) amino-acids (Arg, Phe, Trp, Met, Met.SO, Tyr and His), (2) carnitines and (3) phosphatidylcholines (PCs). Proteins included in the In gene expression, age, maternal education, KIDMED score, week day and hour of sample collection were re- sponsible for at least 2% of the variance in more than 5% of the transcripts each, with the KIDMED score showing the largest influence. MiRNAs were mainly influenced by the hour of sample collection, age, ancestry, and KIDMED score. Concretely, ancestry and hour of sample collection explained more than 5% of the variance in 4.6% and 8.7% of the miRNAs, respectively. Further- more, the KIDMED score explained more than 2% of variance in 10.6 % of miRNAs. Proteins were mostly influenced by zBMI, ancestry, and age, where each trait explained more than 5% of the variance in 22.2%, 16.7% and 16.7% of the Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 9 of 16 Gallego-Paüls et al. BMC Medicine Fig. 3 Network representation of the Gaussian graphical model (GGM) of the DNA methylome, proteins, serum and urine metabolites with high intra-individual variability measured in 157 children from five European countries. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). Gaussian graphical model networks identify few CpG- metabolite change dependencies The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Fig. 3 Network representation of the Gaussian graphical model (GGM) of the DNA methylome, proteins, serum and urine metabolites with high intra-individual variability measured in 157 children from five European countries. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Serum metabolites were mostly affected by ancestry and age, explaining more than 5% of the variance in 24.3% and 16.4% of the metabolites, respectively. By contrast, ancestry and age explained more than 5% of variance in just 2.3% and 9.1% of the urine metabo- lites, respectively. Our results showed that time to last meal explained more than 2% of the variance in only a 10.7% of the serum metabolites. Among the metab- olites most influenced by sex, BMI and hours of fast- ing, we found sphingomyelin C18:1 and tyrosine for proteins, respectively. Among the proteins largely ex- plained by zBMI were leptin and insulin. Similarly, these same proteins showed sex differences, found in higher concentrations in females. Ancestry explained 25–12% of the variability in these proteins (in order of magnitude): interleukin (IL)8, TNF alfa, BAFF, in- sulin and HGF (see Additional file 2). Insulin ap- peared to be the only protein significatively influenced by the hours of fasting and CRP was asso- ciated with having a cold. proteins, respectively. Among the proteins largely ex- plained by zBMI were leptin and insulin. Similarly, these same proteins showed sex differences, found in higher concentrations in females. Ancestry explained 25–12% of the variability in these proteins (in order of magnitude): interleukin (IL)8, TNF alfa, BAFF, in- sulin and HGF (see Additional file 2). Insulin ap- peared to be the only protein significatively influenced by the hours of fasting and CRP was asso- ciated with having a cold. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 10 of 16 irect associations between features from Fig. 4 Main connected components of the Gaussian graphical model (GGM) network that involve direct associations between features from different omics layers. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. Gaussian graphical model networks identify few CpG- metabolite change dependencies The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Fig. 4 Main connected components of the Gaussian graphical model (GGM) network that involve direct associations between features from different omics layers. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM sex; glucose and 4-deoxyerythronic acid for BMI z- score; and alanine for hours of fasting. metabolites were somewhat in the middle, with strong heterogeneity between features. Consequently, DNA methylation and serum metabolites (targeted assay) will better inform epidemiological studies that rely on single measurements to compare individuals in the search of biomarkers, whereas less stable omics profiles such as gene expression will give more reliable information to studies that assess individual trajectories over time (mul- tiple time point measurements). We evidenced that vari- ability of omics features comes from several sources. Besides technical or analytical variability, which we tried to control for, we identified physiological patterns in intra-individual variability through inter-omics network analysis. In all omics profiles, features with high inter- individual variability were identified, which can be as- cribed to biological (between individuals) variability. While it is always preferable to adhere to standard sam- ple collection conditions, this is not always possible, and omics features with substantial biological variation are potentially robust enough to yield meaningful findings in Discussion The current study offers a multi-omics perspective of medium-term omics variability in childhood. We mod- elled the variability of 6 different types of omics data (blood DNA methylation, gene expression, miRNA, proteome, serum and urine metabolomes) for 156 chil- dren from five European countries at two time points with a 6-month interval, and found a large range of intra- and inter-individual variability between and within each omics profiles. We pointed out that overall intra- individual variation accounted for the largest part of the total variation in all omics. While DNA methylation and serum metabolites exhibited stronger stability over time for many features (median inter-individual variability: 37.6 and 43.4%, respectively), gene expression was the less stable omics profile in average (median inter- individual variability: 6.6%) and proteins and urinary Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 11 of 16 Fig. 5 Violin plots showing multi-omics variability decomposed by biological traits and sample collection parameters measured in the study. Labels correspond to omics features mostly explained by each variable. Abbreviations. Proteome: IL: interleukin; Apo A1: apolipoprotein A1; RA: receptor antagonist; CRP: c-reactive protein. Serum metabolome: C: acylcarnitine; SM: sphingomyelin; PC: phosphatidylcholine; lysoPC: lysophosphatidylcholine Fig. 5 Violin plots showing multi-omics variability decomposed by biological traits and sample collection parameters measured in the study. Labels correspond to omics features mostly explained by each variable. Abbreviations. Proteome: IL: interleukin; Apo A1: apolipoprotein A1; RA: receptor antagonist; CRP: c-reactive protein. Serum metabolome: C: acylcarnitine; SM: sphingomyelin; PC: phosphatidylcholine; lysoPC: lysophosphatidylcholine which are regions 0–2 kb from CpG islands (CpG rich regions) [33]. On the other hand, CpG sites with high intra-individual variability were enriched in open sea areas, which are isolated CpG sites in the genome that have been linked to chromosomal instability and loss of imprinting [33, 34]. Recent studies point out that pheno- typically relevant CpGs tend to be located in CpGs shores [13, 16]. These CpGs with high inter-individual variability are especially relevant for large-scale epi- demiological studies since these probes could be used as reliable biomarker candidates [16]. Our results reinforce that strong methylation differences between individuals spite of collection inconsistencies. The small proportions of variability attributed to cohort demonstrated that standardized sample collection protocols can produce robust results in large-scale omics studies across differ- ent countries. Discussion Our results, in contrast, attributed the majority of the variation to intra- individual effects, potentially due to RNA quality that might differ between visits A and B. Effort has to be put on the initial sample preparation (DNA, RNA extraction and quality) and its harmonization across different cen- tres or time point collection, since it strongly determines the quality of omics measurements and might hinder real biological response. To our knowledge, only two previous studies have estimated miRNA variability in terms of intra- and inter-individual effects, but these studied a longer time span (5 years) [26] or a daily time span (48 h) in cerebrospinal fluid [27]. Despite these dif- ferences in study design, both studies agree that there is diversity within the microRNome in terms of stability of its features. Previous proteome studies in healthy human volunteers have demonstrated moderate inter-individual variability (CV ranging from 30% to 50%) [35, 36], simi- lar to the urine proteome in seven adult donors [37]. A study that estimated intra-individual variation of plasma adipokines concluded that they may be useful bio- markers of inflammation in population-based studies of obesity-related disease due to their stability over time [38]. These corroborate our results for IL-1β, IL-6, IL-8, leptin, adiponectin, hepatocyte growth factor (HGF) and CRP which presented the highest stability in our study. Finally, metabolomics studies comparing serum and urine metabolomes corroborate in children and adult populations strongly corroborate our findings that the serum metabolome is more stable and captures more inter-individual specific variance, compared to the urine metabolome [39–43]. A few recent studies have reported changes in multiple omics profiles in clinical settings and after dietary well- ness coaching intervention or physical activity [30, 66, 67], revealing omics signatures that may serve as poten- tial diagnostic markers. However, variability of omics profiles in healthy population and “normal” living condi- tions remains under-studied. Large observational studies would benefit enormously from this information, be- cause it allows the interpretation of changes that do not match a pattern. This is especially important for early- life studies that focus on the origin of diseases in chil- dren because omics profiles are able to capture very early and subtle molecular responses, even before physiological manifestations appear. Discussion Biological traits and sample collection var- iables, easy to collect in cohort studies, might help to account for the unwanted variability, in particular for metabolomics. We found, in the case of the methylome, that the most stable CpG sites were enriched in functional methylation regions of the genome. CpG sites with the highest inter- individual variability were enriched for CpG shores, Page 12 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 12 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 already appear in childhood as previously reported [18], even in our population of similar European ancestry children. On the other hand, gene expression may re- quire repeat sampling to account for intra-individual and technical variability, in order to generate stable enough markers to be deployed in epidemiological stud- ies. Previous studies in healthy individuals show that gene expression profiles are mostly stable and repeatable in the short/medium term (< 5% of transcripts with high intra-individual variability) [20–22, 24]. Our results, in contrast, attributed the majority of the variation to intra- individual effects, potentially due to RNA quality that might differ between visits A and B. Effort has to be put on the initial sample preparation (DNA, RNA extraction and quality) and its harmonization across different cen- tres or time point collection, since it strongly determines the quality of omics measurements and might hinder real biological response. To our knowledge, only two previous studies have estimated miRNA variability in terms of intra- and inter-individual effects, but these studied a longer time span (5 years) [26] or a daily time span (48 h) in cerebrospinal fluid [27]. Despite these dif- ferences in study design, both studies agree that there is diversity within the microRNome in terms of stability of its features. Previous proteome studies in healthy human volunteers have demonstrated moderate inter-individual variability (CV ranging from 30% to 50%) [35, 36], simi- lar to the urine proteome in seven adult donors [37]. A study that estimated intra-individual variation of plasma adipokines concluded that they may be useful bio- markers of inflammation in population-based studies of obesity-related disease due to their stability over time [38]. These corroborate our results for IL-1β, IL-6, IL-8, leptin, adiponectin, hepatocyte growth factor (HGF) and CRP which presented the highest stability in our study. Discussion Finally, metabolomics studies comparing serum and urine metabolomes corroborate in children and adult populations strongly corroborate our findings that the serum metabolome is more stable and captures more inter-individual specific variance, compared to the urine metabolome [39–43]. intake and fat storage [61] and the pro-inflammatory cy- tokines IL-6 and IL-1 beta, both known to be elevated in subjects with obesity or with serum lipid concentrations abnormalities, leading to a state of chronic inflammation [62]. Similarly, these same proteins showed sex differ- ences, in particular leptin and IL-1 beta, known to be higher in females and resulting in a higher risk to de- velop obesity than males [63]. Interestingly, circulating IL-8, a pro-inflammatory cytokine, was strongly influ- enced by ancestry (6% of children had Pakistani ances- try) in our cohorts (25% of variance explained). IL-8 was previously found to be influenced by genetic polymor- phisms in an eastern Indian population, potentially driv- ing individual variations in the host’s immune response, in particular to infectious diseases [64]. Intra-individual factors such as hours of fasting and having a cold gave the expected results: insulin appeared to be the only pro- tein significatively influenced by the hours of fasting and CRP was associated with having a cold. This is in line with a previous study that considered the fasting/post- prandial state of the samples and revealed that, on aver- age, it explained less than 2% of the total variance [41]. Associations of serum and urine metabolites with sex, BMI and hours of fasting (only for serum) was also pre- viously reported in the cross-sectional study of 1300 HELIX children [39]. Interestingly, high levels of 4- deoxyerythronic acid in children, found correlated to higher BMI in our study, have been previously related to early-onset type I diabetes although further understand- ing of its metabolism is required [65]. already appear in childhood as previously reported [18], even in our population of similar European ancestry children. On the other hand, gene expression may re- quire repeat sampling to account for intra-individual and technical variability, in order to generate stable enough markers to be deployed in epidemiological stud- ies. Previous studies in healthy individuals show that gene expression profiles are mostly stable and repeatable in the short/medium term (< 5% of transcripts with high intra-individual variability) [20–22, 24]. Discussion Variations in omics profiles due to factors such as BMI, age, physical activity, fasting time and sampling conditions need to be well characterized in order to interpret subtle changes related to environmental exposures such as air pollution or endocrine-disrupting chemicals, the ultimate goal of re- cent exposome initiatives [60]. Omics measurements in our study are of great clinical relevance as they provide the basis for the discovery of new biomarkers of multiple medical conditions such as cancer, heart disease, neuro- logical disorders or inflammatory diseases [61–64]. Here, Biological traits, such as body weight, sex and age, accounted for inter-individual variability in gene expres- sion, similar to results obtained by Hughes et al. (2015) [25], where roughly 2% of total gene expression variation was explained by these traits in placenta samples. Age and hour of sample collection have also been found to be significant sources of variation in blood gene expres- sion patterns of healthy individuals [60]. Previous studies on miRNA variability identified the strong influence of age and sample storage time [15, 23], but the time of the day or dietary intake has not been previously studied to our knowledge. Among the proteins largely explained by zBMI were leptin and insulin, hormones related to food Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 13 of 16 Page 13 of 16 approach provided reduced biological coverage, we are confident that this insured reliable annotation and quan- tification of markers. In contrast, we report high intra- individual effects in a large proportion of genome-wide omics features that are potentially due to technical vari- ability. Despite considering quality control parameters (e.g. filtering for call rate) in order to minimize this ef- fect, we note that precision in the intra-individual vari- ability apportionments can be strengthened by adding technical replicates. This, together with increasing the number of individuals, would be the ideal way to strengthen the biological signal. Moreover, despite hav- ing measured many biological traits in the children under study, there are relevant missing variables, such as recent dietary intake in the last 24 h, which is expected to influence the variability of omics such as the urine metabolome. It would be interesting to study if and how other factors account for variability within omics. Our study could also benefit from a more exhaustive collec- tion of repeated samples across short periods of time. Discussion For example, having morning and night samples in the same individual at different time points, as done previ- ously for metabolomics [77, 78] or miRNAs [27]. we provide insight into which omics features are stable within individuals and demonstrate sufficient inter- individual variation in order to reduce chance findings when conducting epidemiological studies with follow-up for disease outcomes [16]. We emphasize the fact that children from five Euro- pean countries took part in the study. The multiple loca- tions of our participants allow to generalize the influence of factors such as lifestyle habits, seasonal in- fluences and maternal education, which vary greatly be- tween countries/cultures and not many studies consider. A major strength of our study lies in the cross-omics ap- proach. We created a network of interacting omics fea- tures of four different omics layers (DNA methylome, proteome, serum and urine metabolomes) using Gaussian graphical models (GGMs). GGMs circumvent the selec- tion of indirect associations that usually appear between omics measurements, as Pearson correlations are generally high in these data. GGMs are based on partial correlation coefficients and provide an estimate of conditional de- pendencies between variables, elucidating direct associa- tions [54]. This enabled us to obtain a holistic view of the biological significance of our results. By studying the inter- action of multiple omics features, we exploited the data to their full potential for further disease prediction and pre- vention studies [56]. Moreover, it allowed us to go a step further by identifying interactions of features across omics layers, which is the main goal of the recent discipline in- teractome within systems biology. Despite not finding many dependencies across omics, a few metabolites were related to groups of CpGs. These included TMAO, a com- pound generated by the gut microbiota from diet-derived components, hence with high variability and strongly de- termined by diet, gut microbial flora and drug administra- tion [65]. Elevated levels of TMAO positively correlate to cardiovascular disease through the development of athero- sclerosis in previous studies [68–73]. Variation in four CpGs was related to glucose in urine (pool morning and night urine), which level depends on lifestyle factors like diet and exercise behaviours and its dysregulation related to obesity and type 2 diabetes (T2D) in children [74]. Interestingly, two of these CpGs are within genes related to obesity and T2D [53]. Discussion This supports previous studies on GGMs that show their ability to reconstruct metabolic pathways [54, 75, 76], including in this case across omics. Conclusions d We assessed omics profiles variability over the medium- term in child cohorts from the general population, using a multi-omics approach. We found large heterogeneity within and between omics profiles. Intra-individual vari- ability presented the highest median variability in all cases. The cross-omics analysis we performed provides global insights into how different omics features vary over time, within and between individuals and among cohorts from different countries. This study thereby pro- vides a valuable framework for future epidemiological studies that aim to detect omics signatures linked to disease, environmental exposures or both. Abbreviations BMI: Body mass index; CRP: C-reactive protein; CV: Coefficient of variation; FDR: False discovery rate; GGM: Gaussian graphical model; HELIX: Human Early Life Exposome; ICC: Interclass correlation coefficient; IL: Interleukin; KIDMED score: Mediterranean diet quality index; miRNA: MicroRNA; PC: Phosphatidylcholine; PCCs: Partial correlation coefficients; Q1, 2, 3, 4: Quartile 1, 2, 3, 4; QC: Quality control; SD: Standard deviation; SVA: Surrogate variable analysis; TC: Transcript cluster; TMAO: Trimethylamine oxide Our study had some limitations. In the first place, we studied omics variability in 156 children and a larger population size would have provided greater statistical power to our model. However, this sample size allowed us to analyse 6 different molecular layers in the same in- dividual and at the same time-point, twice, data rarely obtained in the past. 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Acknowledgements e would like to thank all the families for their generous contributio We would like to thank all the families for their generous contribution. Availability of data and materials The summarized results generated during the current study are available in supplementary material. The raw data supporting the current study are available from the corresponding author on request subject to ethical and legislative review. 5. Barker DJP. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–7. https://doi.org/10.1111/j.1365-2796.2007.01809.x. 5. Barker DJP. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–7. https://doi.org/10.1111/j.1365-2796.2007.01809.x. Declarations 6. Maitre L, de Bont J, Casas M, Robinson O, Aasvang GM, Agier L, et al. Human Early Life Exposome (HELIX) study: a European population-based exposome cohort. BMJ Open. 2018;8:e021311. 6. Maitre L, de Bont J, Casas M, Robinson O, Aasvang GM, Agier L, et al. Human Early Life Exposome (HELIX) study: a European population-based exposome cohort. BMJ Open. 2018;8:e021311. Authors’ contributions B RS C RG Authors contributions LM, MB, JW, RS, MC, JRG, HK and MV designed the omics study in HELIX. The following authors participated in omics data acquisition and quality control: AC, CR-A, MB (DNA methylation), XE, MV-U (transcriptomics), ES, EB (proteo- mics), CEL, APS, LM, HK, MC (metabolomics). JW, RS, BH, RG, MV and LCh are the PIs of the cohorts or participated in sample and data acquisition. CH-F, LM, MB, JU, JRG, XB and JB, performed statistical analyses and JRG functional enrichment analyses. The project was coordinated by MV. MG-P wrote the original draft of the paper and LM, MB, MV, JRG, CH-F and HK contributed to reviewing and editing the manuscript. All authors read and approved the manuscript. Funding Th d The study has received funding from the European Community’s Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project) and the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project). y g p y Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project) and the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project). Additionally, BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/ N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Rhea project was finan- cially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV- 1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in pre- school children, in Heraklion district, Crete, Greece: 2011- 2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Govern- ment (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (MS16/00128). LM is funded by a Juan de la Cierva-Incorporación fellowship (IJC2018-035394-I) awarded by the Spanish Ministerio de Economía, Industria y Competitividad. Received: 30 March 2021 Accepted: 8 June 2021 Received: 30 March 2021 Accepted: 8 June 2021 Consent for publication Not applicable. Consent for publication Not applicable. Competing interests Th h d l h The authors declare that they have no competing interests. MC is currently affiliated to AstraZeneca but the company had no role in the design, conduct, or analysis of the H2020-EU funded project. Author details 1 1ISGlobal, Barcelona, Spain. 2Universitat Pompeu Fabra (UPF), Barcelona, Spain. 3Consorcio de Investigacion Biomedica en Red de Epidemiologia y Salud Publica (CIBERESP), Madrid, Spain. 4Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain. 5MRC Centre for Environment and Health, School of Public Health, Imperial College London, London, UK. 6Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, South Kensington, London, UK. 7Cancer Metabolism & Systems Toxicology Group, Division of Cancer, Department of Surgery & Cancer and Division of Systems Medicine, Department of Metabolism, Digestion & Reproduction, Imperial College London, London, UK. 8Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK. 9Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, Institute for Advanced Biosciences (IAB), Inserm, CNRS, Université Grenoble Alpes, Grenoble, France. 10Université de Paris, Centre for Research in Epidemiology and Statistics (CRESS), INSERM, INRAE, F-75004 Paris, France. 11Department of Environmental Sciences, Vytautas Magnus University, Kaunas, Lithuania. 12Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. 13Medicine Genomics Group, Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER), University of Santiago de Compostela, CEGEN-PRB3, Santiago de Compostela, Spain. 14Galician Foundation of Genomic Medicine, Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Servicio Gallego de Salud (SERGAS), Santiago de Compostela, Galicia, Spain. 15Oncology Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK. 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https://openalex.org/W4321607103	https://www.researchsquare.com/article/rs-2294147/latest.pdf	English	null	Uveal metastasis arising from thyroid neoplasms: a systemic review of case reports	Research Square (Research Square)	2,023	cc-by	4,946	Uveal metastasis arising from thyroid neoplasms: a systemic review of case reports Mirsaeed Abdollahi Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. Hooman Nateghian Research Center for Evidence‑Based Medicine, Iranian EBM Centre: A Joanna Briggs Institute Affiliated Group, Tabriz University of Medical Sciences, Tabriz, Iran Navid Sobhi Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. Ali Jafarizadeh Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. https://orcid.org/0000- 0003-4922-1923 Research Article Keywords: Uvea, Choroid, Iris, Ciliary Body, Metastasis, Thyroid Neoplasm Posted Date: May 3rd, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2294147/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Uveal metastasis arising from thyroid neoplasms: a systemic review of case reports Mirsaeed Abdollahi Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. Hooman Nateghian Research Center for Evidence‑Based Medicine, Iranian EBM Centre: A Joanna Briggs Institute Affiliated Group, Tabriz University of Medical Sciences, Tabriz, Iran Navid Sobhi Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. Ali Jafarizadeh Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. https://orcid.org/0000- 0003-4922-1923 Research Article Keywords: Uvea, Choroid, Iris, Ciliary Body, Metastasis, Thyroid Neoplasm Posted Date: May 3rd, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2294147/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Uveal metastasis arising from thyroid neoplasms: a systemic review of case reports Research Center for Evidence‑Based Medicine, Iranian EBM Centre: A Joanna Briggs Institute Affiliated Group, Tabriz University of Medical Sciences, Tabriz, Iran N id S bhi DOI: https://doi.org/10.21203/rs.3.rs-2294147/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at European Journal of Ophthalmology on March 5th, 2024. See the published version at https://doi.org/10.1177/11206721241236528. Page 1/15 Page 1/15 Abstract Purpose: We systematically reviewed the case report literature to identify cases of uveal metastases (UM) originating from Thyroid Cancers (TCs) and evaluate this condition's demographic, ophthalmologic, and oncologic characteristics. Methods: Web of Science, Medline, and Scopus databases were searched for case reports or series reporting uveal metastasis originating from a thyroid neoplasm. Articles published in any language from inception through November 2022 were searched and screened independently by two reviewers. The quality of the included studies was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Case Reports. Results: A total of 1049 records were detected. After removing duplicates and screening articles based on title, abstract, and full text, 46 cases from 43 studies were identified. The mean (SD) age of patients at the time of UM diagnosis was 56.521 (±15.68) years (range, 20–83 years), and 16 cases (34.78%) were reported in the elderly (˃64 years). Male patients consisted 56.52% (n=26) of the cases. Sixteen cases happened in the right eye, 19 in the left eye, and 11 cases in both eyes. In total, 39 cases (84.78%) had choroidal involvement. Papillary carcinoma was the most reported type of thyroid cancer in the patients (16/46), followed by follicular (15/46) and medullary (10/46) carcinomas of the thyroid. Conclusion: Uveal metastases may appear in metastatic thyroid neoplasms. Therefore, physicians should maintain suspicion when ocular symptoms accompany a neck mass or a history of previous thyroid malignancies. 2. Methods This systematic review is based on the Preferred Reporting Items for Systematic Reviews and Meta- analysis (PRISMA) guidelines (15). The protocol of this study was registered on the International Prospective Register of Systematic Reviews database (PROSPERO). The registration code was CRD42022366663. 2.1. Search strategy and study selection Web of Science, Medline, and Scopus databases were searched for relevant studies published in any language from inception through August 2022. Major medical subject heading (MESH) and related keywords were used to build the search strategy brought in Supplementary File 1. After excluding records not meeting the inclusion criteria, the reference sections of studies selected for full-text retrieval were explored to specify other potentially related investigations. Our comprehensive analysis included both published and unpublished studies to reduce the risk of publication bias. Two reviewers independently reviewed the titles, abstracts, and full-text articles to decide on study inclusion. Disagreements and uncertainties were resolved through discussion and the help of a third reviewer. 1. Introduction Thyroid cancers (TCs) are the most common endocrine malignancies, and cancers are growing in the United States. TCs were evaluated at 43,800 as new cases in 2021, with a 300 percent increase in global incidence over the last four decades (1, 2). In most geographic regions and demographic groups, it is three times more common in women. TC affects a younger population than most other cancers, and the mean age at diagnosis is around 40 years old (3). Histologically, primary TCs are classified into four types: well-differentiated epithelial thyroid cancers, including papillary thyroid cancers (PTC) and follicular thyroid cancers (FTC), poorly differentiated epithelial thyroid cancers, medullary thyroid cancers (MTC), and rare thyroid tumors, such as sarcoma and lymphoma (4). PTC and FTC account for 95% of all thyroid cancer cases, and PTC is the most common type of TCs (5). PTC metastasizes to regional lymph nodes in 30 to 40% of cases. However, distant metastases can occur in 1-4 percent of patients (2). Male gender, older age, histologic grade, completeness of surgical resection, and extrathyroidal and lymph node involvement at the initial examination are all risk factors for distant metastases (6, 7). The lungs and the bones are the most common sites for distant metastasis in TCs cases (8), and intraocular involvements are less common. However, one percent of all metastatic intraocular involvements originated from TCs (9). Page 2/15 The most prevalent intraocular metastatic lesions are uveal metastases, and up to 10% of individuals with metastatic malignancies have ophthalmic involvement (10). Because of the hematogenous spreading of tumor emboli from distant locations, the choroid is the most common site in the uveal tract impacted by metastases (88%) (11). unilateral choroidal metastases (CM) are three times more common than bilateral metastases (12). blurred vision, decreased VA (visual acuity), floaters, flashes, and pain are the most frequent symptoms of uveal metastases (13). Numerous diagnostic methods, such as ultrasonography (US), multicolor imaging, enhanced depth imaging optical coherence tomography (EDI-OCT), OCT angiography (OCTA), fluorescein angiography (FA), and indocyanine green (ICGA), can now be used to examine particular features of CM (14). Because there was no previous study similar to this title, we conducted a systematic review and meta- analysis of all relevant case reports of patients with uveal metastases originating from the TCs. This study aimed to evaluate the worldwide distribution, epidemiology, clinical findings, and factors associated with this condition. 3.1. Study selection and study characteristics A PRISMA flow diagram of selected studies is presented in Figure 1. A total of 1049 records were identified. After removing duplicates and screening articles based on title and abstract, 58 articles remained. The remaining 58 articles were reviewed in full, and finally, 46 cases of choroidal metastases related to thyroid neoplasms were identified from 43 studies from 18 countries (Table 1). All but three studies were in English. A detailed extraction table can be accessed in Supplementary File 2. 2.2. Eligibility criteria Page 3/15 Case reports/series of proven choroidal metastasis caused by thyroid neoplasms diagnosed were included in this systematic review. Diagnosis of choroidal metastasis should have been based on ophthalmoscopic examination and valid pathologic findings. All types of thyroid neoplasms were included. Poorly described cases, conference abstracts, editorials, review articles, or case series without primary data or where the analysis was pooled without the description of individual patient data were excluded. Studies with unrelated subjects or not evaluating the association of studied parameters were also excluded. excluded. Studies with unrelated subjects or not evaluating the association of studied parameters were also excluded. 2.4. Quality of studies Two authors independently assessed the quality of the studies using the JBI Critical Appraisal Checklist for Case Reports (16). Any inconsistencies between these two reviewers were resolved through discussion with a third investigator. 2.5. Statistical analysis Given the descriptive nature of this review, we used descriptive statistics to report demographics and clinical characteristics, with means and standard deviation for continuous variables and frequencies and percentages for categorical variables. 3.2 Demographic characteristics of the patients The mean (SD) age of patients at the time of UM diagnosis was 56.521 (±15.68) years (range, 20–83 years), and 16 cases (34.78%) were reported in the elderly (˃64 years). Male patients consisted 56.52% (n=26) of the cases. Past ocular problems were reported in only eight cases, three of which had no history of ocular problems. The conditions of five other patients included cataract surgery, keratoconjunctivitis, non-proliferative diabetic retinopathy, retinal venous thrombosis, and recurrent uveitis. A summary of patients’ demographics can be seen in Table 1. 2.3. Data extraction Unreported or missing data were sought through contact with the corresponding authors of the pertaining articles. A standardized excel form was used to extract key data from each study, including first author name, country of publication, year of publication, investigation type, patient demographics, comorbidities, primary eye symptoms, time to develop choroidal metastases after the cancer diagnosis, type of thyroid neoplasm, presence of other systemic metastasis, history of eye diseases, and ocular outcomes. Data extraction was done by one researcher and crosschecked by another. 3.4 Ocular Outcomes Four of 11 studies describing the ocular outcomes before and after intervention for UM reported enucleation. In two cases, VA degraded to light perception and in another to finger counting. However, in three cases, no reduction in VA was reported after the treatment for UM, which was radioiodine therapy (17-19). In another case presented by Fabrini et al., the patient's VA improved after the intervention. Both eyes of this patient were affected and after treatment with episcleral 106Ru plaque brachytherapy, followed by intravitreal injection of bevacizumab a week later, post-treatment VA of both eyes was better than pre-treatment status (20/20 vs. 20/25 in the right eye and 20/40 vs. 20/70 in the left eye)(20). 3.5 Quality assessment findings The quality of the studies was assessed using the JBI Critical Appraisal Checklist for Case Reports (16). Most studies had adequate quality and presented the minimum required information. Quality assessment of individual studies is described in detail in Supplementary File 3. No study was excluded based on the results of the quality appraisal. 3.3 Clinical characteristics of the patients Page 4/15 Among 40 cases that clearly stated the interval between diagnosing uveal metastasis and thyroid neoplasm, seven cases of UM were identified as the first symptom of thyroid neoplasms. More than half of the UM cases (22/40) were diagnosed in less than five years of being diagnosed with thyroid cancer. Although in other cases, it had taken more years for thyroid cancer to metastasize and be identified. Theinterval of diagnosing UM has been reported to be as long as 35 years in one case. Sixteen cases happened in only the right eye, 19 happened only in the left eye, and 11 cases in both eyes. The distribution of involved uveal parts and other characteristics of uveal metastases are detailed in Table 2. In total, 39 cases (84.78%) had choroidal involvement. In 35 patients, decreased visual acuity was reported among the first symptoms of eye complications, and UM was incidentally found in three patients. Retinal detachment was reported in 35 patients. 3.3 Characteristics of the neoplasms and metastases Characteristics of thyroid neoplasms metastasizing to the uveal layer are detailed in Table 3. Papillary carcinoma was the patients' most reported type of thyroid cancer (16/46). In 40 cases that reported concomitant metastases, only one reported UM as the only metastasis. The most frequently reported sites for concomitant metastases in these patients were the lungs (36), bones (26), mediastinum (13), liver (10), skin (10), and brain (8). Of 32 studies describing the location of lesions in the eye, the temporal side of the choroid (12) and posterior pole (10) were the most common locations for metastatic lesions (Table 2). 4. Discussion Page 5/15 Page 5/15 Uveal metastases are the most common intraocular malignancies, and up to 10% of patients with metastatic cancer have intraocular involvement (21, 22). One percent of all uveal metastases originated from TCs, in which the PTC is the most common type (5, 9, 23). In our study, 34% of patients with uveal metastases had PTC, and 32% had FTC. MTC accounts for 5–8% of all TCs (24), while in our study, 21% of patients with uveal metastases had MTC, which means the ratio of MTC in uveal metastases was higher than expected. As life expectancy rises and treatment options improve, the number of patients with intraocular involvement will increase (13). In this study, patients were reported from 20 different countries. Most were reported from European countries and the United States, high-income countries with high facilities and life expectancies. In this study, 56% of patients with uveal metastases as the primary site of the thyroid were males, and the difference between the two genders was trivial. Welch et al. (25) studied 1,111 patients with UM from all primary sites and reported that 36% of patients were males. Furthermore, In a study by d’Abbadie et al. (12) ,123 patients with CM were examined, and 20% of patients with CM with any primary sites were males. This difference between our study and d’Abbadie’s and Welch’s can be due to the different populations of these studies because the most common primary site for CM is the breast, which is more common in females. d’Abbadie et al. (12) reported that 75% of patients have unilateral choroidal metastases, which is three times more common than bilateral metastases; also, we found that 75% (35 patients) had unilateral uveal metastases. In our study, there was no remarkable difference in right or left eye involvement, the reason for which may be the similar hematogenous route from the thyroid to the eyes. Uveal metastases arising from the gastrointestinal tract are more commonly reported in the left eye. This laterality may be due to the more direct pathway from the aorta to the left carotid artery (26). In uveal metastases cases, the ophthalmic symptoms, such as decreased VA, may be prior to other symptoms. In our study, 15% of patients had ophthalmic symptoms prior to the diagnosis of TC. As similar, Welch et al. 5. Limitations And Strengths This study systematically searched for relevant cases with an inclusive search strategy and thoroughly examined the cases to extract findings to inform oncology and ophthalmology practices. However, the findings of this study were limited by the small population and incomplete reporting in many cases. 4. Discussion Similarly, we found that the first symptom of uveal involvement in 82% of patients decreased VA or blurred vision. Therefore, this may be due to the high involvement of the postquatorial region and the frequent development of subretinal fluid in CM. Furthermore, 9 of 13 patients with iris or ciliary body metastases had decreased visual acuity, mainly due to seeding to the anterior chamber or the development of secondary cataracts, as these lesions cause inflammatory symptoms more frequently than choroidal lesions (33, 34). Management of patients with uveal metastases, especially CM, depends on the systemic status and the number and location of the lesions. Treatment options include systemic chemotherapy, immunotherapy, hormone therapy, external beam radiation therapy (EBRT), photodynamic therapy (PDT), brachytherapy with 125I episcleral insertion, and enucleation. The treatment of choice remains EBRT. As with other forms of radiation, it induces DNA damage to the rapidly growing tumor cells (35, 36). Observation may be considered for small peripheral metastatic lesions in patients with poor systemic status. Enucleation is usually considered for blind painful eyes, loss of vision, and persistent symptoms (32, 37). Despite all these treatment options, there are limited data regarding treatment efficacy. 6. Conclusion Even though uveal metastases from thyroid neoplasms are relatively rare, they might still threaten the vision of every patient with thyroid cancer. Therefore, physicians should maintain suspicion when ocular symptoms accompany a neck mass or a history of previous thyroid malignancies. 4. Discussion (25) reported that 7% of patients diagnosed with TCs had ophthalmic symptoms prior to diagnosis. In this study, the mean interval of uveal metastases detection after proven TCs, was seven years; however, the range was too broad and the population too small to generalize the mean. Besic et al. (27), in 2013, in a study on 22 patients with CM of thyroidal origin, reported that the mean interval from diagnosis of TC was 10.4 years. Furthermore, Welch et al. (25) reported that the mean interval of UM from thyroidal origin was 13 years, which is relatively longer. In our study, 84% of patients had choroidal involvement, the most commonly involved part, followed by the iris and ciliary body. Our findings are similar to previous studies (11, 28-30). The bigger share of CM from UM might be due to the rich blood supply to the choroid via the posterior ciliary arteries. This facilitates a greater volume of blood flow and is associated with a greater risk of metastatic emboli Page 6/15 Page 6/15 compared to the anterior ciliary vessels that supply blood to the iris. In addition, the upper temporal and posterior pole were the most common locations for CM. Furthermore, Freedman et al., in a review of 141 eyes, reported that 40% of CM are located in the macular region, which may be due to the different blood flow of the macular area (31). compared to the anterior ciliary vessels that supply blood to the iris. In addition, the upper temporal and posterior pole were the most common locations for CM. Furthermore, Freedman et al., in a review of 141 eyes, reported that 40% of CM are located in the macular region, which may be due to the different blood flow of the macular area (31). CM has been reported to appear as a white or yellow dome-shaped mass associated with subretinal fluid with orange variations in the cases of TCs(32). In our study, in the few cases that reported the ophthalmoscopy findings, the most commonly reported appearance was white, yellow, and orange lesions. In a study by shields et al. (13), among 479 eyes with choroidal metastases, 92% of the CM with any primary sites were located posterior to the equator. On the other hand, Besic et al. (27) reported that patients with CM typically presented with blurred vision and decreased VA in 70-81%. Acknowledgments The authors would like to thank Tabriz University of Medical Sciences for their support. Authors' contributions M.A and H.N directed the project, drafted the manuscript, and developed the theoretical framework. A.J and N.S designed the research and assisted in the interpretation of the findings. M.A and H.N searched the databases, selected the articles, and extracted the data. A.J performed the analysis and interpreted the findings. All authors helped to shape this study with their expertise and feedback. Declarations Page 7/15 Funding This study received no particular grant from any funding agency. 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Ferry AP, Font RL. Carcinoma metastatic to the eye and orbit. I. A clinicopathologic study of 227 ases. Arch Ophthalmol. 1974;92(4):276-86. 0. Celebi AR, Kilavuzoglu AE, Altiparmak UE, Cosar CB, Ozkiris A. Iris metastasis of gastric denocarcinoma. World J Surg Oncol. 2016;14:71. 1. Freedman MI, Folk JC. References Metastatic tumors to the eye and orbit: patient survival and clinical haracteristics. Archives of Ophthalmology. 1987;105(9):1215-9. 27. Besic N, Luznik Z. Choroidal and orbital metastases from thyroid cancer. Thyroid. 2013;23(5):543-51. 28. Shields CL, Welch RJ, Malik K, Acaba-Berrocal LA, Selzer EB, Newman JH, et al. Uveal Metastasis: Clinical Features and Survival Outcome of 2214 Tumors in 1111 Patients Based on Primary Tumor Origin. Middle East Afr J Ophthalmol. 2018;25(2):81-90. 29. Ferry AP, Font RL. Carcinoma metastatic to the eye and orbit. I. A clinicopathologic study of 227 cases. Arch Ophthalmol. 1974;92(4):276-86. 30. Celebi AR, Kilavuzoglu AE, Altiparmak UE, Cosar CB, Ozkiris A. Iris metastasis of gastric adenocarcinoma. World J Surg Oncol. 2016;14:71. 31. Freedman MI, Folk JC. Metastatic tumors to the eye and orbit: patient survival and clinical characteristics. Archives of Ophthalmology. 1987;105(9):1215-9. Page 10/15 Page 10/15 Page 10/15 32. Konstantinidis L, Damato B. Intraocular metastases—a review. The Asia-Pacific Journal of Ophthalmology. 2017;6(2):208-14. 33. Shields JA, Shields CL, Kiratli H, DE POTTER P. Metastatic tumors to the iris in 40 patients. American journal of ophthalmology. 1995;119(4):422-30. 34. Giuliari GP, Sadaka A. Uveal metastatic disease: current and new treatment options (review). Oncol Rep. 2012;27(3):603-7. 35. Kanthan GL, Jayamohan J, Yip D, Conway RM. Management of metastatic carcinoma of the uveal tract: an evidence-based analysis. Clin Exp Ophthalmol. 2007;35(6):553-65. 36. Paul Chan RV, Young LH. Treatment options for metastatic tumors to the choroid. Semin Ophthalmol. 2005;20(4):207-16. 37. Al Hassan MS, El Ansari W, Alater A, Darweesh A, Abdelaal A. Choroidal metastasis as initial presentation of aggressive medullary thyroid carcinoma with widespread mediastinal, brain, pituitary, bone, lung, and liver metastasis: Case report and literature review. International journal of surgery case reports. 2021;87:106419. Tables Table 1. Characteristics of the included case-reports (n=46) Table 1. Characteristics of the included case-reports (n=46) Page 11/15 Page 11/15 Characteristics Quantity (%) Year of publication 1938 1 (2.17%) 1980-89 3 (6.52%) 1990-99 6 (13.04%) 2000-2009 13 (28.26%) 2010-2019 19 (41.30%) 2020-2022 4 (8.70%) Country United States 9 (19.57%) Turkey 4 (8.70%) Tunisia 4 (8.70%) India 4 (8.70%) United Kingdom 4 (8.70%) France 3 (6.52%) Switzerland 2 (4.35%) Spain 2 (4.35%) Italy 2 (4.35%) Germany 2 (4.35%) Slovenia 1 (2.17%) Greece 1 (2.17%) Israel 1 (2.17%) Qatar 1 (2.17%) Romania 1 (2.17%) Denmark 1 (2.17%) Portugal 1 (2.17%) Netherlands 1 (2.17%) Australia 1 (2.17%) Norway 1 (2.17%) Age* ˂25 1 (2.17%) 25-64 29 (63.03%) ˃64 16 (34.78%) Gender Male 26 (56.52%) Female 20 (43.48%) Age at the time of diagnosis of uveal metastasis able 2. Clinical characteristics of uveal metastases in patients (n=46 Table 2. Clinical characteristics of uveal metastases in patients (n=46) Page 12/15 Characteristic Quantity (%) Laterality Left 19 (41.30%) Right 16 (34.78%) Bilateral 11 (25.58%) Involved uveal parts Choroid 33 (71.73%) Iris 5 (10.86%) Choroid and iris 3 (6.52%) Ciliary body 2 (4.34%) Choroid and ciliary body 2 (4.34%) Choroid, iris, and ciliary body 1 (2.17%) Location of the metastasis in the eye Choroid Posterior pole 10 (21.73%) Nasal side 1. Superior nasal 2. Inferior nasal 3. Superior and inferior nasal 6 (13.04%) 2 2 2 Temporal side 1. Superior temporal 2. Inferior temporal 3. Tables Superior and inferior temporal 12 (26.08%) 5 2 5 Diffused 1 (2.17%) Iris Inferior 1 (2.17%) Inferior nasal 1 (2.17%) Superior temporal 1 (2.17%) Not reported 14 (30.43%) Primary ocular symptoms Decreased VA 31 (67.39%) Blurred visual field 3 (6.52%) Incidentally found 3 (6.52%) Pain and decreased VA 2 (4.35%) Light flashes 2 (4.35%) Pain 1 (2.17%) Corneal ulceration (referred for another ocular condition) 1 (2.17%) Decreased VA and altered color vision 1 (2.17%) Decreased VA and diplopia in the right eye 1 (2.17%) Not reported 1 (2.17%) Retinal detachment Yes 24 (52.17%) No 2 (4.35%) Not reported 20 (43 48%) Page 13/15 6-10 10 (21.73%) ˃10 7 (15.21%) Not reported 21 (45.65%) 6-10 10 (21.73%) ˃10 7 (15.21%) Not reported 21 (45.65%) VA: Visual Acuity VA: Visual Acuity al Acuity diameter of the largest lesion was used for determining the lesion size Clinical characteristics of thyroid neoplasms in patients (n=46) Characteristic Quantity (%) Type of thyroid neoplasm Papillary carcinoma 16 (34.78%) Follicular carcinoma 15 (32.60%) Medullary carcinoma 10 (21.73%) squamous cell carcinoma 1 (2.17%) Hürthle cell carcinoma 1 (2.17%) anaplastic thyroid carcinoma 1 (2.17%) insular thyroid neoplasm 1 (2.17%) Not specified 1 (2.17%) Interval of diagnosis of uveal metastasis after thyroid neoplasm (years) prior to diagnosis (as a first symptom) 7 (15.21%) ˂1 6 (13.04%) 1-4 9 (19.56%) 5-9 8 (17.39%) 10-19 7 (15.21%) ˃20 3 (6.52%) Not reported 6 (13.04%) Presence of concomitant metastases Present 39 (84.7%) None 1 (2.17%) Not reported 6 (13.04%) Sites of concomitant metastases lungs 36 (90%) Bones 26 (65%) Mediastinum 13 (32.50%) Liver 10 (25%) Skin 10 (25%) brain 8 (20%) Lymph nodes 6 (15%) metastatic sites could be detected in one person, therefore each percentage o he number of patients in whom that organ was involved divided to the numbe y eter of the largest lesion was used for determining the lesion size Several metastatic sites could be detected in one person, therefore each percentage only reflects the number of patients in whom that organ was involved divided to the number of all cases that reported the presence of other metastatic sites (n=40) Several metastatic sites could be detected in one person, therefore each percentage only reflects the number of patients in whom that organ was involved divided to the number of all cases that reported the presence of other metastatic sites (n=40) Figure 1 The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. Figures Page 14/15 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SF1searchstrategyandexcludedarticlesatthefulltextlevel.docx SF2extrcationtable.xlsx SF3qualtityappraisal46patients.xlsx Page 15/15
https://openalex.org/W2913597780	https://europepmc.org/articles/pmc6373955?pdf=render	English	null	Dynamic stability and stepping strategies of young healthy adults walking on an oscillating treadmill	PloS one	2,019	cc-by	6,662	Dynamic stability and stepping strategies of young healthy adults walking on an oscillating treadmill Tanya OnushkoID1, Timothy BoergerID2, Jacob Van Dehy3, Brian D. Schmit1 1 Department of Biomedical Engineering, Marquette University, Milwaukee, WI, United States of America, 2 Department of Physical Therapy, Marquette University, Milwaukee, WI, United States of America, 3 Leidos, Inc. Reston, VA, United States of America tanya.onushko@marquette.edu * tanya.onushko@marquette.edu Editor: Eric R. Anson, University of Rochester, UNITED STATES Received: August 24, 2018 Accepted: January 29, 2019 Published: February 13, 2019 Copyright: © 2019 Onushko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Onushko T, Boerger T, Van Dehy J, Schmit BD (2019) Dynamic stability and stepping strategies of young healthy adults walking on an oscillating treadmill. PLoS ONE 14(2): e0212207. https://doi.org/10.1371/journal.pone.0212207 Editor: Eric R. Anson, University of Rochester, UNITED STATES Editor: Eric R. Anson, University of Rochester, UNITED STATES Abstract Understanding how people modify their stepping to maintain gait stability may provide infor- mation on fall risk and help to understand strategies used to reduce loss of balance. The pur- pose of this study was to identify the stepping strategies healthy young individuals select to maintain balance while walking on a destabilizing surface in various directions. A treadmill mounted on top of a 6 degree-of-freedom motion base was used to generate support sur- face oscillations in different degrees of freedom and amplitudes. Fifteen healthy young adults (21.3 ± 1.4 years) walked at self-selected speeds while continuous sinusoidal oscilla- tions were imposed to the support surface in a one degree of freedom: rotation or translation in the mediolateral (ML) direction and rotation or translation in the anteroposterior (AP) direction, with each condition repeated at three different amplitudes. We compared step width, length, and frequency and the mean and variability of margin of stability (MoS) during each experimental walking condition with a control condition, in which the support surface was stationary. Subjects chose a common strategy of increasing step width (p < 0.001) and decreasing step length (p = 0.008) while increasing mediolateral MoS (p < 0.001), particu- larly during oscillations that challenged frontal plane control, with rotations of the walking surface producing the greatest changes to stepping. RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * tanya.onushko@marquette.edu Methods Subjects We recruited fifteen young adults (8 males) with average age, height and weight of 21.3 (SD 1.4) years, 1.7 (SD 0.1) m and 68.8 (SD 10.7) kg, respectively. Informed consent was obtained from all participants prior to participating. All experimental procedures were approved by the Institutional Review Board of Marquette University. Walking strategies of healthy young adults in challenging environments destabilizing environments is necessary to understand fundamental stepping strategies (i.e. spatiotemporal gait parameters) used to reduce the loss of balance. Maintaining mediolateral (ML) stability during gait is an active control process. It requires the center of mass (CoM) motion (i.e. velocity and position) stays within the base of support (BoS). Based on theoretical models of walking, adjusting foot placement to control the CoM is an efficient technique for modifying balance while walking [11]. Healthy adults take shorter, quicker, and wider steps to increase the BoS and limit CoM motion when walking in an unsta- ble environment [10, 12, 13]; a strategy that appears to be an efficient technique for stabilizing gait based on theoretical models of walking [14]. Dynamic balance can be assessed using the margin of stability (MoS). The MoS takes into consideration the CoM position and velocity with respect to the BoS, such that increasing the distance between the CoM and BoS theoretically increases dynamic stability [11]. In response to balance perturbations, healthy adults will effectively adjust their stepping to maintain or increase the MoS [10, 12, 15–17]. For example, when walking on a self-paced treadmill, healthy adults chose to alter their step parameters (walking with wider, shorter and more frequent steps) rather than alter gait speed in order to increase the ML MoS [12]. Previous studies have examined different factors that influence MoS, such as visual perturbations [10], perturbation intensity [12], or direction [17]. Yet, no study has evaluated how continuous rotations com- pared to translations of the walking surface affects spatiotemporal gait parameters and ML dynamic stability. In this study, we examined changes in spatiotemporal gait parameters and ML dynamic sta- bility in response to sinusoidal oscillations of different amplitude, type, and direction imposed to the walking surface. We hypothesized that participants would walk with wider, shorter and quicker steps, increased ML MoS, and MoS variability for all imposed oscillations. Specifically, we hypothesized that these changes: 1) would be the largest during ML compared to antero- posterior (AP) oscillations, 2) would be largest during rotations compared to translations of the walking surface, and 3) would scale with increasing oscillation amplitude. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Introduction A better understanding of the adjustments in spatiotemporal gait parameters individuals make to maintain stable gait on different walking surfaces may provide information on fall risk. Walking in challenging environments can increase the risk and concerns for falling causing individuals to adopt a more cautious gait pattern [1–4]. When gait stability is challenged, such as transitioning between different surfaces [5, 6] or walking on irregular surfaces [7–10], indi- viduals will adjust their foot placement to increase stability and decrease fall risk. During daily activities, individuals encounter a wide variety of environments that challenge stability and stepping strategies could vary depending on size and direction of the destabilizing perturba- tion. Determining how people adjust foot placement while walking in a broader range of Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 1 / 12 Equipment Subjects walked on a custom-designed treadmill system (Fig 1A). A Woodway treadmill (Woodway USA Inc., Waukesha, WI, USA) was mounted on a six-axis motion base (Moog Inc., East Aurora, NY, USA) allowing for movement in 6 degrees-of-freedom (Fig 1B). A cus- tom-written LabVIEW program (National Instruments Corp., Austin, TX, USA) was used to control the oscillations of the motion base system. Subjects wore a safety harness during all walking trials, which did not provide weight support. Kinematic data were collected at 120 Hz from a 14-camera infrared motion capture system (NaturalPoint Inc., Corvallis, OR, USA). Subjects wore reflective markers attached to anatomi- cal landmarks of the anterior superior iliac spine bilaterally, sacrum, greater trochanters, medial and lateral knee joints, medial and lateral ankle malleoli, and first and fifth metatarsal heads bilaterally. The locations of the anatomical landmarks were found by manual palpation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 2 / 12 Walking strategies of healthy young adults in challenging environments Fig 1. Experimental equipment. (A) Subjects walked on a treadmill mounted on top of a 6 degree-of-freedom motion base. (B) Examples of the degrees of freedom the motion base can move. Fig 1. Experimental equipment. (A) Subjects walked on a treadmill mounted on top of a 6 degree-of-freedom motion base. (B) Examples of the degrees of freedom the motion base can move. https://doi.org/10.1371/journal.pone.0212207.g001 https://doi.org/10.1371/journal.pone.0212207.g001 Marker clusters, consisting of 3 markers attached to plastic plates, were strapped around the thighs and shanks, and taped to the heels of the shoes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Experimental protocol Familiarization. Prior to beginning the experiment, subjects were familiarized with the equipment. We incrementally increased belt speed until the subject self-reported a comfortable walking speed (mean 0.78 ±0.19 m/s). This speed was used for the experimental trials. Subjects then completed a control walking trial, in which they walked at their comfortable pace for 80 seconds when the motion base was stationary. This walking trial served as our control condi- tion to which experimental trials were compared. Experimental trials. The protocol consisted of thirteen experimental conditions. For twelve conditions, we imposed continuous sinusoidal oscillations (0.12 Hz) to the treadmill in one degree-of-freedom (Fig 1B): Pitch, Roll, ML and AP. We tested a low, medium and high amplitude for each degree-of-freedom: ±5, ±10, and ±15 degrees for Pitch and Roll oscilla- tions, and ±8, ±16.5 and ±25 cm for ML and AP oscillations. The motion base velocity and acceleration are provided in Table 1. Briefly, the same velocity and acceleration were applied during the ML and AP translation trials, and the same rotational velocity and acceleration were applied to the Roll and Pitch trials. Slower accelerations/velocities were selected in order to create challenging, yet achievable walking conditions. The thirteenth condition was a com- bination of Roll, Pitch and Yaw (RPY) oscillations with offset frequencies (0.15, 0.16, 0.17 Hz, respectively) at an amplitude of ±8 degrees. Subjects walked for 80 seconds per trial and were given 60 seconds rest between trials. Experimental conditions were presented in random order. All subjects were able to walk during the experimental trials without falling and without holding onto handrails. 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Walking strategies of healthy young adults in challenging environments Table 1. Velocity and acceleration of the motion base during the single degree-of-freedom sinusoidal oscillation trials. Translation Trials Peak Velocity (m/s) Acceleration (m/s2) 8 cm 0.0418 0.0316 16.5 cm 0.0841 0.0634 25 cm 0.1263 0.0952 Rotation Trials Peak Velocity (˚/s) Peak Acceleration (˚/s2) 5 deg 3.6 2.7 10 deg 7.3 5.5 15 deg 10.9 8.2 https://doi.org/10.1371/journal.pone.0212207.t001 Table 1. Velocity and acceleration of the motion base during the single degree-of-freedom sinusoidal oscillation trials. XCoM ¼ CoM þ C _oM ffiffiffiffiffiffiffiffiffiffi ðl=gÞ p fifififififififififi where CoM and C_oM are position and velocity of CoM, respectively, g is acceleration due to gravity, and l is maximum height of the estimated CoM, which was calculated as 1.34 times trochanteric height. The CoM position was estimated using the pelvic segment [19]. During experimental trials, we corrected the MoS by subtracting the velocity factor of treadmill move- ment, _V motion base ffiffiffiffiffiffiffiffiffiffi ðl=gÞ p from XCoM, where _V motion base was either ML or AP linear or tangen- tial velocity of the treadmill [20]. This maintained consistency when comparing the MoS among trials. We calculated the mean and variability (coefficient of variation (CoV)) of the MoSML during the final 60 seconds of each trial. Increasing MoSML indicates the XCoM is within the BoS increasing dynamic stability. Decreasing or negative MoSML indicates the CoM is nearing or outside of the BoS. The MoS variability was used as an indicator of fall risk– increasing MoS variability suggests increased risk of falling [21]. fifififififififififi where CoM and C_oM are position and velocity of CoM, respectively, g is acceleration due to gravity, and l is maximum height of the estimated CoM, which was calculated as 1.34 times trochanteric height. The CoM position was estimated using the pelvic segment [19]. During experimental trials, we corrected the MoS by subtracting the velocity factor of treadmill move- ment, _V motion base ffiffiffiffiffiffiffiffiffiffi ðl=gÞ p from XCoM, where _V motion base was either ML or AP linear or tangen- tial velocity of the treadmill [20]. This maintained consistency when comparing the MoS among trials. We calculated the mean and variability (coefficient of variation (CoV)) of the MoSML during the final 60 seconds of each trial. Increasing MoSML indicates the XCoM is within the BoS increasing dynamic stability. Decreasing or negative MoSML indicates the CoM is nearing or outside of the BoS. The MoS variability was used as an indicator of fall risk– increasing MoS variability suggests increased risk of falling [21]. Data analysis Kinematic and spatiotemporal parameters. Kinematic data were low-pass filtered using a 4th order Butterworth filter with a cut-off frequency of 6 Hz using Visual 3D (C-Motion Inc., Rockville, MD, USA). We used a local coordinate system to define model segments for the pel- vis, thighs, shanks and feet. Kinematic data were then used to automatically approximate heel strikes, which were defined as times when the distance between the pelvis and heel markers was maximum [18], and the events were verified by visual inspection. Step width was calcu- lated as ML distance between heel markers at moment of heel-contact. We approximated step length as the AP distance between heel markers at moment of heel-contact. Both step width and length were normalized to the subject’s leg length. During Roll, Pitch and RPY trials, step width and length were calculated using local coordinates with respect to the motion base (i.e. the angle of the base was taken into account). Step frequency was calculated as the average of inverse step duration for each step. These analyses were performed for the final 60 seconds of each trial, and were conducted in Matlab (The Mathworks, Natick, MA, USA). Dynamic stability. We calculated the ML margin of stability (MoSML) to quantify dynamic stability in the ML direction using previous methods [11, 13]. The MoSML is the min- imum difference between the extrapolated center of mass (XCoM) and BoS (MoSML = BoS – XCoMML) for each step. The lateral malleolus marker defined the lateral boundary of the BoS. The XCoM was calculated using:fifififififififififi Results Subjects made significant changes to foot placement during walking surface oscillations (Fig 2). We observed a main effect of walking surface oscillation for step frequency (F13,182 = 8.66, p < 0.001, ηp 2 = 0.382), step width (F13,182 = 16.24, p < 0.001, ηp 2 = 0.537), and step length (F13,182 = 32.46, p = 0.008, ηp 2 = 0.699). Subjects took quicker steps during RPY (p = 0.017), Pitch (low: p = 0.001; medium: p < 0.001; high: p = 0.013), Roll (medium: p = 0.002; high: p = 0.001), and ML oscillations (low: p = 0.001; medium: p < 0.001. respectively) compared to control walking. Subjects also increased step width for RPY (p < 0.001), Roll (medium: p < 0.001; high: p = 0.001), and ML oscillations (medium: p = 0.003). For all oscillating condi- tions, subjects walked with significantly shorter steps compared with the control condition (p < 0.001 for all comparisons, except: medium Roll, p = 0.007 and low AP, p = 0.007). Subjects walked with larger MoSML during the oscillations than the control condition (F13,182 = 15.2, p < 0.001, ηp 2 = 0.521; Fig 3A). Simple contrasts revealed that MoSML was greater for RPY, high Pitch, Roll and ML oscillations compared with the control condition (p < 0.001, p = 0.001, p = 0.001, p < 0.001, respectively). Subjects also exhibited greater MoSML variability during walking surface oscillations (Fig 3B). We found a main effect of walking surface oscillation on MoSML (F13,128 = 4.62, p = 0.006, ηp 2 = 0.213) variability. Sub- jects walked with greater MoSML variability during RPY (p < 0.001), high Roll (p < 0.001), low ML (p < 0.001) and medium (p = 0.004) and high (p = 0.009) AP oscillations. We also compared Type (Rotation vs. Translation), Direction (ML vs. AP) and Amplitude (low, medium and high) on stepping strategies and dynamic stability. The statistical results are reported in Table 2. We observed a main effect of Type and Amplitude for step width, length and frequency (p < 0.05). Subjects walked with wider, shorter and quicker steps during rotat- ing oscillations (Pitch and Roll) compared to translating oscillations (ML and AP). These parameters also scaled with the oscillation amplitude. High amplitude oscillations resulted in the widest, shortest and quickest steps, and vice versa. We only observed a main effect of Direction on the step width. Statistics A one-way repeated measures ANOVA was conducted to compare the effect of walking sur- face oscillation on step frequency, step width, and step length and mean MoSML and CoV of PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 4 / 12 Walking strategies of healthy young adults in challenging environments MoSML. The Greenhouse-Geisser correction factor was used when Mauchly’s test of sphericity was violated. When a significant effect of walking surface oscillation was found, simple con- trasts were conducted to test whether the dynamic walking condition differed from the control walking condition. We also conducted a multi-factor repeated measures ANOVA to compare oscillation Type (Rotation, Translation), Direction (AP, ML) and Amplitude (low, medium, high) on the dependent variables. For this analysis, we excluded the Control and RPY condi- tions. All statistics were performed using IBM SPSS Statistics 21.0 (IBM, New York, NY, USA). The α level was set at 0.05 and a Bonferroni correction (α = 0.0038) was applied for post-hoc multiple comparisons. Data are reported as means (SD). Only significant main effects and contrasts are presented, unless otherwise noted. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Results Subjects took wider steps during the ML directions (Roll and ML) compared to the AP directions (Pitch and AP). The interaction of Type x Direction and Type x Amplitude were significant for step length and frequency only. Post-hoc comparisons for Type x Direction interaction showed that subjects walked with shorter and quicker steps for the rotation oscillations compared to the translation oscillations, particularly for Pitch. Simi- larly, post-hoc comparisons for Type x Amplitude interaction showed subjects walked with shorter and quicker steps during rotation oscillations compared to translation oscillations at each amplitude (p < 0.05 for each comparison). For dynamic stability, we observed significant main effects of Type and Amplitude on the MoSML. Subjects walked with greater MoSML during rotation oscillations (Pitch and Roll) compared to translation oscillations. The MoSML also scaled with the oscillation amplitude– PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 5 / 12 the MoSML increased with increasing oscillation amplitude. We observed significant interac- tion effects between Type and Direction on MoS. Post-hoc comparisons showed a larger M S d i i i h AP d ML di i d l i i AP ML Fig 2. Step parameters. Step frequency (A), step width (B) and step length (C) for all walking conditions are shown for following conditions: control walking condition (C), roll, pitch, yaw combination (RPY), pitch (PL, PM, PH), roll (RL, RM, RH), mediolateral (MLL, MLM, MLH) and anteroposterior (APL, APM, APH) oscillations at low (L), medium (M) and high (H) amplitudes. Asterisks represent significant difference (p < 0.05) with respect to the control condition. https://doi.org/10.1371/journal.pone.0212207.g002 Walking strategies of healthy young adults in challenging environments Walking strategies of healthy young adults in challenging environments Fig 2. Step parameters. Step frequency (A), step width (B) and step length (C) for all walking conditions are shown for following conditions: control walking condition (C), roll, pitch, yaw combination (RPY), pitch (PL, PM, PH), roll (RL, RM, RH), mediolateral (MLL, MLM, MLH) and anteroposterior (APL, APM, APH) oscillations at low (L), medium (M) and high (H) amplitudes. Asterisks represent significant difference (p < 0.05) with respect to the control condition. Fig 2. Step parameters. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Results Step frequency (A), step width (B) and step length (C) for all walking conditions are shown for following conditions: control walking condition (C), roll, pitch, yaw combination (RPY), pitch (PL, PM, PH), roll (RL, RM, RH), mediolateral (MLL, MLM, MLH) and anteroposterior (APL, APM, APH) oscillations at low (L), medium (M) and high (H) amplitudes. Asterisks represent significant difference (p < 0.05) with respect to the control condition. https://doi.org/10.1371/journal.pone.0212207.g002 https://doi.org/10.1371/journal.pone.0212207.g002 the MoSML increased with increasing oscillation amplitude. We observed significant interac- tion effects between Type and Direction on MoS. Post-hoc comparisons showed a larger MoSML during rotation in the AP and ML-directions compared to translation in AP or ML (p < 0.05 for both comparisons). Lastly, we also observed a significant interaction between PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 6 / 12 Walking strategies of healthy young adults in challenging environments Type and Amplitude on the MoS. Post-hoc comparisons showed a larger MoSML during rota- tion oscillations at each amplitude compared to the translation oscillations (p < 0.05 for all comparisons). Fig 3. Dynamic stability. The mean MoSML (A) and the coefficient of variation of MoSML (B) are shown for following conditions: control walking condition (C), roll, pitch, yaw combination (RPY), pitch (PL, PM, PH), roll (RL, RM, RH), mediolateral (MLL, MLM, MLH) and anteroposterior (APL, APM, APH) oscillations at low (L), medium (M) and high (H) amplitudes. Asterisks represent significant difference (p < 0.05) with respect to the control condition. https://doi.org/10.1371/journal.pone.0212207.g003 Fig 3. Dynamic stability. The mean MoSML (A) and the coefficient of variation of MoSML (B) are shown for following conditions: control walking condition (C), roll, pitch, yaw combination (RPY), pitch (PL, PM, PH), roll (RL, RM, RH), mediolateral (MLL, MLM, MLH) and anteroposterior (APL, APM, APH) oscillations at low (L), medium (M) and high (H) amplitudes. Asterisks represent significant difference (p < 0.05) with respect to the control condition. Type and Amplitude on the MoS. Post-hoc comparisons showed a larger MoSML during rota- tion oscillations at each amplitude compared to the translation oscillations (p < 0.05 for all comparisons). Table 2. Statistical results. Significance accepted at p < 0.05 and are indicated in bold font. https://doi.org/10.1371/journal.pone.0212207.t002 Significance accepted at p < 0.05 and are indicated in bold font. s://doi.org/10.1371/journal.pone.0212207.t002 Discussion The aim of this study was to determine how continuous oscillations of the walking surface at differing amplitudes, types and directions affects stepping strategies and dynamic balance in healthy young adults. Compared to level walking, healthy adults took wider steps particularly for trials in the ML direction (Roll and ML translation). The oscillation amplitude and type (rotation trials Roll and Pitch) applied to the walking surface resulted in an increase in step fre- quency and decrease in step lengths, with rotations applied in the AP direction (Pitch) result- ing in the greatest change in step length and frequency. Dynamic stability was also dependent on the type of movement. Rotation oscillations caused the largest increase in ML MoS com- pared to the translation oscillations. As reported in similar studies, the observed stepping adjustments in response to different walking surface oscillations suggest that this is a natural strategy to increase dynamic stability, and potentially reduce loss of balance [10, 12, 13, 15, 17, 22]. From the current study, the increase in ML MoS and its relationship to loss of balance may be interpreted in several ways. First, an increased dynamic stability during the oscillating conditions suggests the participants were more stable, and thus, less likely to fall, especially during the more challenging walking conditions (i.e. RPY, RollH). Participants walked with wider steps to increase the distance between the XCoM and BoS thereby increasing ML MoS compared to the control walking condition. A second interpretation is that young healthy adults walked with wider steps to increase ML MoS, but their risk for falls may still be the same as the control walking condition. In this latter scenario, the walking environment itself imposes a challenge to balance and healthy adults naturally will adjust their stepping to maintain dynamic stability. Another potential interpretation may be that subjects favored control of frontal plane bal- ance over sagittal plane balance. Shortening step length can also be regarded as a strategy to decrease risk of falling when walking in unstable conditions [23]. For example, shortening step length will move the CoM near or even in front of the BoS (i.e. in front of the leading foot), which will reduce a chance of backwards loss of balance but at the cost of potentially inducing a forward loss of balance [12, 24, 25]. Results Dependent Measure Type Direction Amplitude Type x Direction Type x Amplitude Direction x Amplitude Step width F = 5.2 p = 0.038 ηp 2 = 0.27 F = 13.3 p = 0.003 ηp 2 = 0.48 F = 7.8 p = 0.002 ηp 2 = 0.36 F = 0.95 p = 0.347 ηp 2 = 0.06 F = 0.27 p = 0.769 ηp 2 = 0.02 F = 1.89 p = 0.169 ηp 2 = 0.12 Step length F = 28.4 p < 0.001 ηp 2 = 0.67 F = 3.36 p = 0.088 ηp 2 = 0.19 F = 58.7 p < 0.001 ηp 2 = 0.81 F = 32.2 p < 0.001 ηp 2 = 0.72 F = 17.8 p < 0.001 ηp 2 = 0.56 F = 1.4 p = 0.270 ηp 2 = 0.09 Step frequency F = 17.0 p = 0.001 ηp 2 = 0.55 F = 2.59 p = 0.130 ηp 2 = 0.16 F = 25.9 p < 0.001 ηp 2 = 0.65 F = 16.8 p = 0.001 ηp 2 = 0.55 F = 8.1 p = 0.002 ηp 2 = 0.37 F = 0.02 p = 0.979 ηp 2 = 0.01 MoSML F = 32.4 p < 0.001 ηp 2 = 0.70 F = 0.04 p = 0.837 ηp 2 = 0.01 F = 13.4 p < 0.001 ηp 2 = 0.49 F = 18.3 p = 0.001 ηp 2 = 0.57 F = 7.4 p = 0.003 ηp 2 = 0.35 F = 1.7 p = 0.209 ηp 2 = 0.11 Significance accepted at p < 0.05 and are indicated in bold font. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 7 / 12 Walking strategies of healthy young adults in challenging environments PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Discussion In the current study, we observed decreased step length, with the shortest step length occurring in the Rotation conditions at high amplitude. Together with taking wider steps, taking shorter steps could imply that subjects are still at risk for loss of balance in the sagittal plane in order to favor better frontal plane control. However, we cannot verify this from the current data because the XCoM model for AP stability does not consider angular momentum, which can influence AP stability, misrepresenting true stability in the sag- ittal plane during up/down hill walking [26]. Furthermore, the changes in knee and hip angles during the Pitch trials affect joint power and stiffness, which violates the assumption that the inverted pendulum model is a passive system [27]. Further work is needed to define the rela- tionship between ML MoS, AP MoS and fall risk when walking on challenging environments. One major finding is that subjects made greater adjustments to foot placement during rotating oscillations. We observed increased step width during the Roll oscillations, and decreased step length and increased step frequency during the Pitch oscillations. Furthermore, these stepping adjustments were more pronounced during the rotating oscillations compared to the translation oscillations for each oscillation amplitude (i.e. low, medium and high). Sloped walking requires a person to alter their ground reaction forces, and therefore joint moments, to maintain or increase stability. For example, during side-sloped walking, subjects will decrease hip joint moments but increase moment about the knee and ankle to keep the CoM stable [28]. While our results are similar to previous studies investigating sloped walking [29–31], it is important to note that we cannot make direct comparisons of the oscillation amplitudes between rotating and translating conditions– 15 degrees of rotation may not have 8 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Walking strategies of healthy young adults in challenging environments the same influence on gait parameters and dynamic stability as 9.8 cm of translation. However, from observation, subjects largely walked in the center of the treadmill, near the rotational axes. This implies this area would have the lowest rotational velocity and acceleration imposed to the person from the motion base. Walking under continuous sinusoidal oscillations of the walking surface increased MoSML variability. Discussion The observed increase in MoSML variability may indicate that imposing sinusoidal oscillations of the walking surface in different directions increases the risk for falling for healthy young adults [21]. While a greater MoS when walking can be considered an indicator of dynamic stability [11], MoS variability may be related to how well people control CoM and foot placement when stability is challenged [16, 32]. In our study, subjects maintained suffi- cient MoSML when walking during the oscillations, even though they exhibited greater MoSML variability. This could suggest that they were able to quickly adjust their gait and keep their bal- ance while walking in a destabilizing environment. Conversely, sinusoidal oscillations of the walking surface could have increased MoS variability while participants remained dynamically stable. For example, walking on a laterally oscillating treadmill, healthy adults were shown to alter their stepping at particular motions of the walking surface resulting in increased step width variability [21]. Methodological considerations Sinusoidal oscillations created a predictable walking surface that could encourage subjects to time their stepping with the oscillations (i.e. to entrain). Two factors, however, reduce the like- lihood of entrainment. First, the average step frequency (1.6 Hz for the control walking condi- tion) was much faster than the imposed sinusoidal frequency (0.12 Hz). Previous studies have shown that dynamic entrainment largely occurs if the period of the perturbation is close to the individual’s preferred cadence [33–35]. Second, rhythmic cueing reduces variability of step- ping [36, 37] and we observed increasing levels of stepping variability during the destabilizing conditions. Walking on elevated surfaces can increase postural threat and raise concern for falling lead- ing to altered gait [38–41]. Walking on elevated surfaces increases the time spent in double support phase, reduces step length, velocity and cadence [38–40]. In our study, subjects walked ~100 cm above the ground. Walking at this height could have increased their fear, causing sub- jects to walk with a “cautious” gait pattern, and may explain why we observed a slower than normal walking speed for healthy young adults. Despite this, our subjects continued to exhibit changes in stepping similar to previous studies [10, 12, 13]. Clinical implications These results enhance our knowledge on how healthy individuals modify their gait in challeng- ing walking conditions and are useful for future studies investigating dynamic balance in bal- ance-impaired individuals. Among clinical populations, walking over uneven terrain increases fall risk and limits mobility [8, 9, 16]. Quantifying dynamic balance of high-fall risk individuals in challenging walking conditions can tell us what factors are important in managing fall risk and how to address rehabilitation concerns. Findings from our study can be used to compare how balance-impaired individuals compensate for the same walking conditions or whether increasing the challenge imposed to balance affects balance-impaired individuals similarly. Project administration: Tanya Onushko Project administration: Tanya Onushko. Software: Tanya Onushko, Jacob Van Dehy. Software: Tanya Onushko, Jacob Van Dehy. Supervision: Brian D. Schmit. Visualization: Tanya Onushko. Writing – original draft: Tanya Onushko. Writing – original draft: Tanya Onushko. Writing – review & editing: Tanya Onushko, Timothy Boerger, Jacob Van Dehy, Brian D. Schmit. Conclusion This study demonstrates that healthy young adults adjust their gait pattern to increase dynamic stability when walking over destabilizing surfaces. Subjects took quicker, shorter and wider 9 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 Walking strategies of healthy young adults in challenging environments steps to increase dynamic stability when oscillations were imposed to the walking surface and were largely influenced by rotations and amplitude of oscillation. These results suggest that healthy young adults use a generalized gait strategy to maintain dynamic stability when their balance is challenged. steps to increase dynamic stability when oscillations were imposed to the walking surface and were largely influenced by rotations and amplitude of oscillation. These results suggest that healthy young adults use a generalized gait strategy to maintain dynamic stability when their balance is challenged. Author Contributions Author Contributions Conceptualization: Tanya Onushko, Brian D. Schmit. Data curation: Tanya Onushko. Formal analysis: Tanya Onushko. Investigation: Tanya Onushko, Timothy Boerger, Jacob Van Dehy. Methodology: Tanya Onushko. Project administration: Tanya Onushko. Software: Tanya Onushko, Jacob Van Dehy. Supervision: Brian D. Schmit. Visualization: Tanya Onushko. Writing – original draft: Tanya Onushko. Writing – review & editing: Tanya Onushko, Timothy Boerger, Jacob Van Dehy, Brian D. Schmit. Conceptualization: Tanya Onushko, Brian D. Schmit. Project administration: Tanya Onushko. Project administration: Tanya Onushko. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212207 February 13, 2019 References 1. Winter DA, Patla AE, Frank JS, Walt SE. Biomechanical walking pattern changes in the fit and healthy elderly. Phys Ther. 1990; 70(6):340–7. Epub 1990/06/01. PMID: 2345777. 2. Menz HB, Lord SR, Fitzpatrick RC. Age-related differences in walking stability. Age Ageing. 2003; 32 (2):137–42. Epub 2003/03/05. PMID: 12615555. 3. Terrier P, Reynard F. 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W2623537605.txt	https://online.unisc.br/seer/index.php/redes/article/download/9352/pdf	en		Extensão Rural Agroecológica: experiências e limites	DOAJ (DOAJ: Directory of Open Access Journals)	2,017	cc-by	10,658	DOI: 10.17058/redes.v22i2.9352 Extensão Rural Agroecológica: experiências e limites FRANCISCO ROBERTO CAPORAL Professor Adjunto da Universidade Federal Rural de Pernambuco OLIVO DAMBRÓS Universidade Tecnológica Federal do Paraná RESUMO O artigo inicia com abordagem teórica sobre a construção de uma proposta de Extensão Rural Agroecológica (ERA), em seguida faz link entre os aspectos teóricos da ERA e dados empíricos de uma ATER em rede, analisando nove projetos de Acompanhamento Técnico e Extensão Rural desenvolvidos junto a agricultores familiares e assentados da reforma agrária no estado do Paraná. Nesta análise, faz-se um breve histórico sobre o modelo de Extensão Rural até a nova ATER no que se refere à participação das organizações sociais e descreve sucintamente os nove projetos/experiências ressaltando aspectos, como: relação projeto e processo; gestão e organização; formação de equipes de ATER e instrumentos metodológicos utilizados na transição agroecológica dos sistemas familiares, grupais e de comunidades rurais. Por fim, apresenta alguns impactos das ações executadas e conclui destacando avanços e limites para desenvolver uma proposta teórica de Extensão Rural Agroecológica. Palavras-chave: Extensão Rural Agroecológica. Extensão Rural. Agroecologia. AGROECOLOGICAL RURAL EXTENSION: EXPERIENCES AND LIMITS ABSTRACT The article begins with a theoretical approach on the construction of a proposal of Rural Agroecological Extension (ERA), then links the theoretical aspects of the ERA and empirical data of a network ATER, analyzing nine projects of Technical Accompaniment and Rural Extension developed together to family farmers and settlers of agrarian reform in the state of Paraná. In this analysis, a brief history is made of the Rural Extension model to the new ATER with regard to the participation of social organizations and briefly describes the nine projects / experiences highlighting aspects such as: project and process relationship; Management and organization; Training of ATER teams and methodological tools used in the agroecological transition of family, group and rural community systems. Finally, it presents some impacts of the executed actions and concludes highlighting advances and limits to develop a theoretical proposal of Agroecological Rural Extension. Keywords: Agroecological Rural Extension. Rural Extension. Agroecology. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 275 Francisco Roberto Caporal e Olivo Dambrós EXTENSIÓN RURAL AGROECOLÓGICA: EXPERIENCIAS Y LÍMITES RESUMEN El artículo comienza con una aproximación teórica a la construcción de una propuesta de Extensión Rural Agroecológica (ERA), y a continuación, hace el vínculo entre los aspectos teóricos de la ERA y los datos empíricos de una ATER en red, a partir del análisis de nueve proyectos de asesoría técnica y extensión rural desarrollados en conjunto con los agricultores familiares y asentados de la reforma agraria en el estado de Paraná. En este análisis, se hace una breve historia del modelo de Extensión Rural hacia la nueva ATER en lo que respecta a la participación de las organizaciones sociales y describe los nueve proyectos / experiencias destacando aspectos tales como: relación entre el proceso y el proyecto; gestión y organización; capacitación de los equipos de ATER e instrumentos metodológicos utilizados en la transición agroecológica de los sistemas de producción familiares o grupales y los sistemas de las comunidades rurales. Finalmente, se presentan algunas repercusiones de las acciones y concluye destacando los avances y límites para desarrollar una propuesta teórica de Extensión Rural Agroecológica. Palabras clave: Extensión Rural Agroecológica. Extensión Rural. Agroecología. 1 INTRODUÇÃO O presente artigo nasce de pesquisas realizadas pelos autores e que foram materializadas em suas teses de doutorado, defendidas, respectivamente em 1998 e em 2014. O texto procura colocar um pouco de luz sobre a proposta teórica de Extensão Rural Agroecológica (ERA) em contraponto à Extensão Rural Convencional que vem sendo praticada no Brasil desde os anos 40 e como esta proposta teórica se concretiza na ação prática da extensão rural, o que se analisa a partir de uma pesquisa realizada no estado do Paraná, cujas experiências estudadas podem ser extrapoladas para a realidade brasileira. Desde os anos 60 do século XX, a partir da obra Comunicação ou Extensão? (FREIRE, 2006), a extensão rural institucionalizada no Brasil, vem sendo objeto de críticas, tanto por se tratar de uma prática autoritária de transferência de tecnologias, como por ser uma ação acrítica frente às realidades socioculturais, ambientais e econômicas dos grupos sociais com quem atuava. Ademais, o modelo de intervenção extensionista priorizou suas orientações a partir da base técnica que conformou os chamados pacotes tecnológicos da Revolução Verde. De fato, o que se viu ao longo dos últimos 60 anos foi uma ação extensionista difusionista e excludente, que contribuiu, decisivamente, para a construção da agricultura industrial capitalista, enquanto deixou à margem uma expressiva população rural que se via impossibilitada de adotar as tecnologias recomendadas pelos extensionistas rurais ou, mesmo, impedida de ter acesso ao crédito rural que foi instituído como instrumento de apoio à adoção das tecnologias. Neste segmento de excluídos, encontram-se não só os agricultores de pequeno porte, como também os quilombolas, os indígenas, os ribeirinhos e tantas outras categorias sociais mais empobrecidas do meio rural. Ao longo do período, a extensão rural brasileira tornou-se alvo de recorrentes críticas à prática extensionista. Entre elas, aquelas que buscavam evidenciar os problemas socioambientais decorrentes da difusão e adoção dos pacotes tecnológicos caraterizados pelos monocultivos baseados no uso de Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 276 Extensão Rural Agroecológica: experiências e limites sementes melhoradas, híbridas e, finalmente, transgênicas, além da mecanização pesada, da irrigação irresponsável e mal conduzida e do uso intensivo de fertilizantes químicos e de agrotóxicos. Na produção animal não foi diferente, saímos de modelos de produção extensiva, para dar suporte a diferentes pacotes de produção intensiva em larga escala, com o uso de raças selecionadas para diferentes finalidades o que implicou no uso crescente de rações (com grande uso de milho e soja) e de toda uma bateria de medicamentos como os anabolizantes, hormônicos, antibióticos, etc. Em ambos os casos gerou-se uma intensa subordinação e dependência dos agricultores adotantes das “novas” tecnologias e formas de manejos ao setor agroindustrial. Portanto, a agropecuária que se tornou hegemônica com o apoio técnico da extensão rural (e, obviamente, da pesquisa agropecuária e do ensino das ciências agrárias) pronto se mostrou insustentável do ponto de vista social e ambiental, sendo uma das causas do êxodo rural massivo ocorrido nos anos 60 e 70, assim como pela diferenciação social hoje presente no campo. Ao mesmo tempo, o modelo de produção agropecuária que se fez dominante trouxe consigo um enorme leque de externalidades negativas do ponto de vista ambiental, como: redução da biodiversidade, extinção de espécies e variedades de plantas e raças de animais, deterioração dos solos agrícolas (erosão, salinização, desertificação), assim como a contaminação das águas superficiais e subterrâneas, do ar e da comida produzida mediante o modelo tecnológico da Revolução Verde. Todos os aspectos acima mencionados levaram à introdução de mudanças no discurso extensionista que passou a incorporar a necessidade de mudanças de paradigma, não só quanto à forma de intervenção, cobrando a mudança do modelo difusionista para uma forma de intervenção mais democrática e participativa que incluísse o respeito aos conhecimentos locais (indígenas, tradicionais), como, também, na base tecnológica levada ao campo, reclamando por tecnologias menos agressivas ao meio ambiente, ecologicamente responsáveis, cultural e socialmente adaptadas, economicamente viáveis para os setores que ficaram à margem da modernização tecnológica e adequadas às especificidades edafoclimáticas dos diferentes ecossistemas e agroecossistemas. Por outro lado, cabe ressaltar que a partir do anos 1980 a extensão rural se vê enfrentada ao movimento que defendia as agriculturas alternativas que defendiam uma agricultura mais limpa, sem o uso de fertilizantes químicos de síntese e agrotóxicos. Todas as evidências dos problemas antes enunciados e das demandas postas por setores da sociedade encontraram eco no sistema brasileiro de extensão rural de modo que em meados dos anos 80 nasceu um movimento chamado de “repensar da extensão rural” que envolveu não só organizações de representação de agricultores e movimentos sociais, como também a própria entidade de coordenação nacional, à época a Empresa Brasileira de Assistência Técnica e Extensão Rural (Embrater). Ficava evidente que o extensionismo rural estatal havia acusado o golpe e reconhecia a necessidade de mudanças em suas práticas. Não obstante, a extinção da Embrater, em 1990, colocou por terra não só o esforço de democratização das formas de intervenção e de mudanças técnicas que a entidade vinha capitaneando, como desarticulou o sistema de extensão na medida em que cada estado da federação passou a estabelecer suas próprias diretrizes, o que, na maioria das vezes, contribuiu para manter em nível dos estados o modelo Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 277 Francisco Roberto Caporal e Olivo Dambrós transferencista que vinha sendo criticado, embora os discursos oficiais das entidades públicas de Ater evidenciassem algumas expressões incorporadas a partir dos debates anteriores aos anos 1990, tais como: desenvolvimento sustentável, proteção ao meio ambiente, metodologias participativas, foco na agricultura familiar, entre outros discursos que apontavam ara mudanças na sua prática. De todo modo, as mudanças demandadas ao aparato extensionista estavam longe de se concretizar, por diferentes razões, entre as quais, as posições ideológicas dominantes dentro das instituições públicas de extensão rural, notadamente conservadoras, bem como as resistências dos próprios extensionistas formados a partir dos cânones da Revolução Verde (CAPORAL, 1991). Retomando o debate em torno das mudanças necessárias para que a extensão respondesse positivamente ao que vinha sendo demandado pela sociedade, foi publicado, em 1994, um artigo intitulado Por uma Nova Extensão Rural: fugindo da obsolescência (CAPORAL; COSTA BEBER, 1994). Nele se fazia um alerta: “Não podemos nos deixar levar pelo imobilismo conservador que continua aprisionando as organizações públicas de extensão rural.” (CAPORAL; COSTA BEBER, 1994, p.70) E se enfatizava que era necessário que a ATER enfrentasse os desafios socioambientais presentes no campo, e, para isso, a extensão rural deveria “revolucionar suas bases teórico-conceituais e seus referenciais metodológicos”, fugindo do modelo convencional, já que para enfrentar estes novos desafios não basta que os extensionistas sejam bons difusores de tecnologias, mas exige que sejam bons mediadores, articuladores e que suas intervenções sejam mais democráticas, dialógicas e participativas. Nasceria daí a noção de Extensão Rural Agroecológica (ERA), que foi construída a partir da compreensão de que ante os desafios do desenvolvimento sustentável, os aparatos públicos de extensão terão que transformar sua prática convencional para que possam atender às novas exigências da sociedade. A crise socioambiental, gerada pelos estilos convencionais de desenvolvimento rural e de extensão, recomendava uma clara ruptura com o modelo extensionista baseado na Teoria da Difusão de Inovações e nos tradicionais pacotes da Revolução Verde. Assim, a proposta teórica da ERA foi construída tomando como referência as exigências de uma ação diferenciada de Extensão Rural, que fosse social e ambientalmente responsável (CAPORAL, 1998)1. 1 Embora hoje muita gente fale e escreva sobre Extensão Rural Agroecológica, sem falsa modéstia, é preciso lembrar que a construção conceitual da ERA foi iniciada pelo autor citado, em 1994 e se consolidou no capítulo VIII de sua tese de doutorado, em 1998. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 278 Extensão Rural Agroecológica: experiências e limites Concretamente, vai ser somente em 2003 que a Agroecologia como ciência norteadora das ações extensionistas vai aparecer em uma política do governo federal, a Política Nacional de Assistência Técnica e Extensão Rural (Pnater).2 Posta em marcha em 2004, a Pnater partia das críticas históricas à extensão rural brasileira e incorporava um novo arsenal teórico e metodológico, dando consistência à orientação das ações extensionistas com base em uma política pública. Era a hora de levar à prática a proposta teórica da Extensão Rural Agroecológica (BRASIL, 2004). Não obstante os esforços no sentido da sua efetivação na prática extensionista, pronto evidenciou-se que havia obstáculos objetivos que dificultavam ou impediam as mudanças nas práticas das instituições públicas de extensão rural. Em um primeiro balanço, evidenciou-se que havia uma força de “inercia” e uma reação interna às instituições, fruto de questões estruturais, políticas, ideológicas e mesmo de formação dos extensionistas que impediam as mudanças. (CAPORAL; RAMOS, 2006). Investigações posteriores confirmaram esta tendência conservadora. (CAPORAL, 2014). Ademais, para evidenciar empiricamente as dificuldades de implementação da ERA no cotidiano das ações extensionistas foi realizada uma pesquisa no estado do Paraná a qual trouxe evidências sobre os avanços e dificuldades presentes na vida real para colocar na prática das entidades e dos extensionistas o conjunto de recomendações da Pnater, em especial a partir do ano de 2010 quando entrou em vigor a Lei nº 12.188 conhecida como Lei de Ater (BRASIL, 2010). Assim, neste artigo, além de uma abordagem teórica sobre a proposta de uma Extensão Rural Agroecológica, os autores dedicam uma atenção especial para a análise de experiências concretas de extensão rural levadas à prática em diferentes territórios, evidenciando aspectos positivos e limites objetivos para se por em marcha um modelo de extensão rural diferenciado do convencional, mais democrático, dialógico e participativo e tecnicamente orientado pela Agroecologia. 2 Cabe lembrar que, entre 1999 e 2002, a Empresa de Ater do Rio Grande do Sul, ASCAR/EMATER-RS, desenvolveu um grande esforço no sentido de implementar a proposta da ERA na extensão rural daquele estado, tendo obtido avanços significativos, dentre os quais vale destacar os Seminários de Agroecologia que foram o embrião para a criação da Associação Brasileira de Agroecologia. 2 ALGUNS ELEMENTOS TEÓRICOS PARA UMA EXTENSÃO RURAL AGROECOLÓGICA Como visto antes, a proposta teórica da Extensão Rural Agroecológica nasce em um ambiente de mudanças no qual se destaca o paradigma ecológico como um imperativo para os modelos de desenvolvimento rural e da agricultura. Neste contexto, um dos elementos centrais passou a ser a Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 279 Francisco Roberto Caporal e Olivo Dambrós busca pela ecologização da agricultura. Esse processo, que foi considerado como sendo a “segunda transição agroecológica” do século XX, se caracteriza, entre outras coisas, pela “politização ecológica”, tanto da agricultura como dos alimentos, de modo que “os movimentos ecologistas e afins passam a exercer uma influência crescente sobre as políticas agrárias e alimentares”. (BUTTEL, 1995, p.11) Neste marco, nasceu a proposta de desenvolvimento sustentável, expressão que até hoje não logrou alcançar consenso sobre o que é e como se pode chegar a este ideal de sustentabilidade socioambiental. Neste sentido, existem várias tendências e discursos em disputa, cada uma das quais propondo, desde sua posição teórica e ideológica, a perspectiva de “outro estilo de desenvolvimento”, que surge mais como adaptação ou oposição aos modelos convencionais que, propriamente, como uma concepção teórica acabada que pudesse superar a anterior. Entre as correntes de pensamento sobre a sustentabilidade na agricultura é possível destacar duas que, em nossa opinião, se constituem nos extremos de um processo de ecologização agrícola. Ambas querem garantir “nosso futuro comum” neste planeta finito, ainda que para isso apresentem diferentes alternativas. Por um lado, encontramos a corrente liberal, “oficialista”, que está preocupada com a manutenção e incremento das taxas de crescimento econômico e da produção e produtividade agrícola, subordinando a estas variáveis os aspectos ambientais e sociais do desenvolvimento, que ficam em segundo plano. Trata-se da chamada alternativa “ecotecnocrática” do desenvolvimento sustentável, a qual se faz operativa, quanto ao desenvolvimento agrícola, mediante a proposição de um processo de intensificação verde (SEVILLA GUZMÁN; ALONSO MIELGO, 1995). A outra corrente que se pode destacar, parte de una visão crítica e defende que o desenvolvimento para ser sustentável deve atender, entre outros requisitos, os de equidade social, produtividade, estabilidade e sustentabilidade ambiental. Neste caso, aparecem como aspectos fundamentais para esta corrente de pensamento, as noções de variabilidade espacial dos agroecosistemas; de coevolução do homem com seu meio ambiente; de reconhecimento das diferentes estruturas culturais; da importância da biodiversidade; assim como a necessidade da distribuição da riqueza como alternativa ao crescimento econômico ilimitado. Dados tais condicionantes teóricos, sua proposta operativa para a agricultura sustentável implicaria na necessária integração, em níveis de igualdade, dos objetivos econômicos, sociais e ambientais do desenvolvimento, assim como na observância das variáveis culturais, políticas e éticas da sustentabilidade. Trata-se, neste caso, da perspectiva teórica e conceitual oferecida pela ciência da Agroecologia. Logo, uma extensão rural que possa contribuir para o desenvolvimento rural mais sustentável deve trabalhar no apoio à implementação de tipos de agriculturas mais sustentáveis, o que não é possível a partir da difusão de pacotes tecnológicos verdes, mas sim seguindo os ensinamentos da Agroecologia e, portanto, tratar-se-ia de uma Extensão Rural Agroecológica. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 280 Extensão Rural Agroecológica: experiências e limites 3 A AGROECOLOGIA COMO BASE TEÓRICA-CONCEITUAL PARA A AÇÃO EXTENSIONISTA Não se pretende neste texto repetir as bases conceituais e epistemológicas da Agroecologia, mas sim interessa reafirmar que a Agroecologia vem se fortalecendo como uma ciência capaz de contribuir tanto para os estudos do desenvolvimento rural, como para o estabelecimento de uma nova forma de ver e entender o desenvolvimento agrícola na perspectiva da sustentabilidade. O que se deseja aqui é resgatar alguns aspectos que, em nosso entendimento, justificam sua adoção como orientação para a prática da extensão rural. Para iniciar, cabe recordar que, desde uma perspectiva normativa, a Agroecologia aparece como um conjunto de [...] ideias ambientais e de sentimento social acerca da agricultura, cujo conteúdo trata da produção, mas também da sustentabilidade ecológica dos sistemas de produção ainda que mais estritamente, Agroecologia se refere ao estudo de fenômenos puramente ecológicos que ocorrem nos campos de cultivo, tais como as relações entre parasitas e hospedeiros, competições entre plantas, etc. (HECHT, 1989, p.28) Assim mesmo, se trata de um novo enfoque para o estudo e manejo de sistemas agrícolas e dos sistemas agroalimentares, oferecendo um marco teórico cujo fim é contribuir para a compreensão da insustentabilidade dos modelos de sistemas agrícolas convencionais da Revolução Verde e dos sistemas agroalimentares globalizados instituídos pelas sociedades capitalistas. Este mesmo marco teórico aparece como orientador para os processos de transição no sentido da construção de agriculturas mais sustentáveis. Na perspectiva da extensão rural e suas práticas, a Agroecologia pode contribuir para a análise dos processos agrícolas de uma maneira mais ampla, ou seja, ver a agricultura desde um enfoque sistêmico, destacando a sustentabilidade inerente aos ciclos naturais e às interações biológicas (ALTIERI, 1995). Mas, além disto, a Agroecologia se constitui como uma disciplina científica orientada ao estudo da agricultura desde uma perspectiva ecológica, que pretende que o manejo ecológico dos recursos naturais corresponda a um enfoque holístico, de modo que, mediante a aplicação de uma estratégia sistêmica, se possa reconduzir o curso alterado da coevolução social e ecológica mediante um controle das forças produtivas, que ataque seletivamente as forças degradantes - de produção e de consumo - causadoras da atual crise ecológica. (SEVILLA GUZMÁN, 1995, p. 24) Logo, para a abordagem das questões estritamente agrícolas ou agropecuárias e florestais a Agroecologia adota os agroecossistemas como unidades fundamentais de estudo, ademais de entender o agroecossistema como uma unidade onde coevoluem culturas específicas e suas respectivas formas de interação com o ambiente natural, a partir de suas diferentes cosmovisões. Assim mesmo, enfatiza a importância dos componentes de diversidade: biodiversidade ecológica e sociocultural. De igual forma destaca a importância do desenvolvimento local e do conhecimento dos agricultores, que aparecem como a base de um potencial endógeno capaz de impulsionar um modelo de desenvolvimento mais sustentável. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 281 Francisco Roberto Caporal e Olivo Dambrós Assim, como já se demonstrou em outro lugar (CAPORAL, 2016), a Agroecologia pode contribuir para a resolução do quebra-cabeça proposto pelos “Cinco axiomas da sustentabilidade” (HEINBERG, 2007) no que tange à agricultura e ao sistema agroalimentar. Como também já foi demonstrado, a Agroecologia pode contribuir para o decrescimento sustentável na agricultura a partir de ações, práticas e tecnologias que modifiquem os atuais padrões do metabolismo social dominante nas formas industriais de agricultura (GONZÁLEZ DE MOLINA, 2012). Esta nova perspectiva teórica não poderia se fazer operativa com base no modelo tradicional de transferência de tecnologias e inovações, adotado pela extensão rural convencional, de modo que uma Extensão Rural Agroecológica seria um instrumento fundamental no campo das políticas públicas capaz de dar suporte a uma transição agroecológica. Neste sentido, é necessário observar que, desde o ponto de vista agroecológico, os processos de transição lançam mãos dos ensinamentos das ciências agrárias, ainda que se proponha uma forma distinta de intervenção nos agroecossistemas, partindo de uma perspectiva de desenvolvimento local autossustentável, que é oposta ao modelo hegemônico de desenvolvimento rural e agrícola. Ademais, desde a Agroecologia se propõe a necessidade de mudar a ênfase convencional das ciências agrárias, tendo em conta as interações complexas entre pessoas, cultivos, solos, animais, etc., que têm lugar dentro de cada agroecossistema e de forma diferenciada entre eles.3 Como se pode ver, segundo esta corrente agroecológica, a intervenção extensionista, em apoio ao desenvolvimento agrícola e rural, deveria seguir uma trajetória distinta daquela que foi seguida tanto pelo modelo difusionista convencional como por aqueles enfoques da agricultura sustentável baseados na “intensificação verde” ou na simples substituição de insumos e práticas ambientalmente arriscadas e agressivas para o meio ambiente por outras mais amigáveis. Isto seria insuficiente para dar conta da resolução ou minimização da crise socioambiental em curso. Tal trajetória distinta deveria considerar, em primeiro lugar, a noção de sistemas e o enfoque holístico, propostos pela Agroecologia. Isso determinaria a necessidade de uma visão dos agroecossistemas como uma totalidade, o que implica não só na exigência de aproximações inter e intradisciplinares, mas, sobretudo, a necessidade de uma clara consciência sobre a importância dos atores sociais como parte desse todo. 3 Para melhor entender a Agroecologia como alternativa científica aos modelos convencionais de desenvolvimento agrícola basta ver as premissas identificadas por Richard Norgaard ao tratar sobre os aspectos epistemológicos da Agroecologia. (NORGAARD, 1989, p.46-47) Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 282 Extensão Rural Agroecológica: experiências e limites Em segundo lugar, ao reconhecer a existência de uma estreita relação entre a evolução das culturas dos diferentes grupos sociais e de suas relações com ambiente natural leva a que as ações extensionistas deveriam partir de estudos das realidades locais e dos saberes dos grupos sociais ali presentes. Assim, as premissas acima expostas são claras ao apontar a existência de uma estreita relação entre a evolução da cultura dos grupos sociais e o respectivo estilo de uso dos recursos que ocorre em cada lugar. Isto é, cultura e ambiente se influem um ao outro, permanentemente. Portanto, o estudo de agroecossistemas deve levar em conta estas relações. Ou seja, o processo de coevolução. Assim, ao contrário dos enfoques convencionais, segundo os quais os extensionistas eram formados para destruir a subcultura camponesa considerada “atrasada” e responsável pelos obstáculos ao progresso (FONSECA, 1985), desde a perspectiva agroecológica, se exige não só o respeito à diversidade cultural como a necessidade de integrar os aspectos culturais, socioeconômicos e ambientais, característicos de cada agroecossistema, no processo de desenho de agroecossistemas mais sustentáveis e de programas e estratégias de desenvolvimento que incluam todos estes fatores. De igual modo, a ênfase no conhecimento local exige que o saber do extensionista não continue sendo considerado como um saber dominante e o único saber válido. A compreensão de que as sociedades (grupos ou comunidades) desenvolveram um tipo de conhecimento próprio, derivado de suas experimentações e segundo suas necessidades históricas e modos de vida específicos, faz com que a Agroecologia proponha a reconstrução de sistemas agrícolas tradicionais a partir dos conhecimentos tradicionais acumulados, sem negar a utilidade das ciências convencionais e da integração de novos conhecimentos, isto é, das inovações tecnológicas necessárias para o avanço no sentido de mais sustentabilidade na agricultura. Assim mesmo, a Agroecologia destaca o papel conjunto que devem jogar agricultores e agentes externos na construção do desenvolvimento e na adaptação de tecnologias adequadas para estas situações locais específicas, de maneira que se restabelece a necessidade de considerar as características de racionalidade próprias que levam os diferentes grupos sociais a imprimirem diferentes estilos de agricultura. Logo, mesmo em realidades particulares onde os conhecimentos tradicionais já não possuem a importância presente nos conhecimentos de sociedades e grupos com maior “densidade cultural”, entende-se que os conhecimentos locais devem servir aos extensionistas, pelo menos, como hipóteses iniciais de trabalho. Ou seja, parece necessário reconhecer que a ciência formal não é a única forma de conhecimento e que outras formas podem ser tão ou mais importantes que aquela, na hora de estabelecer estratégias de desenvolvimento agrícola e rural. Em terceiro lugar, cabe destacar a natureza do desenvolvimento proposto pela Agroecologia, começando pelos elementos que oferece para estabelecer-se uma crítica “científica” ao modelo de “modernização” da agricultura, com sua tendência à simplificação tanto da biodiversidade biológica como da diversidade cultural e sua tentativa de homogeneização dos modos de vida e da agricultura. Neste sentido, os princípios da Agroecologia nos ajudam a consolidar a crítica à extensão rural convencional quando defende que a mudança social deve ser imposta desde fora, normalmente baseando-se nos avanços da ciência e das Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 283 Francisco Roberto Caporal e Olivo Dambrós tecnologias, que tenta a ruptura das relações e condições de vida de populações que seus ideólogos consideravam atrasadas. Por outro lado, seguindo a perspectiva agroecológica, a extensão rural deveria tratar de potencializar estilos de desenvolvimento endógeno, ou seja, potencializar o uso dos meios e recursos disponíveis localmente, assim como os elementos relativos às formas históricas de organização, e a potencialidade dos elementos culturais, sociais, político e econômicos já existentes. Deste modo, as estratégias de uma Extensão Rural Agroecológica não podem orientar-se simplesmente pela acumulação de metas de crescimento econômico, de produção e de produtividade. Ao contrário, devem assumir uma orientação pluridimensional, que inclua os desejos e necessidades de mudança das condições econômicas dos diferentes grupos e que, ao mesmo tempo, possa garantir a segurança alimentar, melhores níveis de educação, de saúde e de bem estar social, sem menosprezar aspectos relativos à busca por mais equidade social e de gênero, que devem ser tratados transversalmente como parte do processo de transição agroecológica. Trata-se, pois, de um enfoque capaz de contribuir, efetivamente, para que a extensão rural possa atuar na construção de outros estilos de desenvolvimento. Isto não significa nenhum retrocesso, como pensam alguns, senão que se trata de implementar processos que correspondam às dimensões econômica, ambiental, social, cultural, política e ética da sustentabilidade. De todo o exposto até aqui, pode-se concluir que a adoção do enfoque agroecológico é um caminho importante na mudança da extensão rural se deseja caminhar na direção a uma Extensão Rural Agroecológica. 4 SOBRE O CONCEITO DE EXTENSÃO RURAL AGROECOLÓGICA Historicamente, a extensão rural foi entendida como, uma deliberada intervenção, de natureza pública ou privada, num espaço rural dado (uma propriedade, uma fazenda, uma comunidade, um povoado, uma microbacia hidrográfica, etc.), realizada por agentes externos, ou por indivíduos do próprio meio, que seria orientada à realização de mudanças no processo produtivo agrosilvopastoril ou em outros processos de natureza social, cultural e econômico, inerentes ao modo de vida da população rural implicada. Trata-se, pois, de uma intervenção intencionada, movida por objetivos normativos e levada a cabo através de um processo comunicativo que envolve inúmeros atores, possuidores de diferentes conhecimentos e situados em posições assimétricas de poder. Este, sem embargo, é um conceito insuficiente ao ser examinado à luz das novas propostas do desenvolvimento sustentável, da agricultura sustentável e da Agroecologia. Assim, a partir de nossas pesquisas, concluiu-se sobre a necessidade de superar a noção convencional de extensão rural introduzindo em seu conceito elementos relativos aos objetivos de equidade e de sustentabilidade. Desde a perspectiva ecotecnocrática, antes enunciada, provavelmente bastaria acrescentar ao conceito acima exposto, a expressão “mudanças sustentáveis”, para dar à atividade extensionista uma nova imagem. Entretanto, desde uma perspectiva agroecológica, orientada a transição a estilos de Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 284 Extensão Rural Agroecológica: experiências e limites desenvolvimento e de agricultura mais sustentáveis, se necessita ter em conta um conjunto de aspectos complementares e explicativos. Neste sentido, construiu-se o conceito de Extensão Rural Agroecológica ou “extensão ecosocial”4, como uma contribuição para o avanço dos estudos e das práticas extensionistas. O novo conceito nasce a partir de algumas palavras-chave que foram encontradas repetidas vezes na investigação (CAPORAL, 1998), as quais apareciam de forma reiterada nos discursos extensionistas e na bibliografia consultada. A apropriação destas palavras permitiu construir o conceito abaixo: a Extensão Rural Agroecológica poderia ser definida como um processo de intervenção de caráter educativo e transformador, baseado em metodologias de investigação-ação participante, que permitam o desenvolvimento de uma prática social mediante a qual os sujeitos do processo buscam a construção e sistematização de conhecimentos que os leve a incidir conscientemente sobre a realidade, com o objeto de alcançar um modelo de desenvolvimento socialmente equitativo e ambientalmente sustentável, adotando os princípios teóricos da Agroecologia como critério para o desenvolvimento e seleção das soluções mais adequadas e compatíveis com as condições específicas de cada agroecossistema e do sistema cultural das pessoas implicadas em seu manejo. (CAPORAL, 1998). 4 A expressão “extensão ecosocial” é utilizada por Sánchez de Puerta (1996), para quem este é um dos “tipos ideais integrados de extensão agrária”. Como “tipo ideal” o autor entende que se trata de um enfoque mais adequado para agências não governamentais, o que não corresponde com o que está ocorrendo na prática concreta, onde agentes do setor público e algumas organizações passaram a adotar as características deste enfoque. Trata-se de um conceito mais amplo do que aquele que vimos anteriormente, já que inclui a necessidade de uma práxis distinta da convencional e implica reconhecer a existência de diferentes conhecimentos e estruturas de poder que influem nos processos de desenvolvimento e de implementação de agriculturas mais sustentáveis. Ademais, recomenda que o manejo dos recursos naturais e a adoção de opções tecnológicas sejam entendidos no marco de processos de natureza construtivista, o que implica na necessidade de pesquisa, aprendizagem e ação e que, portanto, devem ser respeitosos para com as pessoas e suas condições objetivas, seus interesses e necessidades. Assim mesmo, o enfoque agroecossistêmico e a importância que dá aos sujeitos sociais conduzem a um modelo de decisão e ação que contempla o caráter histórico e os aspectos políticos do desenvolvimento local. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 285 Francisco Roberto Caporal e Olivo Dambrós Segundo Caporal (1998), “a adoção de tal conceito, ademais, pode contribuir para fortalecer os processos de resistência que caracterizam as lutas históricas dos camponeses, frente às tendências gerais e ameaças do desenvolvimento capitalista no campo.” Não obstante, segundo o mesmo autor, sua aplicação exige algumas condições nem sempre presentes na extensão rural, como, por exemplo: a) A necessidade de imersão do agente: a compreensão da realidade e da vida das famílias envolvidas no processo de desenvolvimento, o conhecimento dos agroecossistemas e o estabelecimento das estratégias e práticas compatíveis com a realidade, só é possível se o agente de extensão pudesse dispor do tempo suficiente e dedicar a atenção que exige cada situação concreta. Isto não é compatível com a busca de resultados imediatos em termos de aumentos na produção e produtividade, que caracteriza os objetivos centrais da extensão rural convencional. b) O resgate do conhecimento local: para que isso seja possível, é necessária a adoção de metodologias adequadas, que não sejam as metodologias tradicionais utilizadas pela extensão. Assim mesmo, estas devem ajudar no estabelecimento de uma plataforma de negociação, criando oportunidades para a integração do conhecimento local com o conhecimento técnico, de modo que se identifique um conjunto de possíveis alternativas de mudança em direção à sustentabilidade que sejam compatíveis com as condições econômicas, ecológicas e culturais encontradas em cada agroecossistema. c) Participação como direito: a participação não pode ser um processo parcial ou somente vigente quando uma das partes acha que é necessária. Participação, no caso da Extensão Rural Agroecológica, deve ser entendida como um direito e implica na horizontalidade da comunicação e na igualdade de oportunidades para expressar opiniões, selecionar opções possíveis, estabelecer estratégias e caminhos mais adequados e desenvolver as ações de forma conjunta, o que está assentado, necessariamente, em uma igualitária relação entre os atores envolvidos. d) O processo educativo: uma Extensão Rural Agroecológica deve garantir que o processo educativo seja capaz de potencializar o crescimento dos sujeitos como cidadãos e favorecer o empoderamento dos comunitários, de modo que os atores participantes vejam fortalecidas suas capacidades para a ação individual e coletiva, inclusive junto à sociedade maior. Já não se trata de uma educação para a adoção de tecnologias transferidas por um agente que sabe a um agricultor que não sabe, senão que a ERA deve ser entendida e praticada como um processo que permita desenvolver os conhecimentos e ter acesso a informações suficientes que permitam a eleição e a decisão conscientes entre alternativas possíveis, a partir da compreensão de sua própria realidade e das estruturas de dominação pelas quais se vêm afetados. Para isso, será indispensável potencializar o diálogo de saberes. Cabe ao extensionista, além de seu papel como técnico, um importante papel como animador e problematizador. e) Sistematização das experiências: o registro sistematizado dos conhecimentos e das experiências realizadas em terreno passa a ser um processo indispensável, tanto para facilitar sua socialização entre os membros de cada grupo, como para futuras avaliações. Assim mesmo, é necessário conhecer e sistematizar informações sobre os recursos internos disponíveis e suas possibilidades de uso, Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 286 Extensão Rural Agroecológica: experiências e limites assim como os obstáculos externos que devem ser superados. Na nova perspectiva da ERA já não basta o registro referente à adoção de tecnologias e práticas difundidas como ocorre na extensão rural convencional, mas sim o registro dos caminhos trilhados e dos resultados alcançados, de forma a construir bases para novos avanços em direção à sustentabilidade. Estes e outros elementos que poderão ser evidenciados ao logo dos processos de transição serão centrais para que a proposta teórica da Extensão Rural Agroecológica possa vir a ser materializada nas ações concretas do extensionismo rural. 5 SOBRE OS PROJETOS/EXPERIÊNCIAS DE EXTENSÃO RURAL AGROECOLÓGICA DESENVOLVIDOS NO ESTADO DO PARANÁ A sistematização aqui apresentada tem origem na trajetória da Educação Popular desenvolvida por ONGs e movimentos sociais com atuação predominante no Sudoeste do Paraná. Relata a ação prática de 09 (nove) experiências de ATER Agroecológica desenvolvidas nesse estado no período de 2003 a 2014 destacando avanços e limites encontrados para a implementação de uma proposta de Extensão Rural Agroecológica. Os projetos estudados envolvem mais de dez mil famílias, organizadas em centenas de grupos e comunidades rurais, apoiadas por entidades e organizações locais de aproximadamente cem municípios de quase todas as regiões do estado do Paraná e atuação direta de 150 agentes de ATER de formação diversa, majoritariamente, das ciências agrárias. Com exceção do Projeto de Inclusão Social que teve apoio de ONGs internacionais, todas as demais experiências de Ater Agroecológica foram desenvolvidas com recursos públicos do Governo Federal financiados pelo Ministério do Desenvolvimento Agrário e pelo Instituto Nacional de Colonização e Reforma Agraria (INCRA). Descrição resumida dos projetos de ATER Agroecológica Descrevem-se, abaixo, de forma sucinta, os nove projetos de ATER estudados, considerando, principalmente, o período em que foi executado, sua abrangência, a metodologia utilizada, os conteúdos, os objetivos, a origem dos recursos e as parcerias construídas. Projeto de ATER para inclusão social Desenvolvido durante cinco anos (abril de 2003 a abril de 2007), nas regiões Sudoeste, Centro, Vale da Ribeira e Oeste do Paraná, contemplou 1300 famílias articuladas em 120 grupos familiares, distribuídas em 55 municípios do estado. O trabalho de animação e gestão do Programa de Inclusão Social em cada região ficou a cargo de quatro entidades, ou seja: Associação de Produtores Orgânicos (AOPA), na região Metropolitana e Campos Gerais; Cooperativa Iguaçu de Prestação de Serviços (COOPERIGUAÇU), na região Sudoeste; FORUM OESTE da Agricultura Familiar, na região Oeste do Paraná e a Fundação de Estudos Rurais e Ecologia (RURECO), na região Centro Oeste do Paraná. Atuaram nesse projeto 60 profissionais das áreas das ciências agrárias e afins, definidos a partir da indicação das entidades locais nos seus respectivos Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 287 Francisco Roberto Caporal e Olivo Dambrós municípios. A coordenação operacional coube a um Conselho Gestor formado por representantes de cada uma das entidades coordenadoras regionais. Todos os coordenadores assim como os Agentes de ATER eram definidos pelos seus respectivos fóruns de Entidades da Agricultura Familiar. As principais parcerias foram as Cooperativas de Crédito e Interação Solidária (CRESOL), as Cooperativas de Leite da Agricultura Familiar (CLAFs), as Cooperativas da Agricultura Familiar Integrada (COOPAFI), Sindicatos dos Trabalhadores Rurais e algumas prefeituras. O objetivo principal foi apoiar a inclusão social de famílias empobrecidas e a sua participação nos processos de desenvolvimento local. Os principais conteúdos trabalhados foram: transição ecológica, formação, organização social, crédito solidário, gênero e geração. Os recursos para desenvolver as ações eram provenientes de uma ONG da Bélgica denominada de TRIAS, disponibilizados de maneira prática e ágil e fiscalizados por auditorias permanentes do Governo Belga. Havia um valor determinado para os serviços de ATER por família (em torno de 300 reais), e complementado pelas organizações locais o que chegava a um total aproximado de R$ 600,00 (seiscentos reais) por família. Projeto de ATER cooperativada do Sudoeste do Paraná Foi uma continuidade do programa de Inclusão Social desenvolvido de 2007 a 2009. Envolveu 1.500 famílias em 100 comunidades rurais de 30 municípios do Sudoeste. Atuaram no projeto, 30 agentes locais de ATER, quatro agentes de ATER microrregionais (coordenadores) e um coordenador geral. Foram elaborados 1.500 (um mil e quinhentos) Planos de Transição Agroecológica dos sistemas de Produção Familiar, 100 (cem) Planos Comunitários de Desenvolvimento e 04 (quatro) Planos Municipais de ATER. O projeto teve como proponente a Cresol Base Sudoeste. Contou com a parceria da mesma rede de entidades criada no programa de Inclusão Social, articulado por um Conselho Gestor, somado mais três cooperativas de ATER (Coopermarrecas, Cooperpinhais e Cooperfronteira), todas constituídas a partir do programa de Inclusão Social. Os recursos foram provenientes do Ministério do Desenvolvimento Agrário e complementados pelas organizações locais da Agricultura Familiar. O objetivo principal foi desenvolver ações junto às unidades familiares da agricultura familiar cooperativadas do Sudoeste do Paraná e os principais conteúdos eram: transição ecológica, formação, organização social, crédito solidário, gênero e geração, acesso às políticas públicas e comercialização. Projeto de ATER cooperativada no estado Paraná Este projeto foi uma extensão do que havia sido construído no Sudoeste do Paraná, agora expandido para o Estado. Teve a coordenação da União das Cooperativas da Agricultura Familiar e Economia Solidária do Paraná (UNICAFES) e da Agência de Desenvolvimento Regional do Sudoeste. Desenvolveu-se em 55 municípios do Paraná, situados nas regiões Sudoeste, Oeste, Centro, Norte, Sul e Vale do Ribeira, com ações de desenvolvimento técnico e social junto a 4.400 famílias de agricultores familiares. Atuaram no projeto, 55 Agentes Locais de ATER, 12 Agentes de ATER regionais com a função de Coordenar as Equipes, ambos vinculados as Cooperativas de ATER de cada região. À Agência de Desenvolvimento Regional coube o papel de fazer a gestão, apoiada politicamente por um Conselho Gestor, formada por uma REDE de entidades da agricultura familiar, vinculadas à Cresol Baser, ao Sistema de Cooperativas de Leite - SISCLAF, à Central das Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 288 Extensão Rural Agroecológica: experiências e limites Cooperativas de ATER - CENATER e à Central das Cooperativas de Comercialização Coopafi Central. O objetivo do projeto foi desenvolver ações de ATER junto às unidades da agricultura familiar cooperativada do Paraná. Os conteúdos orientadores das ações foram à transição ecológica dos sistemas produtivos, o fortalecimento das organizações da agricultura familiar em especial as cooperativas solidárias, a construção de alternativas de renda que substituam o cultivo do fumo, assim como a produção de alimentos saudáveis na perspectiva do abastecimento das demandas institucionais, em especial, o Programa Nacional de Alimentação Escolar (PNAE). Projeto de ATER junto a assentados - Sudoeste/PR Este projeto de ATER foi financiado com recursos do Programa de Consolidação de Assentamentos do MDA e foi executado pela Cooperiguaçu, de 2004 a 2007, no Assentamento Nova Fartura, localizado no município de Saudade do Iguaçu, região Sudoeste do Paraná. Envolveu 168 famílias de 12 grupos de famílias, em 04 comunidades rurais, com a participação efetiva de 04 Agentes de ATER. Teve como parceiros a Prefeitura Municipal e o Sindicato dos Trabalhadores Rurais de Saudades do Iguaçu. O objetivo principal foi desenvolver ações de acompanhamento técnico junto às famílias assentadas. Os principais conteúdos trabalhados foram: transição ecológica dos sistemas agrários, formação, organização social, crédito solidário, gênero e geração, acesso às políticas públicas e comercialização. Projeto de ATER com assentados da região Centro-oeste do PR Este projeto foi desenvolvido junto aos assentamentos Ireno Alves e Marcos Freire, ambos localizados no município de Rio Bonito do Iguaçu, Região Centro do Estado do Paraná, no período de 2008 a 2011, com recursos do MDA via convênio do Programa de Consolidação de Assentamentos. Envolveu 1.600 famílias assentadas da reforma agrária pertencentes a 29 comunidades rurais, no município de Rio Bonito do Iguaçu. Atuaram 18 agentes de ATER. O objetivo principal foi desenvolver ações de desenvolvimento técnico e social junto às famílias assentadas da Reforma Agrária. Os principais temas trabalhados foram: melhoria da infraestrutura dos assentamentos, aplicação do “Pronaf A”, elaboração de planos de transição agroecológica dos sistemas familiares de produção, práticas ecológicas de sementes crioulas, adubação verde, sistema de produção de leite a pasto e outras. Teve como parceiros as Associações dos Agricultores dos Assentamentos (ACACIAACANF), o Centro de Desenvolvimento Sustentável e Capacitação em Agroecologia (CEAGRO) e a Universidade Federal Fronteira Sul (UFFS), de Laranjeiras do Sul. Projeto “ATER em rede” do território da cidadania no Vale Ribeira – PR O projeto foi financiado pelo Ministério do Desenvolvimento Agrário através da modalidade de financiamento denominada “Chamada Pública de ATER”. Teve início em final de 2010 com previsão de encerrar em 2011, mas foi finalizado em 2012. Envolveu 900 famílias articuladas em 70 grupos localizados em seis municípios do Vale do Ribeira/PR e contou com a atuação direta de uma equipe formada por 12 agentes de ATER, apoiada por assessoria externa. Executado pela Cooperiguaçu em parceira com as cooperativas de crédito (Cresol) e de Comercialização (Coopafi, de Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 289 Francisco Roberto Caporal e Olivo Dambrós Cerro Azul), AOPA, Aprotunas – APRAF - Associação Rio Sul (ambas associações comunitárias de agricultores), Secretaria de Agricultura de Cerro Azul, Prefeitura de Adrianópolis, Sindicatos dos Trabalhadores Rurais e o Departamento de Estudos Rurais (DESER). O objetivo principal foi desenvolver ações de acompanhamento técnico junto às unidades familiares visando criar alternativas para mitigar a migração da mão de obra do campo para os centros urbanos, ampliar a diversificação produtiva das unidades familiares, melhorar a renda familiar, ampliar a articulação e organização das entidades da agricultura e reivindicar melhoria das condições de infraestrutura básica (estradas, saúde e educação) para a população do campo que vive na região do Vale do Ribeira. Projeto de ATER nos assentamentos da região Centro-oeste do PR Iniciado em 2011, o projeto se desenvolveu junto aos Assentamentos Marcos Freire e Ireno Alves, no Município de Rio Bonito do Iguaçu. Envolveu 1.300 famílias e contou com a participação direta de 13 agentes de ATER da Equipe técnica do CEAGRO. O objetivo central foi de construir de forma coletiva, ações de apoio ao desenvolvimento dos assentamentos da reforma agrária, com segurança alimentar e inserindo as famílias no processo de produção e comercialização integrado à dinâmica do desenvolvimento municipal e regional e articulado na organização de um Núcleo de Agroecologia da Rede ECOVIDA. Os temas desenvolvidos contemplaram a produção de alimentos saudáveis e comercialização junto ao PAA (Programa de Aquisição de Alimentos), participação social, conscientização sobre reforma agrária, proteção do meio ambiente, valorização da cultura camponesa e produção familiar e ecológica. Projeto de ATER com assentamentos do Noroeste/PR Esta experiência se refere à chamada pública editada pelo INCRA/MDA. O projeto iniciou em 2011 e foi interrompido pelo INCRA em 2016 no processo de mudança do governo Federal por ocasião do “impeachment” da então presidente Dilma Rosseaf. Contemplava 310 famílias moradoras em três municípios: Querência do Norte, Xambre e Planaltina do Paraná. Participaram desse projeto três agentes com formação nas ciências agrárias. Dentre as organizações, parceiras destacam-se os Sindicatos dos Trabalhadores Rurais, as Cooperativas de Economia Solidária, o MST (Movimento dos Trabalhadores Sem Terra), e as Secretarias municipais das Prefeituras. Os temas desenvolvidos foram a transição agroecológica, a formação e organização social. Projeto de ATER com assentamentos do Norte Pioneiro/PR Esta experiência se refere à chamada pública editada pelo INCRA/MDA. Foi executada pela Cooperiguaçu e teve como objetivo principal o desenvolvimento técnico e social em Assentamentos de Reforma Agrária na região denominada de Norte Pioneiro do Estado do Paraná. O projeto iniciou em 2011 e se desenvolveu até 2016 quando foi interrompida, devido à mudança do governo Federal por ocasião do “impeachment” da então presidente Dilma Rosseaf. Abrange 900 famílias, localizadas em 10 municípios. Participam das ações de forma direta 10 (dez) Agentes de ATER das áreas de Ciências Agrárias e afins. Nas parcerias destacam-se os Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 290 Extensão Rural Agroecológica: experiências e limites Sindicatos dos Trabalhadores Rurais, as Cooperativas de Economia Solidária, o Movimento dos Sem Terra e algumas secretarias municipais de Agricultura. A partir dos estudos vivenciados desses projetos/experiências de ATER investigados, descrevem-se a seguir algumas percepções sobre avanços e limites na perspectiva de construção de uma ATER orientada pelos princípios da Agroecologia e que contribua no desenvolvimento local. Avanços e limites analisados nos projetos de ATER Agroecológica A análise dos avanços e limites para a construção de uma ATER Agroecológica considerou alguns temas relevantes observados no estudo dos projetos descritos acima. Entre eles, destacamos: 1) a gestão dos projetos/experiências de ATER; 2) a formação e estruturação das equipes de ATER; 3) a relação/interação entre projetos e processos; 4) articulação entre executoras de serviços de ATER e organizações locais; e, 5) os instrumentos metodológicos de apoio as ações de campo. Ressalte se que estes temas e considerações dos autores estão fundamentados nas informações oriundas de fontes relacionadas a cada projeto desenvolvido e através da observação participante por um dos autores. Em relação à gestão dos projetos/experiências de ATER Apesar das diferenças entre regiões e públicos trabalhados a gestão dos projetos, de forma geral, seguiu orientação dos colegiados de entidades regionais, microrregionais ou locais da agricultura familiar e assentados da reforma agrária. O envolvimento dos Sindicatos dos Trabalhadores Rurais – STRs, cooperativas de economia solidária, organizações não governamentais - ONGs e associações comunitárias, na gestão, foi importante, porque acrescentaram-se novas sugestões e críticas aos projetos de ATER. Também, observou-se que aumentou a corresponsabilidade destas em relação ao sucesso ou fracasso das ações em seus respectivos locais e empoderou as organizações envolvidas que passaram a sentirse parte do processo. Entre os limites encontrados na gestão dos projetos conveniados, um dos mais importantes se refere ao acesso dos recursos para desenvolver estas ações. Ainda são poucas as fontes financiadoras para projetos com essa concepção. O maior financiador ainda é o governo federal que, entre outras questões limitantes, não dispunha de uma legislação clara sobre repasses de recursos à sociedade civil. Por essa e outras razões houve muitos impasses durante a execução dos projetos acarretando atrasos frequentes na liberação dos recursos para o custeio das ações e interrupções dos mesmos por várias vezes. Sobre a formação e estruturação das equipes de ATER As equipes de ATER eram compostas pelos agentes locais de ATER, coordenadores de equipe, profissionais de apoio técnico e articulador geral do projeto, ambos sob a liderança política de Conselhos Gestores formados pelas entidades da Agricultura Familiar e/ou assentados da reforma agrária daquele local, juntamente com a entidade/empresa executora. A seguir são apresentadas características de cada componente da referia equipe. a) Agente local de ATER desempenhou o papel de animador e organizador local. Na equipe exercia a função de orientar a construção de demandas das Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 291 Francisco Roberto Caporal e Olivo Dambrós famílias, grupos e comunidades rurais; b) Coordenadores de equipes de ATER: no geral são dirigentes locais ou técnicos liberados por uma das entidades parceiras das entidades que acompanham e encaminham as demandas levantadas pelos Agentes de ATER; c) Profissionais de apoio: por formação e/ou função, têm acúmulo técnico e focam suas ações de modo setorial: leite, comercialização, crédito, sementes, educação, saúde, etc. Esses profissionais, geralmente, eram de nível superior ou com “notório saber” e responsáveis diretos pela formação dos agentes e apoiadores de atividades afins, demandadas pelas famílias e suas organizações; d) Articulador geral do Projeto: a função do articulador era fazer a ponte entre as equipes de ATER e as organizações que compunham o Conselho Gestor. É importante esclarecer que esse formato de equipe de ATER foi possível em apenas projetos financiados por ONGs e pelos convênios com o governo federal antes da Lei de ATER (Lei nº 12.188/2010). Após 2010 quando aquela Lei entrou em vigor, esse formato de equipe se tornou inviável. A Lei exige que os profissionais tenham registro em seus respectivos conselhos de classe e dificilmente podem ser definidos pelas organizações locais porque já fazem parte orgânica de entidades executoras destes serviços. Sobre a relação/interação entre projetos e processos Como a maioria das experiências analisadas foi financiada por projetos do MDA (Ministério do Desenvolvimento Agrário), através da Secretaria da Agricultura Familiar (SAF) e do Instituto Nacional de Colonização e Reforma Agrária (INCRA), evidenciam-se algumas dificuldades resultantes, principalmente, das condições predeterminadas para o possível financiamento e até mesmo de condições construídas na elaboração dos projetos. Um dos limites significativamente relevantes foi a desconexão do tempo de execução do projeto e o tempo dos processos que envolvem a ação extensionista. Os projetos têm um tempo cronológico determinado, com poucas possibilidades de ajustes em seu tempo de execução. A maioria deles tiveram a duração prevista de um ano e não foi possível a obtenção de recursos para sua continuidade. Como as organizações (cooperativas, ONGs, ou outras entidades da agricultura familiar) realizam grande parte de seus trabalhos através de projetos, acabam dependendo da disponibilidade de recursos para continuar as ações, de modo que quando cessam os repasses, os projetos são param e se tornam descontinuados. Sobre a formação de redes locais para desenvolver ATER Um dos maiores limites encontrados no processo para a construção de uma proposta de ATER Agroecológica consiste na dificuldade de integrar as organizações locais em rede. A percepção que se teve é que mesmo próximas em termos de história e articuladas informalmente em um mesmo Fórum de Entidades da Agricultura Familiar ou camponesa, ainda guardam reservas entre si e têm dificuldades de se perceberem como partes de um projeto comum. Entretanto, quando foi possível essa articulação, como ocorreu no Projeto de Inclusão Social, as redes locais de ATER tencionaram o poder público a atender demandas planejadas e encaminhadas coletivamente, muitas delas historicamente reivindicadas. Ficou evidente a força de pressão social que as redes locais de entidades exercem sobre o Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 292 Extensão Rural Agroecológica: experiências e limites poder local. Em vários municípios e comunidades rurais passou-se a incluir as famílias rurais menos favorecidas em processos de desenvolvimento local, em democratizar as políticas públicas e orientar recursos que, mesmo escassos, surtiram grande efeito em localidades carentes. Instrumentos metodológicos de planejamento às ações de ATER Os instrumentos metodológicos de planejamento participativo foram fundamentais na execução dos projetos de ATER Agroecológica principalmente quando se tratava de ações que envolviam as praticas de campo. Estes instrumentos incorporam importantes condições e dimensões estratégicas, como o protagonismo coletivo, a decisão da família, a visão sistêmica e a perspectiva transformadora da realidade e da sociedade. Um dos limites encontrados foi a maior dificuldade de adotar estes instrumentos após a Lei de ATER porque, na sua maioria, as metodologias vinham pré-definidas nas Chamadas Públicas. Dessa maneira, apenas os planos comunitários/ou de assentamentos e os planos familiares foram exercidos em todas as experiências, já os planos municipais foram assumidos apenas em um dos projetos estudados. Os principais instrumentos metodológicos utilizados nos projetos foram: a) Os Planos de Vida e Produção Familiar, que se mostraram bastante efetivos para o diagnóstico, planejamento e avaliação dos sistemas de produção, registrando as diferentes dimensões e condições desejadas pela família no processo de transição agroecológica. Neste sentido, o uso de desenhos/croquis se mostrou uma ferramenta adequada ao forte senso prático dos (as) agricultores (as), pois fortalecem a visão e o desenho que eles (as) já têm “em mente” e proporcionam uma visão sistêmica do todo da unidade de produção, incluindo as atividades produtivas, aspectos ambientais, estrutura, localização, etc.; b) Os Planos comunitários (grupais e Planos de Desenvolvimento dos Assentamentos – PDAs) permitem resgatar e entender a história a partir da experiência das pessoas junto aos locais de vivência coletiva habitual, que são as festas locais, jogos de final de semana, locais de encontros de lazer e debates sobre a realidade no seu dia a dia. Em geral, as propostas dos Planos Comunitários incorporaram aquelas dos Planos Familiares dando-lhes uma amplitude maior. Assim, por exemplo, se em nível do Plano Familiar a demanda trata de organização da produção leiteira como uma questão de filiação do produtor à Cooperativa de Leite da Agricultura familiar (CLAF), em nível do Plano Comunitário, a mesma refere-se à necessidade de organizar um serviço de coleta para o transporte da produção leiteira. Por conseguinte, existe, de modo geral, uma correspondência entre os Planos Familiares e os Planos Comunitários; c) Os Planos Municipais de ATER Agroecológica foram elaborados como experiências piloto factíveis de serem realizadas naqueles municípios onde o contexto político fosse favorável com boa relação entre o governo local e as entidades da agricultura familiar e principalmente entre estas. Embora se previsse desencadear um processo mais amplo, esse instrumento, só ocorreu em poucos municípios. Para a execução financeira dos planos municipais, foram constituídos fundos virtuais que permitiam a aplicação de recursos variáveis para uma mesma conta bancária. Assim, enquanto algumas entidades aplicavam recursos em espécie, Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 293 Francisco Roberto Caporal e Olivo Dambrós outras emprestavam um carro, outras uma sala e assim por diante, permitindo a participação de diferentes atores e dentro de suas possibilidades reais. O maior limite para a construção de Planos Municipais de ATER Agroecológica foi a falta de compreensão do trabalho compartilhado. Enquanto estava na fase de diagnóstico e planejamento a participação e construção coletiva era animada, mas ao chegar no momento operacional, cada entidade voltava a cuidar de suas ações e poucas restavam para cuidar do processo. A maioria abandonava a construção coletiva. 6 CONCLUSÕES E RECOMENDAÇÕES As conclusões que seguem pretendem abrir caminhos a outras reflexões sobre a construção na prática de uma ATER que se oriente pela Agroecologia e que valorize e reconheça um Brasil Rural de muitas etnias, saberes, organizações locais e comunitárias, historicamente relegadas pela sociedade urbana–industrial. De forma geral, os projetos estudados permitem afirmar que o exercício da superação da Extensão Rural Convencional rumo a uma Extensão Rural Agroecológica ainda encontra muitos limites para sua implantação de forma massiva. A pressão cotidiana do modelo agroquímico e industrial com sua multiplicidade de atores e propulsores (mídia, lojas agropecuárias, técnicos vendedores, técnicos de empresas integradoras), aliado à presença maciça de técnicos do estado defendendo uma ATER convencional se tornaram obstáculos desafiadores para essa transição. Outros limites encontrados que dificultaram a construção da ATER Agroecológica foram o engessamento metodológico dos projetos financiados pelo governo federal; a distância entre governos e sociedade civil, principalmente em nível local; a falta de pesquisa oficial que oferecesse subsídios para apoiar a transição para estilos de agriculturas mais sustentáveis; e a falta de capacidade técnica instalada com formação para atuar segundo o enfoque agroecológico. Ainda em relação à metodologia, um limite encontrado foi o processo utilizado para monitorar e avaliar as ações dos projetos de ATER estudados. Nas avaliações realizadas pelo governo federal a verificação é feita pelos meios (métodos, cadastros realizados) e não pelos resultados alcançados. Os gestores públicos, principalmente dos projetos financiados através de Chamadas Públicas de ATER, se restringem a avaliar ações pontuais de como o número de participantes nas atividades, o número de reuniões, oficinas e seminários realizados. Não se observou avaliações de resultados junto às famílias de agricultores e suas organizações, como a realização de feiras ecológicas, o aumento da produção de alimentos orgânicos, o fortalecimento das pequenas cooperativas e outros resultados concretos alcançados pelas ações dos projetos. Sobre o debate de questões de gênero e geração é possível afirmar que houve avanços nos projetos estudados. A participação das mulheres como sujeitos sociais desde a sua inclusão nas metas específicas das Chamadas Públicas e demais projetos de ATER desenvolvidos, até a presença delas nas ações de campo dessa política publica mostrou que mulheres e jovens tiveram espaço, embora ainda tímido e distante de conseguir mudanças concretas de protagonismo feminino nos processos. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 294 Extensão Rural Agroecológica: experiências e limites Outro importante avanço constatado foram as articulações em rede que a proposta de ATER Agroecológica gera. Percebeu-se como uma via de duas mãos: tanto as redes locais contribuem para avanços na construção de ATER Agroecológica, como o tema da Agroecologia tem grande efetividade para aglutinar organizações. É possível afirmar que essa aglutinação cumpre um papel importante no tensionamento e redirecionamento de políticas públicas locais para a promoção da agricultura ecológica e do desenvolvimento sustentável. As financiadoras destes serviços de ATER, principalmente com nova base tecnológica são muito restritas. Até final de 2015 o maior financiador era o governo federal. A partir do “impeachment” de Dilma Rosseaf o governo que assumiu ainda não liberou financiamentos no marco da política publica de ATER. O que se observa são alguns editais muito restritos que envolvem Universidades e outros entes públicos, mas distantes de recuperar as ações que estavam curso no governo anterior. Por fim, uma das conclusões mais importantes que em momentos se caracterizou como avanço e em outros se constitui num desafio estratégico, se refere à gestão dos serviços de ATER. Foram impactantes as diferenças entre a gestão dos projetos feita pelas empresas prestadoras de serviços de ATER e quando essa gestão foi feita pelas organizações da sociedade civil. As organizações sociais vivem nesses locais, conhecem essas realidades e são cúmplices desses processos. Enquanto os profissionais são transitórios, temporários e, muitas vezes, alheios aos processos endógenos. O estudo das experiências estudadas permite afirmar que a construção de uma proposta de política pública de Extensão Rural Agroecológica deve ter sua gestão política coordenada pelas organizações sociais e não pelos profissionais de ATER ou suas empresas. Estes podem e devem fazer parte, mas nas ações de assessoramento, facilitação e organização, mas as decisões finais devem ser dos colegiados locais e territoriais. 7 REFERÊNCIAS ARL, V.; DAMBRÓS, O. Da Assistência Técnica e Extensão Rural à construção Social do Conhecimento: um desafio para o desenvolvimento local sustentável. Laranjeiras do Sul, PR: Editora CEAGRO, 2012, 541 p. ALTIERI, M. A. El estado del arte de la agroecología y su contribución al desarrollo rural en América Latina. In: CADENAS MARÍN, A. (ed.): Agricultura y Desarrollo Sostenible. Madrid: MAPA. 1995, p. 151-203. BRASIL. Ministério do Desenvolvimento Agrário. Política Nacional de Assistência Técnica e Extensão Rural. Brasília, MDA/SAF/Dater, 2004. BRASIL. Lei nº 12.188, de 11 de janeiro de 2010. Institui a Política Nacional de Assistência Técnica e Extensão Rural para a Agricultura Familiar e Reforma Agrária – PNATER e o Programa Nacional de Assistência Técnica e Extensão Rural na Agricultura Familiar e na Reforma Agrária – Pronater, altera a Lei n° 8.666, de 21 de junho de 1993, e dá outras providências. Brasília, 2010. Redes - Santa Cruz do Sul: Universidade de Santa Cruz do Sul, v. 22, n. 2, maio-agosto, 2017 295 Francisco Roberto Caporal e Olivo Dambrós BUTTEL, F. H. Transiciones agroecológicas en el siglo XX: análisis preliminar. In: Agricultura y Sociedad, nº 74 (Enero-Marzo). p. 9-37, 1995. CAPORAL, F. R. A Extensão Rural e os Limites à prática dos Extensionistas do Serviço Público. 1991. 133f. Dissertação. (Programa de Pós-Graduação em Extensão Rural) Universidade Federal de Santa Maria, Santa Maria. 1991. CAPORAL, F. R. La extensión agraria del sector público ante los desafíos del desarrollo sostenible: el caso de Rio Grande do Sul, Brasil. 1998. 516f. Tese de Doutorado. (Instituto de Sociología y Estudios Campesinos). Universidad de Córdoba, Córdoba: Espanha, 1998. CAPORAL, F. R. Extensão Rural como política pública: a difícil tarefa de avaliar. In: SAMBUICH, R. H. R. et al. Políticas Agroambientais e Sustentabilidade: desafíos, oportunidades e lições aprendidas. Brasília: Ipea, 2014, p. 19-48 CAPORAL, F.R. Poderá a Agroecologia responder aos cinco axiomas da sustentabilidade? In: Rev. Bras. de Agroecologia, v. 11, n.4, p.390-402, 2016. CAPORAL, F. R.; COSTA BEBER, J. A. Por uma nova extensão rural: fugindo da obsolescencia”. In: Rev. Reforma Agrária, nº 3, vol. 24, set/dez/. Campinas: ABRA, p. 70-90, 1994. CAPORAL, F. R.; RAMOS, L. F. Da extensão rural convencional à extensão rural para o desenvolvimento sustentável. In: MONTEIRO, D. C. C.; MONTEIRO, M. A. (Orgs.). 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https://openalex.org/W2807967913	https://bmcpregnancychildbirth.biomedcentral.com/track/pdf/10.1186/s12884-018-1885-z	English	null	Are women with history of pre-eclampsia starting a new pregnancy in good nutritional status in South Africa and Zimbabwe?	BMC pregnancy and childbirth	2,018	cc-by	8,621	Are women with history of pre-eclampsia starting a new pregnancy in good nutritional status in South Africa and Zimbabwe? Gabriela Cormick1,2, Ana Pilar Betrán3, Janetta Harbron2, Tina Dannemann Purnat4, Catherine Parker5,6,7, David Hall8, Armando H. Seuc9, James M. Roberts10, José M. Belizán1, G. Justus Hofmeyr5,6,7 and on behalf of the Calcium and Pre-eclampsia Study Group Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 https://doi.org/10.1186/s12884-018-1885-z Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 https://doi.org/10.1186/s12884-018-1885-z RESEARCH ARTICLE Open Access Are women with history of pre-eclampsia starting a new pregnancy in good nutritional status in South Africa and Zimbabwe? Gabriela Cormick1,2, Ana Pilar Betrán3, Janetta Harbron2, Tina Dannemann Purnat4, Catherine Parker5,6,7, David Hall8, Armando H. Seuc9, James M. Roberts10, José M. Belizán1, G. Justus Hofmeyr5,6,7 and on behalf of the Calcium and Pre-eclampsia Study Group Open Access Abstract Background: Maternal nutritional status before and during pregnancy is an important contributor to pregnancy outcomes and early child health. The aim of this study was to describe the preconceptional nutritional status and dietary intake during pregnancy in high-risk women from South Africa and Zimbabwe. Methods: This is a prospective observational study, nested to the CAP trial. Anthropometric measurements before and during pregnancy and dietary intake using 24-h recall during pregnancy were assessed. The Intake Distribution Estimation software (PC-SIDE) was used to evaluate nutrient intake adequacy taking the Estimated Average Requirement (EAR) as a cut-off point. Results: Three hundred twelve women who had pre-eclampsia in their last pregnancy and delivered in hospitals from South Africa and Zimbabwe were assessed. 73.7 and 60.2% women in South Africa and Zimbabwe, respectively started their pregnancy with BMI above normal (BMI ≥25) whereas the prevalence of underweight was virtually non-existent. The majority of women had inadequate intakes of micronutrients. Considering food and beverage intake only, none of the micronutrients measured achieved the estimated average requirement. Around 60% of pregnant women reported taking folic acid or iron supplements in South Africa, but almost none did so in Zimbabwe. Conclusion: We found a high prevalence of overweight and obesity and high micronutrient intake inadequacy in pregnant women who had the previous pregnancy complicated with pre-eclampsia. The obesity figures and micronutrient inadequacy are issues of concern that need to be addressed. Pregnant women have regular contacts with the health system; these opportunities could be used to improve diet and nutrition. Trial registration: PACTR201105000267371. Registered 06 December 2010. Trial registration: PACTR201105000267371. Registered 06 December 2010. Keywords: Nutrient intake, Weight, Pregnancy, Supplement, Obesity, BMI * Correspondence: gabmick@yahoo.co.uk 1Instituto de Efectividad Clínica y Sanitaria (IECS-CONICET), Emilio Ravignani, 2024 Buenos Aires, Argentina 2Division of Human Nutrition, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa Full list of author information is available at the end of the article Background the CAP trial with the aim of describing the nutritional status of women from South Africa and Zimbabwe that became pregnant during the CAP trial. More specifically, we aimed to describe levels of overweight and obesity be- fore and during pregnancy, and the adequacy of macronu- trient and micronutrient intake during pregnancy. g Nutrition status of women before and during pregnancy is one of the main contributors to pregnancy outcomes and early child health [1]. In many low and middle-income countries undernutrition and overnutrition coexist in the same population [2]. Obesity is increasing while micronu- trient deficiencies still persist, particularly in the most vulnerable groups such as women and children [3]. Con- sequently, women start pregnancy with higher risks to de- velop complications such as pre-eclampsia, gestational diabetes mellitus, gestational hypertension, depression, fetal macrosomia, stillbirth, preterm birth, birth by caesar- ean section and infant mortality [4–9]. In addition, high maternal body mass index (BMI) has also been associated with delayed breastfeeding, weight retention and in women with gestational diabetes, a higher risk of develop- ing chronic diseases [5]. Inter-pregnancy interval is also an important factor that may influence maternal availabil- ity of nutrients, especially in those populations with exist- ing micronutrient deficiencies [10]. © 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. Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 2 of 10 Page 2 of 10 Participants and methods This is a nested prospective observational study of women from South Africa and Zimbabwe recruited in the CAP trial [15]. The CAP trial is a multi-centre randomized, double-blind placebo-controlled clinical trial with the ob- jective to determine whether calcium supplementation be- fore conception and during the first half of pregnancy reduces the incidence of recurrent pre-eclampsia more ef- fectively than supplementation starting at 20 weeks, which is the current WHO recommendation. In the CAP trial, non-pregnant women with history of pre-eclampsia or eclampsia in their most recent pregnancy were invited to participate as they are at higher risk of developing pre-eclampsia in subsequent pregnancies. Once admitted in the trial, participants were required to attend study sites every 12 weeks for follow up until pregnancy occurred. Pregnant women were followed up throughout their preg- nancy and trial visits were scheduled at 8, 20 and 32 weeks´ gestation. Eligible women were randomized to receive either 500 mg of elemental calcium daily or pla- cebo from recruitment and blinded supplementation con- tinued while participants were non-pregnant or until 20 weeks’ gestation. From 20 weeks’ gestation, all partici- pants received calcium supplements in compliance with WHO guidelines [16]. The CAP trial started in 2011 and recruitment was completed in September 2016. Interest in pre-conceptional interventions to reduce risk factors during pregnancy is growing, although their effectiveness on pregnancy outcomes is less certain [11]. Current WHO Guidelines on antenatal care recommend supplementation with iron and folic acid to all pregnant women, and with calcium and vitamin A to women in specific areas with a high prevalence of deficiency [12]. In populations where calcium intake is low, the WHO recommends supplementation with 1.5–2.0 g elemental calcium/day from 20 weeks´ gestation until the end of pregnancy for the prevention of pre-eclampsia. The WHO Guidelines also report that women receiving counselling on diet and/or exercise are less likely to ex- perience excess weight gain during pregnancy, although the evidence on the impact of other pregnancy outcomes is less certain [12]. Settings and study population Participants were recruited from government secondary or tertiary urban referral hospitals with large obstetric units serving urban and rural populations. The maternity and obstetric units included in the CAP trial were located in Cape Town (1), East London (2) and Johannesburg (1) in South Africa; and in Harare (2), Zimbabwe. Women were eligible for the CAP trial if they had pre-eclampsia or eclampsia in their most recent pregnancy, if they were not pregnant but in a sexual relationship, not using contracep- tion and if they gave informed consent. For admission we reviewed the participant clinical records and accepted the clinical evaluation of pre-eclampsia or eclampsia reported there. Exclusion criteria included: less than 18 years of age; chronic hypertension with persistent proteinuria; cal- cium supplement intake; and history or symptoms of uro- lithiasis, renal disease or parathyroid disease [15]. For a complete list of eligible criteria please refer to the pub- lished protocol [15]. In this analysis, we included women recruited in the CAP trial who became pregnant and In South Africa, the National Health and Nutrition Examination Survey (SANHANES-1) reported a preva- lence of overweight (BMI ≥25 kg/m2) and obesity (BMI ≥30) in women of 24.8 and 39.2%, respectively in 2012 [13]. Data from Zimbabwe in 2000 shows a preva- lence of overweight and obesity in women of 17.4 and 5.7% respectively [14]. However information on over- weight and obesity rates as well as dietary intake during pregnancy is scarce in these countries. Nutrient and sup- plement intake information would be important to bet- ter plan and tailor interventions to improve pregnancy outcomes [10]. We conducted a randomized controlled trial to evaluate the effect of pre-pregnancy calcium supplementation on the incidence of recurrent pre-eclampsia (Calcium and Pre-eclampsia: CAP trial) [15]. This was a multi-country trial conducted in South Africa, Zimbabwe and Argentina. This manuscript presents the results of a sub-analysis of Page 3 of 10 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 3 of 10 reached 20 weeks´ gestation between March 2013 to March 2016. reached 20 weeks´ gestation between March 2013 to March 2016. nutrient intakes have a wide day-to-day variability, data obtained from one single day is not sufficient to describe the usual intake or to assess the proportion of individ- uals with intakes below certain thresholds (e.g. below re- quirements). Dietary assessment The dietary intake of participants was assessed at 20-weeks’ gestation using a triple pass 24-h dietary recall adapted from the method developed by Nelson M. team from the King’s College London after it was piloted in South Africa and Zimbabwe [18]. The 24-h recall is a guided interview to assess food intake of the previous day. CAP trial research nurses were trained in-site in March 2013 to administer the triple pass 24-h recalls and to use the Dietary Assessment Education Kit (DAEK) to assist with the portion size estimation [19]. Xhosa and Zulu translators were trained at the sites that required them. A specific questionnaire was also included to investi- gate supplement intake during pregnancy. Women were asked about frequency and dose of the supplements and medicines. Trial supplementation was not computed in the dietary assessment of this sub-study as it was the intervention being tested and not, otherwise, part of the diet of this group of women. Reported food intakes from the 24-h recall were en- tered and analysed using the Food Finder III computer program, provided by the South African medical re- search council (SAMRC) to obtain daily energy and nu- trient intakes for each participant. If properly conducted, a single day 24-h dietary recall is a reliable method to as- sess individual intake on one day and can be used to es- timate a population mean [20]. However, as food and Anthropometric assessment and clinical data collection Variables used for this sub-study included: age, height, pre-pregnancy weight, number of previous pregnancies and date of birth of last pregnancy complicated with pre-eclampsia. This data were collected at admission. For women who became pregnant, weight during preg- nancy was recorded at 8, 20 and 32 weeks´ gestation during the scheduled trial follow-up visits. Research nurses specially trained for the CAP trial assessed all an- thropometric, clinical and dietary variables at admission and during follow up visits at each participating site. Body weight was measured to the nearest 0.1 kg in light clothing and without shoes. Height was measured to the nearest 0.1 cm and without shoes using a stadi- ometer. Scales and stadiometers were those provided by each hospital and remained the same throughout the study. The Manual of Operations and the Standard Op- erating Procedures (SOPs) provided clear instructions on how women should be weighted and measured. The Estimated Average Requirement (EAR) of carbohy- drates and each micronutrient as recommended by the In- stitute of Medicine (IOM) for pregnant women was used as the cut-off point to assess adequacy of nutrient intake [23]. The Estimated Energy Requirement (EER) for preg- nant women during the second trimester was calculated for each participant using the age, weight and height at admission and according to the Dietary Reference Intake (DRI) formula for adult women [23]. As data of physical activity was not collected the value for sedentary lifestyle was used conservatively. Energy intake obtained from the first 24-h recall assessment was divided by the EER then normalized using PC-SIDE [23, 24]. The 80% of EER dur- ing pregnancy suggested by Goldberg was used to calcu- late the plausibility of energy intake [25]. Protein adequacy was calculated using the EAR of 0.88 g per kg of body weight, using weight at 20 weeks´ gestation [23]. Pre-pregnancy BMI was calculated as weight (kg) di- vided by the square of the body height (m) using measure- ments recorded at admission. Women were classified according to the WHO BMI standards for adults, defined as underweight (BMI < 18.5), normal (18.5 ≤BMI < 25), overweight (BMI ≥25), or obese (BMI ≥30) [17]. Gestational weight gain was calculated by subtracting the weight at 8 weeks´ gestation from the weight at 32 weeks´ gestation, since the participant’s weight at de- livery was not assessed. Settings and study population Statistical models have been developed to better estimate usual nutrient intakes in a population by adjusting for within-individual intake variability [21]. Therefore, in order to estimate the proportion of women with intakes below requirements we used the Intake Dis- tribution Estimation software (PC-SIDE, version 1.0, 2003; Department of Statistics, Iowa State University, Ames) that requires a sample of at least 50 dietary as- sessments that are repeated on a non-consecutive day to the first assessment [22]. For these purposes, a second 24-h dietary recall assessment was administered in a subsample of women on a non-consecutive day after the first 24-h recall. Energy and nutrient intake distributions from the single 24-h recall were thus adjusted by within-person variance obtained from the second 24-h recall assessment and by interview weekday to estimate usual nutrient intake and to calculate the proportion of women with intakes below requirements. Statistical analysis Categorical values were described using percentages and numerical variables using means and standard deviations (SD). Statistical data analyses were performed using the SPSS 23.0 software package (IBM, New York, NY, USA). Dietary intake variables were log transformed and tested for normality using the Anderson-Darling statistical test Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 4 of 10 using the PC-SIDE software. The software performs three steps: adjustments for weekday; transformation to normal- ity using power transformation; and estimation of within-person variance using an error measurement model. before 20 weeks´ gestation, 34 did not attend to the preg- nancy visit at 20 weeks’ gestation, and 102 (18.8%) com- pleted the visits at 20 weeks´ gestation outside the sub-study period. A total of 350 women were eligible for this sub-study, however 38 (10.8%) missed the dietary as- sessment interview. Thus, we present the results of 312 women that completed the dietary assessment at the 20 weeks´ gestation visit. Of these women 224 (71.8%) were from South Africa and 88 (28.2%) from Zimbabwe. Re- peated dietary assessments were obtained from 107 (34.3%) women, 79 from South Africa and 28 from Zimbabwe. before 20 weeks´ gestation, 34 did not attend to the preg- nancy visit at 20 weeks’ gestation, and 102 (18.8%) com- pleted the visits at 20 weeks´ gestation outside the sub-study period. A total of 350 women were eligible for this sub-study, however 38 (10.8%) missed the dietary as- sessment interview. Thus, we present the results of 312 women that completed the dietary assessment at the 20 weeks´ gestation visit. Of these women 224 (71.8%) were from South Africa and 88 (28.2%) from Zimbabwe. Re- peated dietary assessments were obtained from 107 (34.3%) women, 79 from South Africa and 28 from Zimbabwe. Ethics Ethical approval was obtained from appropriate national and institutional ethics review bodies as applicable for each study site, and all participants provided informed written consent. The study was approved by the Re- search Project Review Panel of the UNDP/UNFPA/ UNICEF/WHO/World Bank Special Programme of Re- search, Development and Research Training in Human Reproduction at the Department of Reproductive Health and Research of WHO, and the WHO Research Ethics Review Committee, Geneva, Switzerland. At the time of the assessment, women from South Africa had been in the study an average of 12.5 (SD ±7.4) months and women from Zimbabwe 13.1 (SD ±7.4) months. Their inter-pregnancy interval was 24.5 (SD ± 22.5) months in South Africa and 30.3 (SD ±23.7) months in Zimbabwe (Table 1). Data management procedures were compliant with good clinical practice (GCP) [26]. Results At recruitment, the mean age of women in this sub-study was 29.2 years (SD ± 5.2) in South Africa and 29.3 (SD ± 4.8) in Zimbabwe. Parity was three or more in about 25% of the women (22.8 and 31.8% in South Africa and Zimbabwe, respectively). The mean height was 159.9 cm (SD ±6.4) in South Africa and 161.1 cm (SD ±6.4) in Zimbabwe; and the mean weight before A total of 2187 women were screened in South Africa and Zimbabwe during the sub-study period (March 2013 to March 2016) and 1101 (50.3%) were eligible and ac- cepted to participate (Fig. 1). Of the 1101 participants randomized, 541 (49.1%) became pregnant of whom, 55 (10.0%) had a miscarriage or a pregnancy termination Fig. 1 Flow chart Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 5 of 10 pregnancy was 78.9 kg (SD ±18.2) in South Africa and 69.0 kg (SD ±12.6) in Zimbabwe. The prevalence of overweight was 27.7% in South Africa and 37.5% in Zimbabwe while the prevalence of any degree of obes- ity was 46.0% in South Africa and 22.7% in Zimbabwe (Table 1). In total, 73.7 and 60.2% women in South Africa and Zimbabwe, respectively entered the trial with BMI above normal. On the other hand, the prevalence of underweight was virtually non-existent in both countries. Results Table 1 Participant Characteristics All South Africa Zimbabwe n = 312 % n = 224 % n = 88 % Age (years) Less than 20 1 0.3 0 0 1 1.1 20 to less than 35 255 81.7 182 81.3 73 83 35 and older 56 17.9 42 18.8 14 17.9 Parity 1 121 38.8 93 41.5 28 31.8 2 111 35.6 79 35.3 32 36.4 3 or more 79 25.3 51 22.8 28 31.8 Missing 1 0.3 1 0.4 0 0 Months in study from admission to 20 weeks´ gestation Mean (sd) 312 12.5 (7.4) 224 13.1 (7.4) 88 11.0 (7.4) Less than 6 month 94 30.1 60 26.8 34 38.6 6 to less than 12 month 88 28.2 59 26.3 29 33.0 12 to less than 24 month 97 31.1 79 35.3 18 20.5 24 or more month 32 10.3 25 11.2 7 8.0 Missing 1 0.3 1 0.4 0 0 Months since last birth with PE to 20 weeks´ gestation Mean (sd) 312 24.5 (22.5) 224 30.3 (23.7) 88 27.4 (19.0) Less than 6 month 9 2.9 7 3.1 2 2.3 6 to less than 12 month 54 17.3 39 17.4 15 17.0 12 to less than 24 month 94 30.1 62 27.7 32 36.4 24 or more month 135 43.3 102 45.5 33 37.5 Missing 20 6.4 14 6.3 6 6.8 Anthropometric variables Height at admission - mean (sd) 286 160.3 (6.4) 202 159.9 (6.4) 84 161.1 (6.4) Weight at admission - mean (sd) 302 76.1 (17.4) 217 78.9 (18.2) 85 69.0 (12.6) Weight at week 8 of gestation - mean (sd) 251 77.1 (16.9) 173 80.2 (17.7) 78 70.2 (12.4) Weight at week 20 of gestation - mean (sd) 300 80.2 (17.5) 215 83.5 (18.1) 85 71.9 (12.6) Weight at week 32 of gestation - mean (sd) 224 84.0 (17.0) 158 88.2 (17.5) 66 74.0 (10.1) Mean BMI at admission - mean (sd) 283 29.6 (6.3) 201 30.7 (6.6) 82 26.8 (4.5) Body Mass Index at admission Underweight (BMI < 18.5 kg/m2) 3 1.0 2 0.9 1 1.1 Normal (18.5 BMI < 25 kg/m2) 62 19.9 34 15.2 28 31.8 Overweight (25 BMI < 30 kg/m2) 95 30.4 62 27.7 33 37.5 Obesity I (30 BMI 35 kg/m2) 75 24.0 58 25.9 17 19.3 Obesity II (35 BMI 40 kg/m2) 32 10.3 30 13.4 2 2.3 Obesity III (BMI > 40 kg/m2) 16 5.1 15 6.7 1 1.1 Missing 29 9.3 23 10.3 6 6.8 Table 1 Participant Characteristics (Table 1). Results In total, 73.7 and 60.2% women in South Africa and Zimbabwe, respectively entered the trial with BMI above normal. On the other hand, the prevalence of underweight was virtually non-existent in both countries. pregnancy was 78.9 kg (SD ±18.2) in South Africa and 69.0 kg (SD ±12.6) in Zimbabwe. The prevalence of overweight was 27.7% in South Africa and 37.5% in Zimbabwe while the prevalence of any degree of obes- ity was 46.0% in South Africa and 22.7% in Zimbabwe pregnancy was 78.9 kg (SD ±18.2) in South Africa and 69.0 kg (SD ±12.6) in Zimbabwe. The prevalence of overweight was 27.7% in South Africa and 37.5% in Zimbabwe while the prevalence of any degree of obes- ity was 46.0% in South Africa and 22.7% in Zimbabwe Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 6 of 10 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Gestational weight gain energy intake. The majority of women in both countries had lower than recommended protein intake and a higher intake of carbohydrates (Table 2). We found that women who were initially classified ac- cording to their BMI as normal weight had gained from 8 to 32 weeks’ gestation an average 8.9 kg (SD ± 4.4) in South Africa and 7.4 kg (SD ± 3.3) in Zimbabwe. Those classified as overweight had gained 7.8 kg (SD ± 4.5) in South Africa and 5.8 kg (SD ±3.8) in Zimbabwe and those classified as obese had gained 5.9 kg (SD ± 6.2) in South Africa and 3.1 kg (SD ± 4.1) in Zimbabwe. Macronutrients The average total daily energy intake was 1765.6 kcal (SD ±346.6) in South Africa and 1827.9 (SD ±303.9) in Zimbabwe. Average daily carbohydrate, fat and protein intakes were 230.8 (SD ± 57.5) grams, 59.1 (SD ± 6.4) grams and 54.7 (SD ±7.8) grams in South Africa and 213.6 (SD ± 22.3) grams, 76.4 (SD ± 24.8) grams and 52.9 (SD ± 25.8) grams in Zimbabwe, respectively (Table 2). Most (54.4%) of total energy intake came from carbohy- drates, 27.8% from fats and 12.6% from proteins in South Africa while the percentages in Zimbabwe were 48.0, 36.38 and 11.26% respectively. Micronutrients Adjusted usual nutrient intakes (from food and beverage, excluding supplements) are presented in Table 2. Micro- nutrient inadequacy was highly prevalent in both coun- tries. Almost all women in South Africa had inadequate intakes of folate, calcium, iron and selenium, while the majority also had inadequate intakes of magnesium, zinc, niacin and vitamin E. More than half of the women had inadequate intakes of riboflavin while less than half had inadequate intake of vitamin C. All women in Zimbabwe had inadequate dietary intakes of iron and folate; the majority also had inadequate intakes of calcium, magne- sium, zinc, selenium and riboflavin. The intake of vita- min C and E was however, adequate in the majority of women from Zimbabwe. (Table 2). Supplements Those classified as overweight gained 7.8 kg (SD ± 4.5) in South Africa and 5.8 kg (SD ±3.8) in Zimbabwe of the 7 to 11.5 kg recom- mended for this group and those classified as obese gained 5.9 kg (SD ± 6.2) in South Africa and 3.1 kg (SD ± 4.1) in Zimbabwe of the 5 to 9 kg recommended for this group [12]. Programmes and interventions to reduce obesity before pregnancy and control weight gain during pregnancy would be advisable in view of the findings of this analysis. (22) reported taking vitamin B complex. At 20 weeks of pregnancy, women reported taking these supplements for a mean period of 2.1 to 2.7 months. Other supple- ments reported include calcium gluconate, magnesium sulphate and copper sulphate. Most of the supplements were provided by the hospital. On the other hand, a total of 29 (12.9%) women reported taking multivitamins for a mean period of 1 to 2 months, which are not provided by the hospitals. In Zimbabwe, only one woman reported taking iron supplements during pregnancy. No other types of sup- plements intake were reported what so ever. The energy intake we report is similar to those reported for women in the US National Health and Nutrition Sur- vey in 2010–2011 where the prevalence of overweight and obesity are also higher than 70% [32, 33]. According to Goldberg, if the estimated usual intake is below 80% of the estimated average requirement for a person, this would imply under-reporting. In our study this would imply 37.7% of underreporting in South Africa and 19.1% in Zimbabwe [25]. Fat intake as a percentage of total en- ergy was within the recommended range of 20 to 35% of total energy in South Africa (27.8%), but slightly higher than recommended in Zimbabwe (36.4%). The high fat in- take in Zimbabwe was due to a higher intake of polyunsat- urated fats. In both countries, carbohydrates and protein intake as a percentage of total energy were within the rec- ommended ranges of 46–65% and 10–35% respectively [23]. However, the intake of sugar in South Africa was slightly above the recommended maximum of 10% of total energy. Considering recommended grams of macronutri- ents, the women from both countries had mostly adequate total grams of carbohydrate intakes, but the majority had inadequate grams per kilograms of protein intake. Discussion This study shows that a high proportion of women whose previous pregnancy was complicated by pre-eclampsia in hospitals from South Africa and Zimbabwe started their subsequent pregnancy overweight or obese (73.7% in South Africa and 60.2% in Zimbabwe). In fact, obesity af- fected about 1 in 4 women in Zimbabwe, and as many as 1 in 2 women in South Africa were obese. Furthermore, at 20 weeks´ gestation more than 90% of these women had intakes of micronutrients, like iron, calcium, folate and zinc below requirements. zinc below requirements. Overweight and obesity problems have already been reported in these countries. The prevalence of over- weight or obese women found in our study is higher than the 64% that has been reported for South Africa by the SANHANES-1 and the 54.9 and 25% that the WHO Global Database on Body Mass Index reports for South Africa in 2004 and Zimbabwe in 2006 respectively, but in line with other studies conducted in South Africa that reported 69% [27–29]. The fact that we only included women who had a previous pregnancy complicated with pre-eclampsia could contribute to the higher overweight or obesity prevalence in our study population [14, 15]. A link between obesity and hypertensive disorders of preg- nancy has been reported in the literature. A systematic review concluded that for every 5 to 7 kg/m2 increase in BMI, the risk of developing pre-eclampsia doubles which confirms the relevance and critical importance of devel- oping and implementing special efforts to control the BMI of these women before they become pregnant [30]. We found that women with normal BMIs’ at 8 weeks´ gestation, compared to those with higher BMI, gained more weight at 32 weeks, which is in accordance to rec- ommendations [31]. However, as we only assessed weight up to 32 weeks´ gestation and most gestational weight gain occurs after 20 weeks´ gestation, we would expect that many of these women would exceed the In- stitute of Medicine recommendations. In our study, women classified as normal weight gained from 8 to 32 weeks’ gestation an average 8.9 kg (SD ± 4.4) in South Africa and 7.4 kg (SD ± 3.3) in Zimbabwe of the 11.5 to Overweight and obesity problems have already been reported in these countries. Discussion The prevalence of over- weight or obese women found in our study is higher than the 64% that has been reported for South Africa by the SANHANES-1 and the 54.9 and 25% that the WHO Global Database on Body Mass Index reports for South Africa in 2004 and Zimbabwe in 2006 respectively, but in line with other studies conducted in South Africa that reported 69% [27–29]. The fact that we only included women who had a previous pregnancy complicated with pre-eclampsia could contribute to the higher overweight or obesity prevalence in our study population [14, 15]. A link between obesity and hypertensive disorders of preg- nancy has been reported in the literature. A systematic review concluded that for every 5 to 7 kg/m2 increase in BMI, the risk of developing pre-eclampsia doubles which confirms the relevance and critical importance of devel- oping and implementing special efforts to control the BMI of these women before they become pregnant [30]. The prevalence of inadequate micronutrient intake from food sources was high in both countries. For the most basic micronutrients like iron, calcium, folate and zinc, the percentage of women below requirements was above 90% in both countries. The most common supple- ments taken in South Africa were folic acid, ferrous sulphate, and vitamin C, all issued by the hospital. There is a policy in South Africa to supplement pregnant women with 5 mg of folic acid and 200 mg of ferrous sulphate (equivalent to 40 mg elemental iron) daily, which allowed those women taking the supplements to reach the recommendations [34, 35]. The elemental iron supplementation provided by this policy is in accordance with the WHO guidelines for antenatal care, however folic acid supplementation is more than 10 times the recommended amount [12]. On the other hand, vitamin C is not recommended, as there is no evidence of an im- pact on birth outcomes. In contrast, only one woman in Zimbabwe reported taking supplements and this may be We found that women with normal BMIs’ at 8 weeks´ gestation, compared to those with higher BMI, gained more weight at 32 weeks, which is in accordance to rec- ommendations [31]. However, as we only assessed weight up to 32 weeks´ gestation and most gestational weight gain occurs after 20 weeks´ gestation, we would expect that many of these women would exceed the In- stitute of Medicine recommendations. Supplements In South Africa, 62.9% of women (141) reported taking 5 mg of folic acid supplements, 57.1% (128) reported taking iron supplements with doses ranging between 75 to 400 mg; 24.1% (54) reported taking vitamin C with doses ranging between 100 to 250 mg daily and 9.8% Average daily intake of total sugars was 45.6 (SD ± 40.9) grams in South Africa and 34.5 (SD ±26.0) grams in Zimbabwe representing 10.6 and 7.5% of the total Table 2 Usual intake from foods and beverages, excluding supplements, estimated using repeated 24-h recalls in 312 women and repeated in a sub-sample of 107 women, and percentage of women with usual intake below Estimated Average Requirement (EAR) Estimated Daily Usual Intakeb South Africa Zimbabwe EARa % women with intakes below EAR n = 224 n = 88 South Africa Zimbabwe Energy (kcal) 1765.6 (346.6) 1827.9 (303.9) NAc NA NA EER Estimated Energy Requirement (%) 0.88 (0.20) 0.93 (0.14) 0.8 37.7 19.1 Protein (g) 54.7 (7.8) 52.9 (25.8) d NA NA NA Protein (g/kg) 0.82 (0.43) 0.79 (0.04) 0.88 71.1 98.3 Carbohydrates (g) 230.8 (57.5) 213.6 (22.3) 135 02.9 0.00 Total Sugars (g) 45.4 (19.4) 35.4 (10.7) NA NA NA Fats (g) 59.1 (6.4) 76.4 (24.8) NA NA NA Calcium (mg) 441.0 (97.7) 360.5 (171.4) 800 99.9 97.6 Iron (mg) 9.9 (5.3) 7.9 (1.7) 22 96.9 100.0 Folate (mcg) 253.2 (69.1) 240.6 (46.2) 520 99.9 100.0 Mg (mg) 239.6 (51.1) 262.5 (38.8) 290 83.9 77.3 Zn (mg) 7.3 (1.7) 6.4 (1.6) 9.5 90.4 95.7 Se (mcg) 37.9 (4.3) 37.5 (11.8) 49 99.1 83.7 Riboflavin (mg) 1.3 (0.6) 0.8 (0.5) 1.2 54.0 84.0 Niacin (mg) 13.0 (3.8) 13.0 (3.7) 14 63.8 64.9 Vitamin C (mg) 82.4 (45.1) 109.80 (41.8) 70 46.6 15.6 Vitamin E (mg) 10.1 (3.8) 26.4 (9.1) 12 73.5 03.7 aEAR Estimated average requirement Table 2 Usual intake from foods and beverages, excluding supplements, estimated using repeated 24-h reca repeated in a sub-sample of 107 women, and percentage of women with usual intake below Estimated Ave m foods and beverages, excluding supplements, estimated using repeated 24-h recalls in 312 women and e of 107 women, and percentage of women with usual intake below Estimated Average Requirement (EAR) Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 7 of 10 Page 7 of 10 16 kg recommended for this group. Supplements It is thus important that interventions should focus on increas- ing the intake of affordable protein sources and decreasing sugar intake. Discussion In our study, women classified as normal weight gained from 8 to 32 weeks’ gestation an average 8.9 kg (SD ± 4.4) in South Africa and 7.4 kg (SD ± 3.3) in Zimbabwe of the 11.5 to Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Page 8 of 10 Page 8 of 10 fewer than 50 repeated interview for Zimbabwe so the estimation might not be as accurate as for South Africa. due to the fact that there is no policy to provide supple- ments during pregnancy in Zimbabwe and very few women bought commercial supplements. The inad- equate intakes are worrying as iron and folic acid sup- plementation are known to prevent anaemia, puerperal sepsis, low birth weight and preterm birth; folate to pre- vent neural tube defects, calcium supplementation in areas with low calcium intake in order to reduce risk of pre-eclampsia; vitamin A in deficient areas to prevent night blindness. Regarding the diet a higher intake of vitamin E is usually related to higher intakes of oils and fats whereas vitamin C indicates an increased intake of fruits and vegetables [36, 37]. Acknowledgements We thank the CAP trial researchers at the sites: Emilia Makaza, Eunice Tahuringana and Bothwell Guzha (Harare, Zimbabwe); Catherine Parker and Gift Phoramphai (Johannesburg, South Africa); Patience Moloi, Annemarie Greef and Saadiqa Alie (Cape Town, South Africa); Xoliswa Williams and Pamela Njikelana (East London, South Africa); Erika von Papendorp (Stellenbosch, South Africa) and Nicole Minckas, Ricardo López and Paula Rubinstein. We especially thank all women in South Africa and Zimbabwe who took part in the trial. Conclusion We found a high prevalence of overweight and obesity and high prevalence of inadequate intakes of protein and micronutrients in pregnant women who had a previous pregnancy complicated with pre-eclampsia. Although this group is not representative of the general population of pregnant woman, taking into account the increasing prevalence of overweight and obesity worldwide among young age groups, the obesity and micronutrient inad- equacy figures reported in this study are issues of con- cern that need to be addressed so that maternal and perinatal outcomes are improved [42, 43]. Noticing the differences found in both countries regarding BMI and nutrient intake it would be interesting to explore the reasons behind as it could help to tackle the problem. Limitations We did not use the same scales to measure weight across sites, as weight was not the main outcome of this trial. However, women were assessed with the same scale throughout the study at each participating site. Strengths Strengths of this study include the standardised proce- dures used throughout the trial in all sites. Women in this study had close follow up from the same research team before and during pregnancy. Supplement intakes during pregnancy seem to be essen- tial for these groups of women to achieve requirements of key micronutrients. Policies should be reinforced and reviewed according to the most recent evidence. Preg- nancy is a period when women may have more regular contacts with the health system and, if health care services were integrated for mothers and babies, these regular con- tacts could be maintained after delivery to improve mater- nal health [44]. These opportunities could be used to deliver dietary and nutritional interventions to high-risk women to improve the outcomes of future pregnancies [45]. Furthermore, taking into account that women with a history of pre-eclampsia are at higher risk of developing cardiovascular disease later in life, pregnancy and postna- tal periods could be an ideal time for preventing future health complication from a young age. The 24-h recall dietary assessment method used is subject to less recall bias than other dietary assessment methods, such as diet histories or food frequency check- lists [38]. Major advantages of using 24-h recalls are that high literacy of the respondent is not required and that inter-observer differences are minimised [18]. On the other hand, food frequency questionnaires usually re- quire use of generic memory and higher numeracy skills in the population interviewed to quantify average food intakes over a period of time [39]. Abbreviations BMI B d We did not use any technique to corroborate energy intake, however under-reporting has been described in the literature in women especially in those with high BMI [40]. Abbreviations BMI: Body mass index; CAP: The calcium and pre-eclampsia study; DRI: Dietary reference intake; EAR: Estimated average requirement; EER: Estimated energy requirement; IOM: Institute of Medicine; SAMRC: South African Medical Research Council; SANHANES-1: National Health and Nutrition Examination Survey; WHO: World Health Organization Food data from both countries was analysed using the SAMRC-Food composition database as there is not a local food composition database in Zimbabwe. There were a few cases where foods reported during the assess- ment were not found in the database, and they were added as the most similar item in terms of macronutri- ents and calcium content. Nevertheless, this was only in a few cases and we believe it cannot affect the main re- sults of this sub-study. Although not having a database for Zimbabwe might be a limitation, it prevents showing differences that are related to errors of the food compos- ition tables rather than of the nutrient intake [41]. Competing interests The authors declare that they have no competing interests. Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Argentina Fund for Horizontal Cooperation of the Argentinean Ministry of Foreign Affairs. The Research Council of Norway, through its Centers of Excellence scheme and the University of Bergen (UiB), Norway, and the Centre for Intervention Science in Maternal and Child Health, supported this sub-study (CISMAC; project number 223269). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Argentina Fund for Horizontal Cooperation of the Argentinean Ministry of Foreign Affairs. The Research Council of Norway, through its Centers of Excellence scheme and the University of Bergen (UiB), Norway, and the Centre for Intervention Science in Maternal and Child Health, supported this sub-study (CISMAC; project number 223269). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. 2. Popkin BM. Nutrition in transition: The changing global nutrition challenge. Asia Pac J Clin Nutr. 2001;10:Suppl S13–8. 3. Lindsay KL, Gibney ER, McAuliffe FM. Maternal nutrition among women from sub-Saharan Africa, with a focus on Nigeria, and potential implications for pregnancy outcomes among immigrant populations in developed countries. J Hum Nutr Die. 2012;25(6):534–46. 3. Lindsay KL, Gibney ER, McAuliffe FM. Maternal nutrition among women from sub-Saharan Africa, with a focus on Nigeria, and potential implications for pregnancy outcomes among immigrant populations in developed countries. J Hum Nutr Die. 2012;25(6):534–46. 4. Mission JF, Marshall NE, Caughey AB. Pregnancy risks associated with obesity. Obstet Gynecol Clin N Am. 2015;42(2):335–53. 4. Mission JF, Marshall NE, Caughey AB. Pregnancy risks associated with obesity. Obstet Gynecol Clin N Am. 2015;42(2):335–53. 5. Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis MUR. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013;382(9890):427–51. 5. Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis MUR. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013;382(9890):427–51. Publisher’s Note S N 16. WHO: World Health Organization. In Guideline: Calcium supplementation in pregnant women. Edited by World Health Organization. Geneva: Geneva, World Health Organization, 2013. [http://apps.who.int/iris/bitstream/10665/ 85120/1/9789241505376eng.pdf]. Accessed 29 May 2018. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. WHO Consultation on Obesity. Obesity: preventing and managing the global epidemic: report of a WHO consultation. Geneva: World Health Organization; 1999. Received: 11 August 2017 Accepted: 5 June 2018 Received: 11 August 2017 Accepted: 5 June 2018 Received: 11 August 2017 Accepted: 5 June 2018 23. IOM (Institute of Medicine) and NRC (National Research Council). Weight gain during pregnancy: Reexamining the Guidelines. Washington: The National Academies Press; 2009. Availability of data and materials h d d d l The datasets generated and/or analysed during the current study are not publicly available as the study is still ongoing, but are available from the corresponding author on reasonable request. 6. Marchi J, Berg M, Dencker A, Olander EK, Begley C. Risks associated with obesity in pregnancy, for the mother and baby: a systematic review of reviews. Obes Rev. 2015;16(8):621–38. 6. Marchi J, Berg M, Dencker A, Olander EK, Begley C. Risks associated with obesity in pregnancy, for the mother and baby: a systematic review of reviews. Obes Rev. 2015;16(8):621–38. Authors’ contributions 7. Spradley FT, Palei AC, Granger JP. Increased risk for the development of preeclampsia in obese pregnancies: weighing in on the mechanisms. Am J Physiol Regul Integr Comp Physiol. 2015;309(11):R1326–43. 7. Spradley FT, Palei AC, Granger JP. Increased risk for the development of preeclampsia in obese pregnancies: weighing in on the mechanisms. Am J Physiol Regul Integr Comp Physiol. 2015;309(11):R1326–43. GC, APB, GJH, CP, TDP and JB designed the substudy and interpreted the data; GC and JH performed statistical analysis; GC, APB, JB and JH drafted the manuscript with contributions from DH, AHS, JR, GJH. All named authors critically reviewed and approved the final version. 8. Ramakrishnan U, Grant F, Goldenberg T, Zongrone A, Martorell R. Effect of women's nutrition before and during early pregnancy on maternal and infant outcomes: a systematic review. Paediatr Perinat Epidemiol. 2012; 26(Suppl 1):285–301. Ethics approval and consent to participate Ethical approval was obtained from appropriate national and institutional ethics review bodies as applicable for each study site, and all participants provided informed written consent. The study was approved by the Research Project Review Panel of the United Nations Development Programme (UNDP) - United Nations Population Fund (UNFPA) - United Nations Children’s Emergency Fund (UNICEF) - World Health Organization (WHO) - World Bank Special Programme of Research, Development and Research Training in Human Reproduction at the Department of Reproductive Health and Research of World Health Organization, and the World Health Organization Research Ethics Review Committee, Geneva, Switzerland. Approval has been obtained from the Health Sciences Faculty, Human Research Ethics Committee (HREC) of University of Cape Town (No HREC 457/2010), Health Research Ethics Committee 1, University of Stellenbosch, Cape Town, South Africa, Medical Research Council of Zimbabwe and the Human Research Ethics Committee (Medical), University of the Witwatersrand, Johannesburg, South Africa. 9. Dean SV, Lassi ZS, Imam AM, Bhutta ZA. Preconception care: nutritional risks and interventions. 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Martorell R, Khan LK, Hughes ML, Grummer-Strawn LM. Obesity in women from developing countries. EurJClinNutr. 2000;54:247–52. 15. Hofmeyr GJ. Protocol 11PRT/4028: Long term calcium supplementation in women at high risk of pre-eclampsia: a randomised, placebo-controlled trial (PACTR201105000267371). Lancet. 2011 [http://www.thelancet.com/ protocol-reviews/11PRT-4028]. Accessed 29 May 2018. 1. Young MF, Nguyen PH, Addo OY, Hao W, Nguyen H, Pham H, Martorell R, Ramakrishnan U. The relative influence of maternal nutritional status before and during pregnancy on birth outcomes in Vietnam. Eur J Obstet Gynecol Reprod Biol. 2015;194:223–7. Funding The Calcium and Pre-eclampsia (CAP) study is part of the PRE-EMPT (Pre-eclampsia- Eclampsia, Monitoring, Prevention and Treatment) study and is supported by the University of British Columbia, a grantee of the Bill & Melinda Gates Foundation; United Nations Development Programme (UNDP) - United Nations Population Fund (UNFPA) - United Nations Children'’s Emergency Fund (UNICEF) - World Health Organization (WHO) • World Bank Special Programme of Research, Development and Research Training in Human Reproduction, Department of Reproductive Health and Research; and the The IOWA methodology to estimate usual intake re- quires at least 50 repeated interviews, we obtained re- peated 24-h recall for 107 women, however we obtained Page 9 of 10 Page 9 of 10 Author details 1 1Instituto de Efectividad Clínica y Sanitaria (IECS-CONICET), Emilio Ravignani, 2024 Buenos Aires, Argentina. 2Division of Human Nutrition, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. 3HRP – UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction, Department of Reproductive Health and Research, World Health Organization, Geneva, Switzerland. 4WHO Regional Office for Europe, World Health Organization, Copenhagen, Denmark. 5Effective Care Research Unit, University of the Witwatersrand, Johannesburg, South Africa. 6Effective Care Research Unit, Eastern Cape Department of Health Walter Sisulu University, Mthatha, South Africa. 7Effective Care Research Unit, University of Fort Hare, East London, South Africa. 8Department of Obstetrics and Gynaecology, Stellenbosch University and Tygerberg Hospital, Cape Town, South Africa. 9Epidemiología y Microbiología La Habana, Instituto Nacional de Higiene, Havana, Cuba. 10Magee-Womens Research Institute, Department of Obstetrics and Gynecology, University of Pittsburgh, Pittsburgh, USA. 18. 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Boon N, Hul GB, Stegen JH, Sluijsmans WE, Valle C, Langin D, Viguerie N, Saris WH. An intervention study of the effects of calcium intake on faecal fat excretion, energy metabolism and adipose tissue mRNA expression of lipid- metabolism related proteins. Int J Obes. 2007;31(11):1704–12. Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 Cormick et al. BMC Pregnancy and Childbirth (2018) 18:236 principles of energy physiology: 1. Derivation of cut-off limits to identify under-recording. Eur J Clin Nutr. 1991;45:569–81. principles of energy physiology: 1. Derivation of cut-off limits to identify under-recording. Eur J Clin Nutr. 1991;45:569–81. 26. WHO Technical Report Series. Guidelines for Good Clinical Practice (GCP) for Trials on Pharmaceutical Products, vol. 850. Geneva: World Health Organization; 1995. p. 97–137. 27. World Health Organisation. BMI classifications. Geneva: WHO; 2006. http:// apps.who.int/bmi/index.jsp?introPage=intro_3.html. Accessed 29 May 2016. 28. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the global burden of disease study 2013. Lancet. 2014;384:766–81. burden of disease study 2013. Lancet. 2014;384:766–81. 29. Nieuwoudt M, van der Merwe JL, Harvey J, Hall DR. Pregnancy outcomes in super-obese women - an even bigger problem? A prospective cohort study. S Afr J OG. 2014;20(2):54–9. 30. O’Brien TE, Ray JG, Chan WS. Maternal body mass index and the risk of preeclampsia: a systematic overview. Epidemiology. 2003;14(3):368–74. 31. Rasmussen KM, Catalano PM, Yaktine AL. New guidelines for weight gain during pregnancy: What obstetrician/gynecologists should know. Curr Opin Obstet Gynecol. 2009;21(6):521–6. 32. Trends in energy intake among adults in the United States: findings from NHANES. Am J Clin Nutr. 2013;97(4):848–53. 33. Fryar C, Carrol MD, Ogden CL. Prevalence of Overweight, Obesity, and Extreme Obesity Among Adults: United States, 1960–1962 Through 2011– 2012. Atlanta: National Center for Health Statistics; 2016. Accessed 29 May 2016 34. Standard Treatment Guidelines and Essential Medicines List for South Africa. Hospital Level, Adults. 4th ed. Pretoria: The National Department of Health; 2015. 35. National Department of Health, South Africa. Guidelines for Maternity Carein South Africa A Manual for Clinics, Community Dent HealthCommunity Health Centres and District Hospitals, vol. 172. 4th ed. Pretoria: NDoH; 2015. http://www.health.gov.za/index.php/2014-03-17-09-09-38/policies-and- guidelines/category/230-2015p#. Accessed 29 May 2016 36. Zhao Y, Monahan FJ, McNulty BA, Gibney MJ, Gibney ER. Effect of vitamin E intake from food and supplement sources on plasma α- and γ-tocopherol concentrations in a healthy Irish adult population. Br J Nutr. 2014;112(9): 1575–85. 37. 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https://openalex.org/W3134775266	https://www.nature.com/articles/s41598-021-00685-w.pdf	English	null	Oscillatory visual mechanisms revealed by random temporal sampling	Scientific reports	2,021	cc-by	10,194	Oscillatory visual mechanisms revealed by random temporal sampling OPEN 1,2, Roxanne Ferrandez3 & Justine Massé Martin Arguin 1,2, Roxanne Ferrandez3 & Justine Massé3 It is increasingly apparent that functionally significant neural activity is oscillatory in nature. Demonstrating the implications of this mode of operation for perceptual/cognitive function remains somewhat elusive. This report describes the technique of random temporal sampling for the investigation of visual oscillatory mechanisms. The technique is applied in visual recognition experiments using different stimulus classes (words, familiar objects, novel objects, and faces). Classification images reveal variations of perceptual effectiveness according to the temporal features of stimulus visibility. These classification images are also decomposed into their power and phase spectra. Stimulus classes lead to distinct outcomes and the power spectra of classification images are highly generalizable across individuals. Moreover, stimulus class can be reliably decoded from the power spectrum of individual classification images. These findings and other aspects of the results validate random temporal sampling as a promising new method to study oscillatory visual mechanisms. There is growing evidence suggesting that functionally relevant neural communication takes the form of periodic synchronized firing by groups of neurons1–4. At a macroscopic level, this takes the form of oscillatory activity which can be recorded in humans by electroencephalography (EEG) or magnetoencephalography (MEG5).h There is growing evidence suggesting that functionally relevant neural communication takes the form of periodic synchronized firing by groups of neurons1–4. At a macroscopic level, this takes the form of oscillatory activity which can be recorded in humans by electroencephalography (EEG) or magnetoencephalography (MEG5).h y p g p y g p g p y In vision, investigation of the functional impact of brain oscillations has largely focused on attention. Thus, by recording brain activity with EEG or MEG or by interfering with it using transcranial magnetic stimulation (TMS), it has been determined that spatial attention operates at a rate of about 5–8 Hz and is driven by oscilla- tory brain activity in the high-theta/low-alpha frequency bands6–11. y y g p q y As a general principle, if brain oscillations underlie faculties such as perception and cognition, then this should be manifest in behavioral performances. For instance, perceptual effectiveness should oscillate rapidly through time, in contrast to subjective experience which rather suggests a stable capacity. Empirical evidence in support of perceptual oscillations has been reported based on the periodicity of performance measures in humans carrying out various perceptual tasks12–18. www.nature.com/scientificreports www.nature.com/scientificreports Oscillatory visual mechanisms revealed by random temporal sampling OPEN The capacity of this approach to characterize putative oscil- latory perceptual mechanisms remains limited, however, and a source for more potent and detailed evidence is clearly desirable. The new technique described below offers such advances which will be addressed in the discussion section. Initially proposed by [19; see also20], we have enhanced the test and analysis procedures for the technique of random temporal sampling to make it a powerful tool to investigate oscillatory visual mechanisms. In addition to demonstrating temporal inhomogeneity in processing effectiveness, the experiments reported here also reveal crucial features of the oscillatory mechanisms involved. As shown below, some of these features are shared to a remarkable degree among observers and they vary substantially between stimulus conditions, such that they are diagnostic of the specific task participants perform. gi Adult neurologically intact human observers carried out visual recognition tasks using words, familiar or novel objects, or faces. Throughout their exposure duration (200 ms), the stimuli were sampled by a manipula- tion of target visiblity (controlled by signal-to-noise ratio), which oscillated according to a random function. Specifically, the stimuli displayed were made from the linear combination of a target stimulus (the signal) and a white noise field (the noise; Fig. 1). The signal-to-noise ratio varied according to a complex pattern made from the integration of 5–55 Hz sine waves (in 5 Hz steps) with random amplitudes and phases, thus making the 1Centre interdisciplinaire de recherche sur le cerveau et l’apprentissage (CIRCA), Département de psychologie, Université de Montréal, Montreal, Canada. 2Centre de recherche, Institut Universitaire de Gériatrie de Montréal, Montreal, Canada. 3Département de psychologie, Université de Montréal, Succ. Centre‑ville, C.P. 6128, Montréal, QC H3C 3J7, Canada. email: martin.arguin@umontreal.ca \| https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The stimuli displayed on every trial were made from the addition of two components, the signal and the noise. The ‘signal’ was made from an image of the target stimulus superimposed with a white noise field whose contrast was adjusted to maintain performance at about 50% correct (see “Methods”). The ‘noise’ was made from a separate white noise field with maximum contrast. New white noise fields were generated independently on each trial. The signal-to-noise ratio of the stimuli displayed was varied across their 200-ms exposure duration according to a new random function generated on each trial (see “Methods”). Figure 1. Oscillatory visual mechanisms revealed by random temporal sampling OPEN The stimuli displayed on every trial were made from the addition of two components, the signal and the noise. The ‘signal’ was made from an image of the target stimulus superimposed with a white noise field whose contrast was adjusted to maintain performance at about 50% correct (see “Methods”). The ‘noise’ was made from a separate white noise field with maximum contrast. New white noise fields were generated independently on each trial. The signal-to-noise ratio of the stimuli displayed was varied across their 200-ms exposure duration according to a new random function generated on each trial (see “Methods”). temporal sampling function. A new random temporal sampling function was generated on each trial thereby making them unpredictable.ifi Classification images21 of visual processing efficiency were then constructed by the weighted subtraction of the sampling functions on which participants made errors from those on which they produced a correct response (Fig. 2). For this operation, the sampling functions were either coded as signal-to-noise ratio variations through time (time domain), the power and phase values from which the sampling functions were constructed (Fourier domain), or the local temporal frequency content of these functions through time (time–frequency domain). Processing efficiency refers to the capacity of participants to use the stimulus information available to perform their task correctly.tfii y After converting the processing efficiency metric of the individual raw classification images into Z scores by a bootstrapping operation, these classification images were averaged, smoothed and then submitted to the Pixel test[22; see “Methods” section for details] to determine the points that differed significantly from zero. Resultshi time sampling functions illustrated on the top right and bottom left of the figure are actual functions which were associated with either correct responses or errors in the word recognition task. The raw classification images were obtained from the weighted subtraction of the sums of the sampling functions (coded in the time, Fourier, or time–frequency domain) associated with errors from those associated with correct responses. They were then transformed into Z scores by bootstrapping, smoothing, and submitted to the Pixel test22. ll f h b l f h l h h f h d ‘ ’ Figure 2. Illustration of the variability of the signal-to-noise ratio through time for the target word ‘navet’ (top left) and of the procedures applied to construct the classification images. The random temporal sampling functions generated on each trial were coded as either signal-to-noise ratio variations as a function of time since target onset (time domain), in terms of the amplitude and phase values of the 5–55 Hz (in 5-Hz steps) sinusoidal functions from which the temporal sampling functions were constructed (Fourier domain), or in terms of the instantaneous oscillation frequencies of the signal-to-noise ratio as a function of time since target onset (time– frequency domain). The signal-to-noise ratio vs. time sampling functions illustrated on the top right and bottom left of the figure are actual functions which were associated with either correct responses or errors in the word recognition task. The raw classification images were obtained from the weighted subtraction of the sums of the sampling functions (coded in the time, Fourier, or time–frequency domain) associated with errors from those associated with correct responses. They were then transformed into Z scores by bootstrapping, smoothing, and submitted to the Pixel test22. to SOA but that this effect interacts significantly with the temporal frequency content of the variations of target visibility through time. Again, these observations are verified for all the stimulus classes tested.i i Classification images of static frequencies of stimulus oscillation based on the Fourier descriptors of the temporal profiles of stimulus sampling were also calculated. None of these classification images showed any significant difference from zero (see Supplementary Fig. S1), meaning that they fail to capture the temporal fea- tures of the target which impact processing efficiency. Resultshi The classification images in Fig. 3 show variations of processing efficiency in the time and time–frequency domains for each stimulus class. The time domain classification images (Fig. 3a–d) illustrate processing efficiency as a function of the time elapsed since the beginning of target display (i.e. stimulus onset asynchrony; SOA). The red dotted horizontal lines indicate the significance thresholds for the Pixel test. Every portion of the classification images between those red lines does not differ significantly from zero, meaning that processing efficiency was effectively null, i.e. the target information available at these moments had no impact on performance. Portions of these classification images that are above the top red line indicate that participants were able to use the target information displayed at that particular time to benefit performance. In contrast, portions of the classification images that are below the bottom red line indicate that target information displayed at that time significantly interfered with performance. It is clear from these illustrations that processing efficiency varies substantially throughout the exposure duration of a visual target, whichever the stimulus class.fi Processing efficiency according to a time–frequency representation of the temporal sampling functions is shown in Fig. 3e–h for each stimulus class. In these illustrations, points at which processing efficiency does not significantly differ from zero are in white. Warmer colours are associated with significant positive values of processing effectiveness (i.e. the target information available led to performance improvements) whereas cooler colours are associated with significant negative values (i.e. the target information displayed interfered with performance). It can easily be seen from these figures that visual processing efficiency varies not only according Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ Figure 2. Illustration of the variability of the signal-to-noise ratio through time for the target word ‘navet’ (top left) and of the procedures applied to construct the classification images. The random temporal sampling functions generated on each trial were coded as either signal-to-noise ratio variations as a function of time since target onset (time domain), in terms of the amplitude and phase values of the 5–55 Hz (in 5-Hz steps) sinusoidal functions from which the temporal sampling functions were constructed (Fourier domain), or in terms of the instantaneous oscillation frequencies of the signal-to-noise ratio as a function of time since target onset (time– frequency domain). The signal-to-noise ratio vs. Resultshi Here also, this finding characterizes each of the stimulus classes used in our experiments.i Another important feature of the results that is apparent from the inspection of Fig. 3 is that the classifica- tion images, whether in the time or time–frequency domain, are very different according to stimulus class. This observation suggests that the temporal features that determine perceptual encoding are affected by the particular demands of the task that needs to be performed. This impression is confirmed by the contrast classification images displayed in Figs. 4 and 5. These were constructed by subtracting the classification image obtained with one stimulus class from that of another, with transformation into Z scores by bootstrapping, followed by smooth- ing, and the application of the Pixel test to determine where differences were significantly different from zero. Illustration conventions are as in Fig. 3.i g One important issue we wanted to examine was the degree to which the classification images obtained from the random temporal sampling technique generalized across individuals. In other words, to what degree can the classification image obtained from one participant predict that from another? This question is important not only to determine how representative the present results are of the general population, but also to index the degree to which the mechanisms captured by the classification images shown in Fig. 3 are fundamental, or essential, for the visual system. To address this question, we calculated the agreement between individual classification images using the intraclass correlation coefficient (ICC23). The theoretical upper bound for the ICC is 1 and it has no lower bound. The mean ICC across stimulus classes is very low for both the time (mean ICC = − 0.90) Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ nature.com/scientificreports/ and time–frequency (mean ICC=0 07) classification images Detailed results for each stimulus class as well as Figure 3. Classification images of encoding effectiveness (in Z scores) as a function of the time elapsed since target onset (stimulus onset asynchrony; SOA—panels a–e) and as a joint function of the frequency content of stimulus oscillations and SOA (panels e–h). Stimuli are: words (a,e), familiar objects (b,f), novel objects (c,d), and faces (d,h). Figure 3. Classification images of encoding effectiveness (in Z scores) as a function of the time elapsed since target onset (stimulus onset asynchrony; SOA—panels a–e) and as a joint function of the frequency content of stimulus oscillations and SOA (panels e–h). nd time–frequency (mean ICC = 0.07) classification images. Detailed results for each stimulus class as well as onfidence intervals are shown in Supplementary Table S1. Resultshi Stimuli are: words (a,e), familiar objects (b,f), novel objects (c,d), and faces (d,h). 4 21309 \| https://doi.org/10.1038/s41598-021-00685-w and time–frequency (mean ICC = 0.07) classification images. Detailed results for each stimulus class as well as confidence intervals are shown in Supplementary Table S1. and time–frequency (mean ICC = 0.07) classification images. Detailed results for each stimulus class as well as confidence intervals are shown in Supplementary Table S1. https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 \| www.nature.com/scientificreports/ w.nature.com/scientificreports/ Classification images however, do contain generalizable information which may be revealed once properly tr t d Th F ri r n l r rri d t n indi id l tim d m in l ifi ti n im ll Figure 4. Contrast classification images in the time domain. Each graph expresses the differences in processing effectiveness (in Z scores) as a function the time elapsed since target onset (stimulus onset asynchrony; SOA) for a particular pair of tasks (defined by stimulus class). The ends of the ‘Processing efficiency difference’ scales are labelled by one of the stimulus classes being contrasted and difference values close to one end or the other end favour the corresponding stimulus class. All possible pairs of tasks have been contrasted: (a) words minus familiar objects; (b) words minus novel objects; (c) words minus faces; (d) familiar objects minus novel objects; (e) familiar objects minus faces; (f) novel objects minus faces. Figure 4. Contrast classification images in the time domain. Each graph expresses the differences in processing effectiveness (in Z scores) as a function the time elapsed since target onset (stimulus onset asynchrony; SOA) for a particular pair of tasks (defined by stimulus class). The ends of the ‘Processing efficiency difference’ scales are labelled by one of the stimulus classes being contrasted and difference values close to one end or the other end favour the corresponding stimulus class. All possible pairs of tasks have been contrasted: (a) words minus familiar objects; (b) words minus novel objects; (c) words minus faces; (d) familiar objects minus novel objects; (e) familiar objects minus faces; (f) novel objects minus faces. Classification images however, do contain generalizable information which may be revealed once properly extracted. Thus, Fourier analyses were carried out on individual time domain classification images as well as separately for each stimulus sampling frequency (5–55 Hz in 5-Hz steps) of individual time–frequency classifi- cation images. Resultshi They were thus decomposed into their power and phase spectra, which were then submitted to an assessment of between-subject agreement. This agreement was reasonably high for the phase spectra of the time domain classification images (mean ICC = 0.66 across stimulus classes) but very low for the time–frequency classification images (mean ICC = 0.06 across stimulus classes). In contrast, the power spectra of classification https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 \| www.nature.com/scientificreports/ atu e co /sc e t c epo ts/ Figure 5. Contrast classification images in the time–frequency domain. Each graph expresses the differences in processing effectiveness (in Z scores) as a joint function of the frequency content of stimulus oscillations and SOA for a particular pair of tasks (defined by stimulus class). The ends of the ‘Processing efficiency difference’ scales are labelled by one of the stimulus classes being contrasted and difference values close to one end or the other end favour the corresponding stimulus class. All possible pairs of tasks have been contrasted: (a) words minus familiar objects; (b) words minus novel objects; (c) words minus faces; (d) familiar objects minus novel objects; (e) familiar objects minus faces; (f) novel objects minus faces. Figure 5. Contrast classification images in the time–frequency domain. Each graph expresses the differences in processing effectiveness (in Z scores) as a joint function of the frequency content of stimulus oscillations and SOA for a particular pair of tasks (defined by stimulus class). The ends of the ‘Processing efficiency difference’ scales are labelled by one of the stimulus classes being contrasted and difference values close to one end or the other end favour the corresponding stimulus class. All possible pairs of tasks have been contrasted: (a) words minus familiar objects; (b) words minus novel objects; (c) words minus faces; (d) familiar objects minus novel objects; (e) familiar objects minus faces; (f) novel objects minus faces. images were remarkably stable across participants for both types of classification images and all four stimulus classes (mean ICC across stimulus classes = 0.96 for the time domain; 0.98 for the time–frequency domain). Discussion Four visual recognition experiments using the technique of random temporal sampling were carried out in normal adult observers with different stimulus classes. The classification images constructed from the relation between response accuracy and temporal features of stimulus sampling demonstrate that visual processing efficiency varies markedly throughout stimulus exposure in conjunction with stimulus oscillation frequencies (Fig. 3). These features falsify subjective impressions of the stability of visual capacity through time. They rather support the hypothesis that the neural systems underlying visual processing are oscillatory.i pp yp y y g p g y Somewhat surprisingly, the present results indicate that temporal and time–frequency domain classification images are quite variable across individuals and thus that their means may not constitute an ideal representation of the perceptual mechanisms studied. Indeed, the index of between-subject agreement was very low for both the time and time–frequency classification images.i q yi g A decomposition of these classification images into their power and phase components however, yields a more substantial picture. It demonstrates strikingly consistent power spectra among participants tested with the same stimulus class, with agreement indexes very close to the theoretical maximum of 1. In contrast, between-subject agreement is much lower for the phase spectra of classification images. This means that while their power spec- tra are nearly identical, the time and time–frequency classification images vary across participants because of mismatching phase spectra. These results indicate that different observers engage to the same degree a number of oscillatory mechanisms which are necessary to perform a given task. However, the temporal relations among these oscillations as well as with stimulus events is what varies substantially across observers. It is the latter which is responsible for the poor between-subject consistency of individual classification images.i p p j yi g In addition to excellent between-subject agreement, the power spectra of time and time–frequency classifi- cation images are rather unique for each task. Thus, their patterns differ significantly as a function of stimulus class. Taken together, these two features of the power spectra of classification images make it possible to use them to determine the stimulus class they originate from with significant accuracy. This observation strongly reinforces the idea that random temporal sampling taps fundamental visual mechanisms that are largely shared among neurologically intact adults. Differences among stimulus classes. The capacity of time and time–frequency domain classification images (Figs. www.nature.com/scientificreports/ and stimulus class as factors showed no significant interaction (F(36, 480) = 1.7; ns). This means that the phase spectra of classification images did not differ significantly as a function of the stimulus class participants had to process. Similarly, the phase spectra of time–frequency classification images failed to reveal a task-specific pat- tern. Thus, the ANOVA with frequency in classification image × sampling frequency × stimulus class showed no significant interaction involving the latter factor (sampling frequency × class: F(30, 400) = 1.11; ns; classification image frequency × class: F(36, 480) < 1; 3-way interaction: F(360, 4800) < 1).if g q y y In contrast to the phase spectra, the power spectra of classification images offered distinct patterns depend- ing on the stimulus class participants were required to recognize. Thus, the interaction of classification image frequency × stimulus class for the power spectra of the time domain classification images was highly significant (F(36, 480) = 8.3; p < 0.001). In the same vein, the 3-way interaction of sampling frequency × classification image frequency × stimulus class was significant for the power spectra of time–frequency classification images (F(360, 4800) = 1.3; p < 0.005). p The high between-subject agreement of the power spectra of individual classification images (shown in Fig. 6) along with their distinct patterns according to stimulus class suggests that this data may comprise signature properties that are diagnostic of the particular class of stimuli participants are required to recognize. This was determined with support vector machines (SVMs24) which were trained to map the power spectra of the clas- sification images of individual participants to stimulus class with a leave-one out validation procedure. Out of four possibilities, stimulus class was predicted with 75% accuracy from the power spectra of the time domain classification images and with 52% accuracy using the power spectra of the time–frequency classification images. These correct classification rates are both well above chance (time domain: χ2 (9) = 68.9; p < 0.001; time–fre- quency domain: χ2(9) = 31.0; p < 0.001). Resultshi The ICCs for the phase and power spectra of each type of classification image and for each stimulus class, along with their confidence intervals are shown in Supplementary Table S2.hi i The phase and power spectra of classification images were examined further with respect to their capacity of offering a pattern that was distinct according to the particular stimulus class participants have to recognize. An ANOVA on the phase spectra of time-domain classification images with frequency in classification image https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 \| www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 \| Discussion 4, 5) as well as of their power spectra to discriminate reliably between stimulus classes indicates that the oscillatory mechanisms revealed are different or are engaged differently across experiments. This obser- vation is congruent with the existence of specialized cortical mechanisms for visual word, object, and face recognition25. In turn, since functional specialization for stimulus classes only emerges at relatively high levels in the cortical hierarchy, this might suggest that random temporal sampling may tap neural mechanisms that are several synapses away from receptors.f y p y p A note of caution is required, however. Apart from stimulus class, there were several other differences between the experiments reported (e.g. stimulus sizes, brightness, background intensity, item familiarity, etc.) which are likely to have affected their outcomes. Thus, the present findings should be considered suboptimal for the purpose of characterizing the different oscillatory visual mechanisms involved in the recognition of the stimulus classes used. Our central purpose here is instead to demonstrate that the results obtained with random temporal sampling are highly sensitive to the changes in putative visual oscillatory mechanisms that are brought about by changing the observer’s perceptual experience and/or task. This is clearly the case considering the significant dif- ferences between the classification images obtained with different types of stimulus materials (Figs. 4, 5). Strong support for this conclusion is also provided by the successful classification of the stimulus class participants had to recognize on the basis of the power spectra of their time or time–frequency domain classification images. g p p q yi g Another interesting aspect of the present results that may be noticed from inspection of Figs. 3, 4, 5 is that processing efficiency (i.e. z-scores) is substantially greater in the task of word recognition than for other stimulus Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ l F i t th i ffi i k f d i th ti d ti f l ifi ti Figure 6. Power spectra of the classification images of encoding effectiveness in the time domain (panels a–e) and in the time–frequency domain (panels e–h). Stimuli are: words (a,e), familiar objects (b,f), novel objects (c,d), and faces (d,h). www.nature.com/scientificreports/ Figure 6. Power spectra of the classification images of encoding effectiveness in the time domain (panels a–e) and in the time–frequency domain (panels e–h). Stimuli are: words (a,e), familiar objects (b,f), novel objects (c,d), and faces (d,h). classes. www.nature.com/scientificreports/ A noteworthy finding from the present study is that classification images of processing effectiveness as a function of the static temporal frequencies present in the sampling func- tions (i.e. Fourier domain classification images) revealed no significant effect (see Supplementary Fig. S1). This finding demonstrates the incapacity of static temporal frequencies of stimulus visibility oscillations to modu- late visual recognition performance. This contrasts with the time–frequency classification images (Fig. 3e–h), which show the large impact of transient stimulus oscillations. These observations appear relevant to the issue of whether the neural oscillations that may be captured in humans by EEG/MEG reflect sustained or transient- burst events30. Specifically, the ineffectiveness of the static frequencies constituting the temporal sampling func- tions to modulate performance seems to suggest that the temporal sampling method used here would only impact transient-burst events while producing no evidence for sustained oscillations. h h h d b l f h d d l p p g We must point out however, that the power spectra extracted by Fourier analysis from the individual time and time–frequency classification images do reflect sustained oscillations. As demonstrated above, these patterns of sustained oscillations are extremely similar across individuals and they can effectively serve to determine the stimulus class participants had to recognize.hf p p g This leads us to suggest that the technique of random temporal sampling may effectively tap both transient and sustained oscillatory visual mechanisms and that both provide a significant contribution to accurate performance. Temporal sampling vs. behavioral periodicity paradigms. The above conclusion that sustained oscillatory mechanisms are important for visual performance is congruent with the previous findings obtained from the behavioral periodicity paradigms discussed in the Introduction. Indeed, the demonstration of the periodicity of a particular phenomenon using these methods requires an oscillation that is sustained for some duration. It is unclear whether evidence for transient oscillations can be provided through the measurement of behavioral periodicity and, to our knowledge, no such demonstration is presently available.i Across tasks, the static oscillation frequencies—i.e. those extracted from individual classification images by Fourier analysis—that seem the most engaged range from 5 Hz to about 25 Hz, as can be seen from the frequen- cies in classification images that carry the most power (Fig. 6). However, substantial energy peaks can also be observed in the 35–55 Hz range, particularly in the novel object and face recognition tasks (Fig. 6c,d,g,h). www.nature.com/scientificreports/ may relate to differences among stimulus classes with respect to the amount of information (in the classic sense of “reduction of uncertainty”26) that is required for their accurate recognition.i may relate to differences among stimulus classes with respect to the amount of information (in the classic sense of “reduction of uncertainty”26) that is required for their accurate recognition.i Indeed, word recognition is known to largely proceed through the identification of its constituent letters27, of which only 26 possibilities exist in French. This means that there is a finite number of five-letter strings that may possibly exist (i.e. 265 items) and this number is largely reduced if we only consider items made of allow- able letter sequences (within pairs or triplets) in the language. A further constraint which applies here is that the letter sequences presented must constitute a French word, which are rather few (2048 orthographically distinct five-letter words in the BRULEX database28, which was used here for stimulus selection). These different factors constitute a priori probabilities that human vision most probably applies for word recognition29, thereby reduc- ing the quantity of information that is needed to identify a word compared to stimulus classes such as familiar objects or faces, for which no such constraints apply. For our novel object recognition task, the number of object identities was quite low (n = 6) but it would seem reasonable to argue that the amount of information required for their recognition was higher than for words given their initial unfamiliarity and the use of varied viewpoints.h Then, under the assumption that the rate of information transmission by the visual system is limited, the brief instants of stimulus visibility that were available allowed the encoding of visual information which may have had a greater potential to lead to correct target recognition in the case of words compared to other stimulus classes. Expressed in another way, the account proposed here for the greater processing efficiency for words than for familiar or novel objects, or faces, is that the scores reflecting processing efficiency would be inversely related to the amount of information that is required to accomplish the task correctly. We suggest it would be of interest for future research to investigate this idea. Static versus transient oscillations. Discussion For instance, the processing efficiency peaks for words in the time and time–frequency classification images are of 72.5 and 49.9 respectively, whereas they are of 6.3 and 11.9 for familiar objects, 19.7 and 9.6 for novel objects, and 7.2 and 9.2 for faces. This is an observation we had not anticipated. We speculate that this classes. For instance, the processing efficiency peaks for words in the time and time–frequency classification images are of 72.5 and 49.9 respectively, whereas they are of 6.3 and 11.9 for familiar objects, 19.7 and 9.6 for novel objects, and 7.2 and 9.2 for faces. This is an observation we had not anticipated. We speculate that this https://doi.org/10.1038/s41598-021-00685-w Scientific Reports \| (2021) 11:21309 \| www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ The range of oscillatory frequencies revealed in the present study is substantially greater than those reported in previous studies using a method based on the periodicity of performance indicators[12–18; see “Introduction”]. Most significantly, the highest relevant frequencies documented here seem to reach as high as 55 Hz whereas behavioral periodicity studies have so far detected oscillation frequencies up to a maximum of about 20 Hz18. The sensitivity of the random temporal sampling technique to high frequency functional oscillations is clearly advantageous relative to alternative methods. This advantage is especially obvious when one considers the fun- damental importance of neural oscillations in the gamma frequency band (30 Hz and above) for a vast range of cognitive and perceptual functions1–3, including visual–spatial attention31. g p p g p Two other practical considerations that also favour the temporal sampling technique over behavioural perio- dicity methods are the numbers of trials required to obtain proper data and the level of complexity of the experimental procedures they require. With regards to numbers of trials, periodicity methods may be somewhat prohibitive (several thousands of trials) in comparison to the 1200 trials per participant used here. Moreover, the procedure required to measure periodicity in perceptual performances may be quite complex whereas it is very straightforward with temporal sampling once the method involved in producing stimulus oscillations is in place. Synchronization with stimulus events. What appears critical for the ability of random temporal sam- pling to deliver relevant observations is for perceptual mechanisms to synchronize precisely with stimulus events. Indeed, without synchrony, the timing relations between the state of the visual system and stimulus events would be random. This would prevent classification images, which are based on large collections of trials, from showing variations of processing efficiency according to the timing of stimulus events since this relation Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ would be entirely blurred. To prevent such problems, great care was taken in designing the experiments to pre- sent preparatory signals that would permit participants to precisely anticipate the moment of target onset and to maximize their level of alertness at that instant32,33. Also, the features of the auditory tone preceding this event were selected based on a prior demonstration of their capacity to reset neural oscillations in the visual system34. Future studies will be required to assess the actual value of these preparatory signals. Truly oscillatory mechanisms? Methods Participant Participants. All 44 participants, 29 women and 15 men, were French speaking neurologically intact under- graduate students at Université de Montréal aged between 19 and 35 years old. All add normal or corrected vision. Eight participants took part in the word recognition experiment, whereas distinct groups of 12 partici- pants took part in each of the others, which were conducted later.ht p p The recruitment of participants was initiated only after approval of the studies by the relevant ethics commit- tee of the Université de Montréal (Comité d’éthique de la recherche en éducation et psychologie). All participants gave their informed consent to participate and all procedures were carried out in accordance with the relevant guidelines and regulations. Materials and stimuli. All experiments were run on an HPZ230 computer equipped with an NVIDIA GeForce GTX970 videocard and an ASUS VG248QR HD monitor with maximum luminance of 200 cd/m2 and a 120 Hz refresh rate. All stimuli were achromatic and all manipulations of intensity were linear. Experiments were programmed in Matlab [Mathworks Inc.] and made the use of the Psychophysics toolbox38. Observation distance was 57 cm, which was stabilized by having participants rest their head on a chin rest.i y g p p For the word recognition experiment, stimuli were 600 five-letter French common words with a mean fre- quency of 157 per million28. Words were printed in Tahoma (x-height of 0.76 deg) in black letters over a grey (half of maximum intensity) background. For the familiar object recognition experiment, stimuli were greyscale photographs (from the Bank of Stand- ardized Stimuli; BOSS39) of 300 objects shown in front of a white background. The items were selected based on the published performance statistics for BOSS images so that it would be easy for participants to recognize them and find their name. The maximum horizontal extent of the stimuli was 17.4 deg of visual angle and their maximal vertical extent was 17.1 deg.h The stimuli used for the novel object recognition task were simple randomly generated 3-D shapes with two interconnected medial axes around each of which a cylindrical shape of variable diameter was built (Fig. 7; this stimulus class has been used previously by40. www.nature.com/scientificreports/ In the present study, we have used variations in visual processing effective- ness according to the temporal features of stimulus sampling as a way to probe putative oscillatory mechanisms in the human visual system. One question that may be asked with respect to the evidence obtained is whether it truly reflects visual mechanisms that are oscillatory. Indeed, there is an ongoing debate in the literature as to whether the oscillatory neural activity resulting from exposure to rhythmic stimuli reflects the response of an actual oscillator (i.e. “a system capable of generating sustained rhythmic behaviour by itself”35 p. 2) or simply neural entrainment by the periodicity of the stimulus; that is, a steady-state response35–37.h y p y y p There are three aspects to the observations reported here that are relevant to the issue and which militate for an interpretation of our results in terms of visual mechanisms that are actual oscillators.i p One is that the Fourier domain classification images showed that the stable rhythmic features of stimulus sampling had no significant impact on behavioral performance (Supplementary Fig. S1). Such an impact is precisely what would have been predicted by the hypothesis of neural entrainment.f p y p y yp Second, the temporal frequency content of stimulus sampling mainly had transient effects on behavioural performance, as shown by the time–frequency classification images (Fig. 3e–h). This observation is incompatible with the notion of neural entrainment. Take for instance the response to 50 Hz stimulus oscillations in Fig. 3f, which is the time–frequency classification image for familiar objects. At some SOAs, these oscillations have no impact on performance but at others, they are associated to either increased or decreased processing effective- ness. We see no way in which such a pattern could be accounted by neural entrainment.hii Third, and finally, the power spectra of the time and time–frequency domain classification images of indi- vidual participants, which probably constitute the most potent observations from the present study, are not directly related to stimulus oscillations. They rather reflect intrinsic properties of the visual processing system that drive the variations of processing effectiveness through time that occur in individual time and time–frequency domain classification images. Note added in proof:. We have recently completed analyses for a new experiment involving one condition that was identical to the word recognition task reported here. These new results closely replicate those reported here in every respect. Methods Participant The stimuli displayed in the experiment were the 2D renderings of these items, lighted from above, covered in a rich achromatic texture and presented in one of four viewpoints, all of which revealed the major features characterizing the shape of the object. Items were shown over a black background and their maximum horizontal and spatial extent was of 9.5 deg. g p g One hundred and thirty greyscale photographs of famous actors and actresses from the face bank of41 were used for the face recognition experiment. The items selected were those which appeared as most likely to be recognized by participants. Each face appeared in frontal view and its expression was either neutral or happy. These photographs were displayed over a white background and their horizontal and vertical spatial extent was of 7.0 deg. Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ Figure 7. Illustration of a subset of the stimuli used in the novel object recognition task. The figure shows one instance of each object of the set. In the experiment, each object could be shown from one of four different viewpoints. Figure 7. Illustration of a subset of the stimuli used in the novel object recognition task. The figure shows one instance of each object of the set. In the experiment, each object could be shown from one of four different viewpoints. In all experiments, the duration of the target stimulus was of 200 ms. Target stimuli were made from the linear summation of two components, the signal and the noise (Fig. 1). The signal was made from the image of the target, as described above, overlaid by a white noise field with a contrast level that was adjusted on a trial by trial basis, as described below, in an attempt to maintain response accuracy to 50% correct. The noise was made from a second, independent white noise field of maximal contrast. Throughout the stimulus duration, the signal- to-noise ratio (SNR), that is the ratio of the weights attributed to the signal and the noise for the construction of the stimulus to be displayed, varied according to a random function constructed by the integration of sine waves with frequencies ranging between 5 and 55 Hz in steps of 5 Hz with random amplitudes and phases. A new, independent SNR function was generated on each trial. Examples of actual temporal sampling functions are illustrated in Fig. 2. Methods Participant The range of the SNR was normalized between 0 and 0.5 and its sum across the sequence of 24 image frames making up the 200 ms stimulus duration (at the refresh rate of 120 Hz) was matched across trials. The overall luminance and contrast of the stimuli were matched across image frames and across trials. Procedure. In all experiments, participants completed a total of 1200 trials over two test sessions of 600 trials each. In all cases, the participant’s task was to identify the target without time pressure. With word stimuli, par- ticipants had to read the word aloud. With familiar objects, they were required to identify the item using a name that was sufficiently specific to demonstrate accurate recognition. With novel objects, they had to indicate orally the digit assigned to it during the learning phase, which is described below. With famous faces, participants were asked to either give the name of the actor/actress, the name of his/her character in a particular movie or his/her specific role in that movie. i In the sequence of trials in the word, familiar object, or face recognition experiments, no item could be repeated before the complete stimulus set had been presented for the same number of trials. In the novel object recognition task, the order of presentation of items was random, with the constraint that each object be presented an equal number of times.i For the novel object recognition experiment, participants first had to be trained with the objects and the name they were assigned, which was a digit ranging from 1 to 6. This began with a familiarization phase during which participants were exposed to a sheet of paper on which the six objects were printed in all four viewpoints along with the digit associated with each object. Once the participant felt having studied the sheet sufficiently, the practice phase followed during which each of the possible 24 stimulus instances were presented eight times in a random trial sequence presenting one non-degraded item at a time until the participant named it. A cor- rect response was followed by a 1000 Hz (50 dB pure tone) auditory feedback whereas errors were followed by a 300 Hz tone. Following each response, whether correct or not, the correct digit identity of the item was displayed on the screen for 500 ms. www.nature.com/scientificreports/ This leads to a sacrifice in the precision of measurements in the frequency domain, which implies sensitivity of the wavelet not just to its particular temporal frequency but to a range of frequencies around it.i j p p q y g q For each mode of coding the SNR time functions, classification images were obtained for each individual participant. This was done by the weighted subtraction of the sums of the temporal features (i.e. SNR as a func- tion of time, Fourier descriptors of these SNR functions, or time–frequency representation of SNR functions) of the sampling functions associated to errors from those associated to correct responses. These raw classification images were then transformed in Z scores by a bootstrapping operation whereby the sampling functions were randomly assigned to response accuracies while allowing for repetition, and from which classification images were constructed. The mean and standard deviation of 1000 such random classification images for an individual participant served as reference to transform the values from his/her raw classification image into Z scores.i p pi g Once transformed to a common scale, the individual classification images were averaged, smoothed, and then submitted to a two-way Pixel test22 with α = 0.05 to determine the points in classification images which differed significantly from zero. The Pixel test is derived from random field theory and has been applied for about the last 30 years for the analysis of brain imaging data. Its purpose is to establish the Z value that will serve as the signifi- cance criterion for a Z-scored image. Among others, factors that will affect the Pixel test is the spatial correlation inherent in the data set, the dimensionality of the latter, and the width of the filter used to smooth the data. In the case of the classification images reported here, the smoothing filter was Gaussian and had a full width at half maximum (FWHM) of 0.6 unit in the time domain and of 1.5 units in the time–frequency domain. Since we wanted to identify data points that were either significantly above or below zero, the Pixel test was two-way. Thus, to achieve an overall α of 0.05, the input given to the algorithm performing the Pixel test requested α = 0.025. The criterion Z score obtained was then used in its positive value to identify points that were significantly above 0 and in its negative value (i.e. www.nature.com/scientificreports/ minimum vs. maximum luminance) elements. The first experimental block began with a white noise contrast exactly at the middle of the available range for the first 10 trials. Starting on trial 11, the average response accuracy for the 10 preceding trials was determined. If accuracy was exactly 50%, white noise contrast remained the same for the following trial. If accuracy was below 50%, white noise amplitude was reduced by one step (see below) whereas it was increased by one step if accuracy was above 50%. Initial step size was of 16 contrast levels and this value was halved every time the direction of adjustment was reversed, down to a minimum of 1. The state of the algorithm adjusting this white noise contrast was maintained across consecutive blocks of trials. Data analysis. Response accuracy was averaged across participants separately for each experiment. The average percentages of correct responses are; words: 60.5%; familiar objects: 49.2%; novel objects: 56.0%; and faces: 30.1%. As indicated above, the contrast of the white noise mask that was part of the “signal” portion of the stimuli displayed was adjusted to maintain response accuracy to about 50% correct. The means of noise levels for each experiment are; words: 111.3; familiar objects: 119.3; novel objects: 119.9; and faces: 11.0, over a possible maxi- mum of 128.hi The main analyses pertained to the construction of classification images representing how response accu- racy was affected by various temporal features of the sampling functions of the target stimuli (see Fig. 2). The temporal features which were analysed are: 1—SNR amplitude as a function of time (time domain) from target onset (or stimulus onset asynchrony; SOA), 2—the Fourier descriptors for these functions (Fourier domain) and, 3—time–frequency representations of these same functions (time–frequency domain). The Fourier descriptors are the amplitude and phase of the temporal frequencies (5–55 Hz in 5 Hz steps) used to build individual SNR time functions (see above). The time–frequency representations of the SNR functions on individual trials were calculated with a wavelet analysis using three-cycle complex Morlet wavelets varying in temporal frequency from 5 to 55 Hz in 5 Hz steps42. The number of cycles in the Morlet wavelet serving as the kernel in wavelet analyses was chosen to offer high precision in the time domain, which is the crucial issue we wanted to investigate. Methods Participant To move to the experimental phase, participants had to obtain at least 90% correct responses in a practice block.i p p In all experiments, the time course of each trial was as follows. A square white noise field of 18° to a side centred on the middle of the display monitor was first presented for 1250 ms. A white fixation cross was then added at the centre of the monitor for 250 ms, which then disappeared. This offset was followed 150 ms later by a 900 Hz–60 dB–14 ms pure tone announcing the onset of the target stimulus 100 ms later. The target stimulus was presented in the middle of the screen for a duration of 200 ms during which the SNR varied according to a random function, as described above. Following its offset, only the background white noise mask remained vis- ible until the participant’s response. A 500 ms delay then followed prior to the beginning of the following trial. Response accuracy on experimental trials was maintained to about 50% correct using a staircase procedure which automatically adjusted the contrast of the white noise superimposed on the target image as part of the “signal” portion of the stimulus to be displayed (see above). The available range of white noise contrast had 128 levels, the lowest producing a zero-contrast (i.e. null) mask and the highest being made of black and white (i.e. Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ Received: 15 February 2021; Accepted: 12 October 2021 Received: 15 February 2021; Accepted: 12 October 2021 References 1. Buzsàki, G. Rhythms of the Brain (Oxford University Press, 2006). 1. Buzsàki, G. Rhythms of the Brain (Oxford University Press, 2006). y f y 2. Cannon, J. et al. Neurosystems: Brain rhythms and cognitive processing. Eur. J. Neurosci. 39, 705–719 (2014). . Cannon, J. et al. Neurosystems: Brain rhythms and cognitive pro 2. Cannon, J. et al. Neurosystems: Brain rhythms and cognitive processing. Eur. J. Neurosci. 39, 705–719 3. Fries, P. Rythms for cognition: Communication through coherence. Neuron 88(1), 220–235 (2015). y y g p g 3. Fries, P. 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The involvement of endogenous neural oscillations in the processing o than a regular repetition of evoked neural responses. Front. Neurosci. 12, 95. https://doi.org/10.3389/fnins 37. Zoefel, B., Ten Oever, S. & Sack, A. T. The involvement of endogenous neural oscillations in the processing of rhythmic input: More than a regular repetition of evoked neural responses. Front. Neurosci. 12, 95. https://doi.org/10.3389/fnins.2018.00095 (2018). 7. Zoefel, B., Ten Oever, S. & Sack, A. T. The involvement of endogenous neural oscillations in the processing of rhythmic input: More than a regular repetition of evoked neural responses. Front. Neurosci. 12, 95. https://doi.org/10.3389/fnins.2018.00095 (2018). 8. Kleiner, M. et al. www.nature.com/scientificreports/ Zcrit − 1) to identify points significantly below 0.i i Analyses of the agreement, or consistency of classification images across participants having taken part in the same experiment were carried out using the intraclass correlation coefficient (ICC23). The same procedure was followed to analyse the consistency among participants of the power and phase spectra of their classification images. The ICC assesses the similarity between participants of a group in their pattern of results across an array of measurements. The upper bound of the ICC is one and it has no lower limit. Supplementary Tables S1 and S2 report every ICC discussed in the present article, along with their 95% confidence intervals.hi i The analysis of raw individual classification images into their power and phase components was performed by one-dimensional fast Fourier transforms applied to either the time domain classification images or to Z score amplitude variations through time separately for each temporal frequency represented in time–frequency clas- sification images (i.e. 5–55 Hz in 5-Hz steps). i g Mixed-factor analyses of variance (ANOVAs) were conducted to examine whether the phase and power spectra of the time and time–frequency domain classification images showed different patterns according to stimulus class. Given the specificity of the question addressed by these analyses, only the relevant interactions are reported in “Results” section. Stimulus class was decoded from the power spectra of individual time domain and time–frequency domain classification images using linear support vector machines (SVMs24) and a leave-one-out cross-validation pro- cedure. Thus, the classification images of all but one participant were presented to the SVM for it to learn the mapping from classification images to stimulus class. Then, the classification image from the participant having been left out of the learning phase was presented to the SVM for it to determine which of the four stimulus classes had been presented. This process was repeated by leaving out a different participant on each iteration until it had iterated through all participants. Classification accuracy was determined from the percentage of iterations on Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ which the SVM determined correctly the stimulus class processed by the left-out participant. Chi-square analyses were used to assess whether classification accuracy deviated significantly from chance. which the SVM determined correctly the stimulus class processed by the left-out participant. Chi-square analyses were used to assess whether classification accuracy deviated significantly from chance. Acknowledgements d b f g Supported by grants from the Fonds de Recherche Québec—Nature et Technologie and the Natural Sciences and Engineering Research Council of Canada (NSERC) to Martin Arguin and a summer research scholarship to Roxanne Ferrandez from NSERC. We are grateful to Prof. Charles E. Connor for the software for the generation of novel objects. Thanks to Robert Haccoun and Geneviève Mageau for statistical counsel. References What’s new in psychtoolbox-3. Perception 36(14), 1–16 (2007). g p p p g ( 38. Kleiner, M. et al. What’s new in psychtoolbox-3. Perception 36(14), 1–16 (2007).h 38. Kleiner, M. et al. What’s new in psychtoolbox-3. Perception 36(14), 1–16 (2007).h p y p 9. Brodeur, M. B. et al. The Bank of Standardized Stimuli (BOSS): Comparison between French and English norms. Behav. Res Methods 44, 961–970 (2012). 40. Yamane, Y., Carlson, E. T., Bowman, K. C., Wang, Z. & Connor, C. E. A neural code for three-dimensional object shape in macaque inferotemporal cortex. Nat. Neurosci. 11(11), 1352–1360. https://doi.org/10.1038/nn.2202 (2008). 1. Butler, S., Blais, C., Gosselin, F., Bub, D. & Fiset, D. Recognizing famous people. Attention Perception Psychophys. 72(6), 1444 https://doi.org/10.3758/APP.72.6.1444 (2010).h p g ( ) 42. Cohen, M. X. Analyzing Neural Time Series Data: Theory and Practice (Massachusetts Institute of Technology, Massachusetts, 2014). Scientific Reports \| (2021) 11:21309 \| https://doi.org/10.1038/s41598-021-00685-w www.nature.com/scientificreports/ Author contributions M.A. was responsible for conceiving and designing the work reported, programming the software for conduct- ing the experiments and analysing the data, data collection for the word recognition experiment, advanced data analyses for all experiments and preparing the manuscript. R.F. prepared the stimulus sets and performed the data collection for the familiar object and the face recognition tasks as well as the preliminary data analyses for these experiments. J.M. prepared the stimulus set and performed the data collection for the novel object recognition experiment and the preliminary data analyses. Competing interests h The authors declare no competing interests. Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-00685-w. Correspondence and requests for materials should be addressed to M.A. 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. 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https://openalex.org/W4214944982	https://europepmc.org/articles/pmc6352081?pdf=render	English	null	Acknowledgement to Reviewers of Foods in 2013	Foods	2,014	cc-by	1,079	Acknowledgement to Reviewers of Foods in 2018 Foods Editorial Office MDPI, St. Alban-Anlage 66, 4052 Basel, Switzerland Published: 17 January 2019 Rigorous peer-review is the corner-stone of high-quality academic publishing. The editorial team greatly appreciates the reviewers who contributed their knowledge and expertise to the journal’s editorial process over the past 12 months. In 2018, a total of 211 papers were published in the journal, with a median time to first decision of 15.8 days and a median time to publication of 35 days. The editors would like to express their sincere gratitude to the following reviewers for their cooperation and dedication in 2018: www.mdpi.com/journal/foods 2 of 4 Foods 2019, 8, 32 ds 2019, 8, 32 Duconseille, Anne Dufossé, Laurent Dugo, Giacomo Duke, Colin Dykes, Gary Elias, Miguel Eller, Fred Ersan, Sevcan Escolà-Gil, Joan Carles Estruch, Ramon Etzel, Mark Fakhr, Mohamed Fang, Zhongxiang Faour-Klingbeil, Dima Farzaneh, Vahid Feke, Donald L. Fernandes, Donald Fernandes, José Oliveira Ferrer-Gallego, Raul Fijan, Sabina Filteau, Marie Fodor, Marietta Francis, Leigh Franzoi, Marco Gan, Ren-You Ganesan, Palanivel Ganzle, Michael Garbowska, Monika Gawlik-Dziki, Urszula Genovese, Alessandro Gentry, Terry Ghosh, Abhijit Giacalone, Davide Giacometti, Jasminka Giannakourou, Maria C. Giaouris, Efstathios D. Gimeno Alcañiz, José Vicente Giuffrè, Angelo Maria Golding, John B. González-Domínguez, Raúl Gradeci, Klodian Grafenauer, Sara Graikou, Konstantia Grech, Amanda Groh, Isabel Anna Maria Grosso, Giuseppe Gueimonde, Miquel Guerrero, Raul F. Hackett, Mark Haldar, Sumanto Harrington, Robert J. Harrison, Sabine Hauge, Sigrun J. Hefni, Mohammed Hernández, Francisca Hill, Arthur R. Horner, Harry T. Hsieh, Changwei Hungerford, James Hurtaud, Catherine Jimenez-Flores, Rafael Kaddoumi, Amal Kamiloglu, Senem Kandylis, Panagiotis Karayannakidis, Panayotis Karipidis, Philippos Karoglan, Marko Kassama, Lamin Katayama, Shigeru Kelly, Matthew Kelly, Alan Keyzers, Robert A. Khallouki, Farid Khouryieh, Hanna Kilcawley, Kieran Killeen, Daniel Kim, Mina K. Klempt, Martin Köhler, Karsten Koutelidakis, Antonios E. Krishnaswamy, Kiruba Kulig, Dominika Kulis, Michael Kurek, Marcin Kus, Piotr Kuzma, Lukasz Kwon, Oran Labra, Massimo Ladero, Victor Lai, Yandong Laming, Donald Richard John Lanza, Massimiliano Lawrence, Mark Lee, Young Seong Legeza, Balázs Leong, Thomas Seak Hou Leong, Sze Ying Li, Fu-Shuang Lim, Beong Ou Livesey, Geoffrey Loizzo, Monica R. Lopez, Gabriela Lücke, Friedrich-Karl Lundén, Janne Duconseille, Anne Dufossé, Laurent Dugo, Giacomo Duke, Colin Dykes, Gary Elias, Miguel Eller, Fred Ersan, Sevcan Escolà-Gil, Joan Carles Estruch, Ramon Etzel, Mark Fakhr, Mohamed Fang, Zhongxiang Faour-Klingbeil, Dima Farzaneh, Vahid Feke, Donald L. Acknowledgement to Reviewers of Foods in 2018 Fernandes, Donald Fernandes, José Oliveira Ferrer-Gallego, Raul Fijan, Sabina Filteau, Marie Fodor, Marietta Francis, Leigh Franzoi, Marco Gan, Ren-You Ganesan, Palanivel Ganzle, Michael Garbowska, Monika Gawlik-Dziki, Urszula Genovese, Alessandro Gentry, Terry Ghosh, Abhijit Giacalone, Davide Giacometti, Jasminka Giannakourou, Maria C. Giaouris, Efstathios D. Gimeno Alcañiz, José Vicente Giuffrè, Angelo Maria Golding, John B. González-Domínguez, Raúl Gradeci, Klodian Grafenauer, Sara Graikou, Konstantia Grech, Amanda Groh, Isabel Anna Maria Grosso, Giuseppe Gueimonde, Miquel Guerrero, Raul F. Hackett, Mark Haldar, Sumanto Harrington, Robert J. Harrison, Sabine Leong, Thomas Seak Hou Guerrero, Raul F. Hackett, Mark Haldar, Sumanto Harrington, Robert J. Harrison, Sabine 3 of 4 Foods 2019, 8, 32 Foods 2019, 8, 32 ds 2019, 8, 32 Lung, Ming-Yeou Maeda, Hayato Magalhães, Júlia M.C.S. Mahapatra, Ajit K. Maleszka, Alicja Malissiova, Eleni Mangia, Nicoletta Pasqualina Marta, Ferreiro-González Martinez, Vicente Martínez, Olaia Marx, Wolfgang Maugeri, Andrea McAuley, Catherine M. McComb, Jacalyn Melini, Valentina Merlino, Valentina Maria Mesías García, Marta Michalska, Anna Mierzwa, Dominik Miglietta, Pier Paolo Migliore, Giuseppina Milham, Paul Miller, Charles Milovic, Vladan Mirzoian, Armen Misciagna, Giovanni Moran, Lara Moreira Martínez, Ramón Felipe Mujaffar, Saheeda Muniz, Juan Muñoz-Alférez, M. José Muriana, Peter Musalgaonkar, Sharmishtha Muschiol, Jan Muzzalupo, Innocenzo Naik, Surabhi Nambeesan, Savithri Narsimhan, Ganesan Navarro-Alarcon, Miguel Nochi, Tomonori Noordergraaf, Wim Nový, Ing. Pavel Núñez Carmona, Estefanía Odeyemi, Olumide A. Ohta, Yoshiji Oikonomou, Evdokia K. Olejar, Kenneth J. Olmedilla-Alonso, Begoña Oracz, Joanna Orfila, Caroline Padalkar, Mugdha Pan, Meixia Papadopoulou, Chrissanthy Papetti, Adele Parveen, Iffat Pashikanti, Srinath Paulsen, Peter Pedrosa, Mercedes M. Pérez-Álvarez, Jose Angel Pérez-Lamela, Concepción Petrova, Olga E. Petruk, Ganna Picozzi, Claudia Pinto, Carmelo García Płotka-Wasylka, Justyna Prester, Ljerka Preti, Raffaella Purchas, Roger W. Pycia, Karolina Raftopoulos, Konstantinos Ragupathy, Subramanyam Raheem, Bamidele Raja, Huzefa A. Raphaelides, Stylianos Reeve, Belinda Ren, Yuan David Reynolds, Joel Ricci, Elena Claire Rijo, Patrícia Rincon, Angela M. Rivera, Tania Rodov, Victor Rodrigues, Nuno Rodríguez-Flores, M. Shantal Rodríguez-López, Pedro Roig-Sagués, Artur Rosenthal, Andrew Rosicka-Kaczmarek, Justyna Ross, Alastair Roullier-Gall, Chloé Ruiz-Capillas, Claudia Rupasinghe, Vasantha Rutkowska, Jaroslawa Ryan, Michael P. Saenz-Navajas, Maria-Pilar Sajdakowska, Marta Salaheen, Serajus Salles, Christian Salvador, María Desamparados Samborska, Katarzyna Sánchez-García, Javier Sandell, Mari Saraiva, Cristina Satake, Masayuki Papadopoulou, Chrissanthy Papetti, Adele Parveen, Iffat Pashikanti, Srinath Paulsen, Peter Pedrosa, Mercedes M. Pérez-Álvarez, Jose Angel Pérez-Lamela, Concepción Petrova, Olga E. Petruk, Ganna Picozzi, Claudia Pinto, Carmelo García Płotka-Wasylka, Justyna Prester, Ljerka Preti, Raffaella Purchas, Roger W. Pycia, Karolina Raftopoulos, Konstantinos Ragupathy, Subramanyam Raheem, Bamidele Raja, Huzefa A. Acknowledgement to Reviewers of Foods in 2018 Raphaelides, Stylianos Reeve, Belinda Ren, Yuan David Reynolds, Joel Ricci, Elena Claire Rijo, Patrícia Rincon, Angela M. Rivera, Tania Rodov, Victor Rodrigues, Nuno Rodríguez-Flores, M. Shantal Rodríguez-López, Pedro Roig-Sagués, Artur Rosenthal, Andrew Rosicka-Kaczmarek, Justyna Ross, Alastair Roullier-Gall, Chloé Ruiz-Capillas, Claudia Rupasinghe, Vasantha Rutkowska, Jaroslawa Ryan, Michael P. Saenz-Navajas, Maria-Pilar Sajdakowska, Marta Salaheen, Serajus Salles, Christian Salvador, María Desamparados Samborska, Katarzyna Sá h G í J i Lung, Ming-Yeou Maeda, Hayato Magalhães, Júlia M.C.S. Mahapatra, Ajit K. Maleszka, Alicja Malissiova, Eleni Mangia, Nicoletta Pasqualina Marta, Ferreiro-González Martinez, Vicente Martínez, Olaia Marx, Wolfgang Maugeri, Andrea McAuley, Catherine M. McComb, Jacalyn Melini, Valentina Merlino, Valentina Maria Mesías García, Marta Michalska, Anna Mierzwa, Dominik Miglietta, Pier Paolo Migliore, Giuseppina Milham, Paul Miller, Charles Milovic, Vladan Mirzoian, Armen Misciagna, Giovanni Moran, Lara Moreira Martínez, Ramón Felipe Mujaffar, Saheeda Muniz, Juan Muñoz-Alférez, M. José Muriana, Peter Musalgaonkar, Sharmishtha Muschiol, Jan Muzzalupo, Innocenzo Naik, Surabhi Nambeesan, Savithri Narsimhan, Ganesan Navarro-Alarcon, Miguel Nochi, Tomonori Noordergraaf, Wim Nový, Ing. Pavel Núñez Carmona, Estefanía Odeyemi, Olumide A. Ohta, Yoshiji Oikonomou, Evdokia K. Olejar, Kenneth J. Olmedilla-Alonso, Begoña Oracz, Joanna Orfila, Caroline Padalkar, Mugdha 4 of 4 Foods 2019, 8, 32 © 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. 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. 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/W3021644519	https://surgical.medresearch.in/index.php/ijoso/article/download/24/47	English	null	Rational use of antibiotics and its guidelines in surgical patients-a retropspective study	Surgical update	2,017	cc-by	3,269	April - June 2017/ Vol 3/ Issue 2 April - June 2017/ Vol 3/ Issue 2 Rational use of antibiotics and its guidelines in surgical patients-a retropspective study Sengupta S 1, Pal B 2, Bala T 3 1Dr Siladri Sengupta, Associate Professor in Surgery & Unit Head, 2 Dr Baneyswar Pal, Assistant Professor, 3Dr Tanuka Bala, Junior Resident, Department of Surgery, ESI-PGIMSR & Medical College Joka, Kolkata, West Bengal, India Address for Correspondence: Dr Siladri Sengupta, Associate Professor in Surgery & Unit Head, ESI-PGIMSR & Medical College Joka, Kolkata, West Bengal, India Abstract Introduction: Rational use of drugs means to prescribe the appropriate drugs in correct dose over an adequate period of time at a lower cost. Irrational use of antibiotic is harmful both for the patient and the society and hence the top cause of mortality and morbidity around the world. So the aim of the present study is to - 1. Avoid adverse effect of irrational use of antibiotics on patient. 2. Avoid emergence of antibiotic resistance. 3. Avoid unnecessary increase of cost of health care. Methods: The study was conducted at the Surgery Unit II of the ESI-PGIMSR and ESIC Medical College, Joka, Kolkata- 700104, West Bengal. The Data was collected on random basis and the SSI was observed between the control and test group after rational and non rational treatment with antibiotics in some selected surgical patient. Results: It was found that in some diseases like acute gall bladder stone, cholecystitis, appendicitis, pancreatitis if some other antibiotics are additionally used besides the recommended as usual antibiotics the significant betterment is achieved in relation to quick recovery of the patients along with minimization of surgical site infection with no other complications. On the other hand in some other diseases like Diabetes foot, hernia, etc. use of antibiotics beyond the recommended antibiotics aggravates some other complications in the patients along with no significant betterment of surgical site infection in Test group. Conclusion: To conclude finally antibiotic resistance is a major problem worldwide and is inevitable and hence its rational use in proper guidelines is the only way of prolonging the useful life of the patients requiring antibiotics. Keywords: Use of antibiotics, Rational, Irrational Keywords: Use of antibiotics, Rational, Irrational Available online at: www.surgicalreview.in 33 \| P a g e Original Research Article for the patient and the society and hence the top cause of mortality and morbidity around the world [16,17]. When considering the role of antibiotics in the peri-operative period it is important to recognize the difference between prophylactic and empirical antibiotic therapy. The goal of prophylaxis is to reduce the post-operative infection assuming where the contamination might have occur but hasn’t occurred [18], whereas in empirical therapy is based on operative findings as leakage of a viscous. or admitted with acute or chronic surgical problems in indoor surgical wards requiring surgical management are included in this study. Exclusion criteria: The patients attending OPD with primary medical problems which require immediate medical management and with coincidental chronic surgical problem which doesn’t require surgical management for the time being are kept outside the study. According to World Health report a study was conducted in 1993-1996 amongst 13 low, middle, and higher income countries and it shows that in 30 % cases of UTI were incorrectly prescribed antibiotics [7,19]. Anti-microbial resistance is a global problem as a result infection fails to respond to standard treatment thereby decreasing the effect of effective antibiotic prolonging illness and risk of death. Under this situation it is always better to avoid the unnecessary complications in the patients due to unwanted and irrational use of antibiotics. So the aim of the present study is to - Study design: To observe the effect of rationally treated antibiotics the patients of pancretitits etc were divided into two group. One group (37) were treated with single recommended antibiotics considered as control whereas in second group (37) were treated with more than recommended antibiotics for recommended periods are labeled as Test group. After the recommended period of treatment the infection of surgical site (SSI) and other side effects were observed and compared. The details of number of patients are mentioned in the table at result section. 1. Avoid adverse effect of irrational use of antibiotics on patient. 1. Avoid adverse effect of irrational use of antibiotics on patient. Similarly to observe the effect of non-rationally treated antibiotics the patients of hernia and etc were also divided into two group. One group were treated with single recommended antibiotics considered as control whereas in second group were treated with more than recommended antibiotics for recommended periods are labeled as Test group. Original Research Article After the recommended period of treatment the infection of surgical site (SSI) and other side effects were observed and compared. The details of number of patients are mentioned in the table at result section. 2. Avoid emergence of antibiotic resistance. 3. Avoid unnecessary increase of cost of health care. Materials Place of study: The study was conducted at the Surgery Unit II of the ESI-PGIMSR and ESIC Medical College, Joka, Kolkata - 700104, West Bengal. Sample collection: The materials employed in this study are randomly selected from the surgical indoor patients admitted between Aug'15 to September ' 16 with various acute infection numbering 103 in total out of which male were 48 and female were 55 . Statistical analysis: The statistical analysis of number of patients having SSI was compared between control group and test group by student’s t test and significance level <0.05 was considered as statistically significant. Inclusion criteria: The surgical patients attending OPD Introduction levels in certain places in India (and around the world) [14], but the problem has remained largely unknown because relatively few studies were published and nationwide surveillance was not being carried out. The fact that antibiotic use is increasing is not, itself, indicative of a problem, but evidence from studies of prescribing patterns suggests that antibiotics are often used in inappropriate ways that mostly results the microbial resistance. But unfortunately the antibiotic resistance surveillance has been limited to small-scale efforts by the Indian Council of Medical Research (ICMR) and Antimicrobial resistance poses a great threat to human health and undermines national economics worldwide [1,2]. It is reported that this resistance is mostly due to consumption of antibiotics [3-5]. It is reported that in European countries, systemic antibiotics are prescribed in the greatest volumes to ambulatory patients, mostly for respiratory tract infections [6]. In Thailand, a study in a tertiary care hospital revealed that only 7.9% of the upper respiratory tract infections (URIs) in the facility were caused by bacteria [7]. Despite this, most URIs are treated with antibiotics by hospitals, health centers, drug stores and patients themselves [8-12]. Liberal use of antibiotics endangers the health of patients without observable clinical benefits, since it neither reduces the rate of complications nor quickens recovery when the illness is caused by a virus [11,13]. some private agencies on a pilot basis. This study is an attempt to study the judicial use of antibiotics which is rather known as Antibiotics smart use. some private agencies on a pilot basis. This study is an attempt to study the judicial use of antibiotics which is rather known as Antibiotics smart use. Resistance against certain antibiotics is already at high Rational use of drugs means to prescribe the appropriate drugs in correct dose over an adequate period of time at a lower cost [15]. Irrational use of antibiotic is harmful both Manuscript Received: 04th October 2016 Reviewed: 12th October 2016 Author Corrected: 20th October 2016 Accepted for Publication: 31st October 2016 International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 33 \| P a g e Available online at: www.surgicalreview.in 33 \| P a g e April - June 2017/ Vol 3/ Issue 2 2455-5436 Available online at: www.surgicalreview.in 34 \| P a g e Original Research Article ( Table –II shows that use of additional antibiotics in test group along with recommended antibiotics failed to reduce the SSI in test group in comparison to control group which were treated only with recommended antibiotics. Rather use of additional antibiotics in test group aggravates some other complications like GI upset and etc.). Original Research Article Original Research Articl Table I: Effect of rationally prescribed antibiotics on different diseases Disease Control Group [37] Test Group [37] Recommended Antibiotic Prescribed (No of patients) SSI observed (No of patients) Additional antibiotic in treatment regime (No of patients) SSI Observed (No of patients) Acute Gall stone Ceftriaxone / Ciprofloxacillin [4] 3 Amikacin & Metrogyl [4] Nil Cholecystitis Ceftriaxone / Ciprofloxacillin [16] 11 Amikacin & Metrogyl [15] 2 Acute Abdomen Ceftriaxone / Ciprofloxacillin [4] 4 Amikacin & Metrogyl [4] Nil Pancreatitis Meropenum [3] 3 Metrogyl [4] 1 Appendicitis Ceftriaxone/ Cefotaxime [6] 4 Amikacin & Metrogyl [6] 1 Colonic Growth Ceftriaxone / Cefotaxime [4] 4 Amikacin & Metrogyl [4] Nil . No within the parenthesis indicates the sample size Table I: Effect of rationally prescribed antibiotics on different diseases ( Table –I shows that use of additional antibiotics in test group along with recommended antibiotics significantly reduce the SSI in test group in comparison to control group which were treated only with recommended antibiotics.) ( Table –I shows that use of additional antibiotics in test group along with recommended antibiotics significantly reduce the SSI in test group in comparison to control group which were treated only with recommended antibiotics.) Table – II: Effect of non-rationally prescribed antibiotics on different diseases Disease Control Group (14) Test Group (15) Recommended Antibiotic Prescribed (No of patients) SSI observed (No of patients) Additional antibiotic in treatment regime (No of patients) SSI Observed (No of patients) Additional complications (No of patients) Diabetic Foot [7] Augmentin [3] 1 Clarithromycin [4] 1 G I upset [3] Breast Abscess [10] Amoxycillin [5] 1 Clarithromycin [5] 4 Drug resistant [4] Hernia & Hydrocoele [12] Ceftriaxone/ Cefotaxime [6] 2 Amoxycilline & Clavulonic acid [6] 2 GI upset [6] .No within the parenthesis indicates the sample size e – II: Effect of non-rationally prescribed antibiotics on different diseases .No within the parenthesis indicates the sample size ( Table –II shows that use of additional antibiotics in test group along with recommended antibiotics failed to reduce the SSI in test group in comparison to control group which were treated only with recommended antibiotics. Rather use of additional antibiotics in test group aggravates some other complications like GI upset and etc.). Available online at: www.surgicalreview.in 35 \| P a g e Result It was found that in some diseases like acute gall bladder stone, cholecystitis, appendicitis, pancreatitis if some other antibiotics are additionally used besides the recommended as usual antibiotics the significant betterment is achieved in relation to quick recovery of the patients along with minimization of surgical site infection with no other complications. Table –I shows that use of additional antibiotics in test group along with recommended antibiotics significantly reduce the SSI in It was found that in some diseases like acute gall bladder stone, cholecystitis, appendicitis, pancreatitis if some other antibiotics are additionally used besides the recommended as usual antibiotics the significant betterment is achieved in relation to quick recovery of the patients along with minimization of surgical site infection with no other complications. Table It was found that in some diseases like acute gall bladder stone, cholecystitis, appendicitis, pancreatitis if some other antibiotics are additionally used besides the recommended as usual antibiotics the significant betterment is achieved in relation to quick recovery of the patients along with minimization of surgical site infection with no other complications. Table –I shows that use of additional antibiotics in test group along with recommended antibiotics significantly reduce the SSI in test group without any side effects or complications in comparison to control group which were treated only with recommended antibiotics. For example if the patients of acute gall stone after surgical removal of stone are treated with amikacin and metrogyl along with ceftriaxone /ciprofloxacilin the SSI was reduced to nil (Test group) in comparison to the patients who are treated only with ceftriaxone/ciprofloxacilin (Control group). On the other hand in some other diseases like Diabetes foot, hernia, etc. use of antibiotics beyond the recommended antibiotics aggravates some other complications in the patients as shown in the Table II along with no significant betterment of surgical site infection in Test group. For example if the patients with diabetic foot after surgical intervention was treated with clarithromycin along with augmentin (Test group) no reduction of surgical site infection was observed rather there is GI upset in such patients which was absent in patients treated only with augmentin (Control group ). International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 34 \| P a g e April - June 2017/ Vol 3/ Issue 2 2455-5436 Original Research Article References 1. World Health Statistics. France: 2011. World Health Organization. 2. Kotwani A, Holloway K, Chaudhury RR. Methodology for surveillance of antimicrobials use among out-patients in Delhi. Indian J Med Res. 2009 May;129(5):555-60. 3. Mathew JL. Pneumococcal vaccination in developing countries: where does science end and commerce begin? Vaccine. 2009;27:4247–51. The present study revealed that judicial use of antibiotics can not only minimize the microbial resistance but also aggravates some other complications. This also demonstrate that non judicial use of antibiotics by using more than single recommended drug has no significant positive effect towards minimization of surgical site infection. This study is thereby is in accordance with many other studies investigated by other researchers worldwide [15-18]. 4. Austin DJ, Kristinsson KG, Anderson RM. The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):1152-6. 5. Laxminarayan R, Malani A, Howard D, Smith D. Washington, DC: 2007. [updated 2007; cited]. Policy responses to the growing threat of antibiotic resistance. Recommendation: On the basis of the result it is recommended that to achieve rational use of antibiotics for management of infection caused by microorganisms the following measures are to be employed. 6. Goossens H. Antibiotic consumption and link to resistance. Clin Microbiol Infect. 2009 Apr;15 Suppl 3:12-5. doi: 10.1111/j.1469-0691.2009.02725.x. 1. Reducing the need for antibiotics by employing - a- Antiseptic cleaning and draping of surgical site. b- Use of dedicated surgical attire, disposable sterile gloves. 7. Apisarnthanarak A, Mundy LM. Correlation of antibiotic use and antimicrobial resistance in Pratumthani, Thailand, 2000 to 2006. Am J Infect Control. 2008 Nov;36(9):681-2. doi: 10.1016/j.ajic.2007.10.022. c- Sterilization of instruments c- Sterilization of instruments d- Isolation of surgical wound from other areas by draping. d- Isolation of surgical wound from other areas by draping. 8. Albrich WC, Monnet DL, Harbarth S. Antibiotic selection pressure and resistance in Streptococcus pneumoniae and Streptococcus pyogenes. Emerg Infect Dis. 2004 Mar;10(3):514-7. 2. Culture sensitivity of organisms to select the appropriate antibiotic and hence the chances of resistance are minimized. 3. Following the antibiotic guidelines like: 3. Following the antibiotic guidelines like: 9. Chokejindachai W. Current situation of antimicrobial resistance in Thailand: a review. Nonthaburi: Health System Research Institute; 2007. A - Is antibiotic necessary? B – Most appropriate Antibiotic C – What dose, route, frequency, duration? D- Is the treatment effective ? 10. Jitraknatee A. Antibiotic values. References In: Niyada Kiatying- Angsulee, Nusaraporn Kessomboon, Usawadee Maleewong, editors. Situation report on drug system 2010: antimicrobial resistance. Bangkok: Drug System Monitoring and Development Center; 2011. Original Research Article established for two major reasons. First, the rational use of medicines as a concept was not always getting translated into practice, and the ASU model was felt to be useful in bridging this gap. Increasing awareness of antimicrobial resistance and promoting the rational use of antibiotics among prescribers and the general public are key to combating the unnecessary use of these drugs [6,16-18]. Discussion antibiotic classes. The fact that antibiotic use is increasing is not, itself, indicative of a problem, but evidence from studies of prescribing patterns suggests that antibiotics are often used in inappropriate ways that mostly results the microbial resistance. The bacterial disease burden in India is among the highest in the world [2]; consequently, antibiotics will play a critical role in limiting morbidity and mortality in the country. Antibiotic use has been increasing steadily in recent years. Between 2005 and 2009, the units of antibiotics sold increased by about 40 per cent. Increased sales of cephalosporins were particularly striking, with sales (in units sold) increasing by 60 per cent over that five-year period, but some increase was seen in most Antibiotics Smart Use (ASU) was introduced in 2007 as an innovative model to promote the rational use of medicines and counteract antimicrobial resistance. It was International Journal of Surgery & Orthopedics International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 35 \| P a g e Available online at: www.surgicalreview.in 35 \| P a g e 2455-5436 Original Research Article 2455-5436 April - June 2017/ Vol 3/ Issue 2 12. Suttajit S, Wagner AK, Tantipidoke R, Ross-Degnan D, Sitthi-amorn C. Patterns, appropriateness, and Original Research Article predictors of antimicrobial prescribing for adults with upper respiratory infections in urban slum communities of Bangkok. Southeast Asian J Trop Med Public Health 2005; 36: 489-97. Day Technical Advisory Committee; European Antibiotic Awareness Day Collaborative Group. European Antibiotic Awareness Day, 2008 - the first Europe-wide public information campaign on prudent antibiotic use: methods and survey of activities in participating countries. Euro Surveill. 2009 Jul 30;14(30):19280. 13. Udomthavornsuk B, Tatsanavivat P, Patjanasoontorn B, Khomthong R, Bhuripanyo K, Saengnipanthkul S, Lumbiganon P, Wiengnond S, Boonma P, Vongsangnak V, et al. Intervention of inappropriate antibiotic use at a university teaching hospital. J Med Assoc Thai. 1991 Oct;74(10):429-36. 16. Selecteddrug used indicator World Health Organization 1 Academic Press (1998). The World Medicins Situation20. 17. Swindell PJ, Reeves DS, Bullock DW, Davies AJ, Spence CE. Audits of antibiotic prescribing in a Bristol hospital. Br Med J (Clin Res Ed). 1983 Jan 8;286(6359):118-22. 14. Pagaiya N, Garner P. Primary care nurses using guidelines in Thailand: a randomized controlled trial. Trop Med Int Health 2005; 10: 471-7 doi: 10.1111/j.1365-3156.2005.01404.x. 18. Gonzales R, Steiner JF, Sande MA. Antibiotic prescribing for adults with colds, upper respiratory tract infections, and bronchitis by ambulatory care physicians. JAMA. 1997 Sep 17;278(11):901-4. 15. Earnshaw S, Monnet DL, Duncan B, O'Toole J, Ekdahl K, Goossens H; European Antibiotic Awareness Available online at: www.surgicalreview.in 37 \| P a g e Sengupta S, Pal B, Bala T. Rational use of antibiotics and its guidelines in surgical patients-a retropspective study.Int J surg Orthopedics. 2017; 3(2):33-37.doi:10.17511/ijoso.2017.i02.02. Conclusion To conclude finally counseling should be performed at ward level. Awareness programs should be launched and seminars should be conducted. News Letters and drug bulletins about the rational use of antibiotics and its effect on community, cost effective prescriptions should be encouraged. All these facts are possible when clinical pharmacist work along with the physician at ward level. 11. Bhavnani D, Phatinawin L, Chantra S, Olsen SJ, Simmerman JM. The influence of rapid influenza diagnostic testing on antibiotic prescribing patterns in rural Thailand. Int J Infect Dis. 2007 Jul;11(4):355-9. Epub 2007 Feb 26. Conflict of interest: None declared. Conflict of interest: None declared. Funding: Nil, Permission from IRB: Yes Funding: Nil, Permission from IRB: Yes International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 36 \| P a g e April - June 2017/ Vol 3/ Issue 2 2455-5436 International Journal of Surgery & Orthopedics How to cite this article? Sengupta S, Pal B, Bala T. Rational use of antibiotics and its guidelines in surgical patients-a retropspective study.Int J surg Orthopedics. 2017; 3(2):33-37.doi:10.17511/ijoso.2017.i02.02. International Journal of Surgery & Orthopedics Available online at: www.surgicalreview.in 37 \| P a g e
https://openalex.org/W2133636629	https://europepmc.org/articles/pmc3079668?pdf=render	English	null	A first generation integrated map of the rainbow trout genome	BMC genomics	2,011	cc-by	8,100	© 2011 Palti 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. RESEARCH ARTICLE Open Access A first generation integrated map of the rainbow trout genome Yniv Palti1†, Carine Genet2†, Ming-Cheng Luo3, Aurélie Charlet2, Guangtu Gao1, Yuqin Hu3, Cecilia Castaño-Sánchez1,4, Kamila Tabet-Canale2,5, Francine Krieg2, Jianbo Yao4, Roger L Vallejo1 and Caird E Rexroad III1 Abstract Background: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. Results: The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. Conclusions: The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout. Correspondence: yniv.palti@ars.usda.gov † Contributed equally 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA Full list of author information is available at the end of the article Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Open Access Background Rainbow trout are cultured on every continent except Antarctica, with 2008 global production estimated at 576,289 metric tons and valued at $2.39 billion [2]. Cou- pling great interest in this species as a research model with the need for genetic improvement for aquaculture production efficiency and product quality justifies the continued development of genome resources facilitating selective breeding. Rainbow trout (Oncorhynchus mykiss) are the most- widely cultivated cold freshwater fish in the world and are considered by many to be the “aquatic lab-rat”. Interests in the utilization of rainbow trout as a model species for genome-related research activities focusing on carcinogenesis, toxicology, comparative immunology, disease ecology, physiology, transgenics, evolutionary genetics, and nutrition have been well documented [1]. The rainbow trout genome is large and complex. Gen- ome size estimates derived from determining the molecu- lar weight of DNA per cell for rainbow trout and other salmonids vary from 2.4 to 3.0 × 109 bp [3,4]. As with most salmonids, rainbow trout experienced a recent genome duplication event resulting in a semi-tetraploid * Correspondence: yniv.palti@ars.usda.gov † Contributed equally 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA Full list of author information is available at the end of the article Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 2 of 9 Page 2 of 9 QTL, the selection of positional candidate genes and the incorporation of marker-assisted selection (MAS) into rainbow trout breeding programs. A major shortcoming of QTL studies is that they are limited to the variation present in a limited number of families and typically do not detect loci with small effects. This can be overcome by whole genome association studies and other approaches, such as genomic selection, that capture the effects of most QTL that contribute to the population- wide variation in a trait. Recently we demonstrated the feasibility of low resolution LD association studies in rainbow trout [40,41]. In the absence of a reference gen- ome sequence assembly, a robust integrated physical and genetic map will provide better resolution than the current genetic maps for ordering of genetic markers and estimating physical distances between markers, thus facilitating future whole genome association studies in rainbow trout. state (i.e. after an autotetraploid event in the salmonids, their genome is undergoing reversion to a diploid state) [5]. Background All ray-finned fishes share an additional (3R) round of ancestral genome duplication in their evolutionary his- tory compared to mammals and birds, but the salmonids’ common ancestor underwent an additional recent (4R) whole genome duplication event and more than half of the loci are still duplicated [6]. In addition, it is estimated that 50% to 60% of the rainbow trout genome contains interspersed repeat sequences (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characteri- zation and assessment of synteny between trout and other fish genomes, submitted). Current genomic resources available for rainbow trout research include multiple bacterial artificial chromosome (BAC) libraries and a BAC fingerprinting physical map [6-8]; a database of ~200,000 BAC end sequences (BES) (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of syn- teny between trout and other fish genomes, submitted); doubled haploid (DH) clonal lines [9-12]; multiple genetic maps based on clonal lines and outbred popula- tions [4,13-16]; large expressed sequence tag (EST) data- bases and a reference transcriptome [17-19]; a microRNAs database [20] and high density DNA micro- arrays [21,22]. The first BAC-based physical map of the rainbow trout genome was recently assembled using DNA finger- prints of 154,439 clones from the 10X HindIII Swanson library [8]. The map contains 4,173 contigs and 9,379 singletons. The physical length of the map contigs was estimated to be approximately 2.0 Gb, which represents approximately 80% of rainbow trout genome. Here we report the construction of the first integrated physical and genetic map of the rainbow trout genome using microsatellites isolated from BAC end sequences and PCR superpools for library screening and identification of BACs that harbor previously mapped markers. This integrated map provides a frame work for a robust com- posite genome map and future reference genome sequence assemblies. Two microsatellite-based genetic maps with medium to high marker densities were recently developed for rainbow trout by INRA [13] and the NCCCWA [16]. The INRA map is based on a panel of two DH gynoge- netic lines. It has more than 900 microsatellites over 31 linkage groups and a total length of 2,750 cM (average resolution of 3 cM). The NCCCWA map is based on a panel of five families that represent the starting genetic material of the NCCCWA selective breeding program. Background It has 1,124 microsatellite loci over 29 linkage groups and a total length of 2,927 cM (average resolution of 2.6 cM). The linkage groups from the two microsatellite genetic maps were anchored to the physical chromo- somes using fluorescent in-situ hybridization and were found to represent 52 chromosome arms [23,24]. Results and Discussion BAC end sequencing (BES) microsatellites BAC end sequencing (BES) microsatellites We screened the BES reads from 184 of the largest BAC fingerprinting contigs and selected 205 microsatellites from 117 contigs for PCR optimization and genotyping (Table 1). Of the 205 markers genotyped, 128 markers appeared to amplify single marker regions and were polymorphic. Ten markers were monomorphic, and 58 markers could not be resolved and unambiguously scored. Fifteen markers generated duplicated patterns, of which 8 could be scored for a single marker region and 1 produced a scorable duplicated pattern. Hence, 7 of the duplicated markers produced a monomorphic or an unresolved pattern for one of the two marker regions. Two of the 128 informative markers could not be assigned to linkage groups (i.e. 126 markers were mapped using the NCCCWA mapping families). The BES reads from which the 126 mapped markers were isolated represent 88 unique BAC FPC contigs. The 205 BES microsatellites are listed in Additional file 1, sheet Qualitative/quantitative trait loci (QTL) mapping experiments in rainbow trout have been very successful because of their high fecundity, external fertilization, and ease of gamete handling and manipulation. Many QTL have been identified for production and life-history traits including resistance to the parasite C. shasta [25], resistance to IHNV [26,27] and to IPNV [28], whirling disease resistance [29], Killer cell-like activity [30], upper thermal tolerance [31,32], embryonic development rate [9,33,34], spawning time [35,36], confinement stress response [37], early maturation [38] and smoltification [39]. The availability of a BAC physical map integrated with the genetic map will facilitate fine mapping of Page 3 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 3 of 9 Table 1 Summary of genotyping results of microsatellite markers isolated from BAC end sequences for integration between the genetic and physical maps Table 1 Summary of genotyping results of microsatellite markers isolated from BAC end sequences for integration between the genetic and physical maps No. of markers identified 205 (from 117 contigs) Informative for linkage analysis 128 (63%; 88 contigs; 129 loci) Mapped to linkage groups 127 loci (98.5%; 88 contigs) PCR optimization failed 58 (28%) Monomorphic in mapping panel 10 (5%) Duplicated 15 (9 informative for mapping and 6 non-informative) Redundancy in contig coverage (optimized, but panel not genotyped) 3 markers PCR of the individual clones and direct sequencing from the BAC DNA. Results and Discussion BAC end sequencing (BES) microsatellites The BAC clones that were positive and their corresponding physical map contigs are listed in Additional file 1, sheet 3. Previously mapped microsatellites The 10x Swanson BAC library was screened with the NCCCWA PCR super-pools using 137 markers that were previously mapped with high confidence to the NCCCWA genetic map representing 25 of the 29 chro- mosomes and the INRA super-pools were screened with 265 markers that were previously mapped onto the INRA genetic map representing all linkage groups. The result of the combined effort was that 146 markers cov- ering all linkage groups were localized to one or two BAC FPC contigs (Table 2). The list of the markers with positive hits is shown in Additional file 1, sheet 2, with the corresponding positive clones and physical map contigs. Single nucleotide polymorphism (SNP) markers Single nucleotide polymorphism (SNP) markers The experimental design and results of SNPs discovery in rainbow trout using a reduced representation library (RRL) were recently published [42]. Of the 183 SNPs that were validated, 167 were polymorphic in the NCCCWA genetic mapping panel and 159 were mapped to chromosomes on the genetic map (Table 3). The HaeIII RRL SNP discovery database was aligned with the BES database (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes, submitted) to find matches that can be useful for the integration of the genetic and physical maps. We found 618 unique matches using SSAHA2 [43]. Assum- ing 48% validation rate for this SNPs database [42] we expect that approximately 300 of the matched SNPs will be useful for integration between the physical and genetic maps. Two of the matching SNPs were among the 183 validated by Castaño-Sánchez et al. [42]. One marker (OMS00144) was among the 159 that were mapped. The other SNP (OMS00174) was not informa- tive for linkage analysis in the NCCCWA panel, but it had two positive hits on end sequences from two BACs that overlap in contig number 431 of the physical map (Additional file 1, sheet 3). 1, with the corresponding PCR primers and conditions for each marker, number of alleles and size range, Gen- Bank accessions, primers sequences and physical map contigs. We have also mapped an additional six BES microsatellites onto linkage groups of the INRA genetic map (Additional file 1, sheet 1). The genetic map I f i f g p Information from 1,486 genetic loci was used for linkage analysis (Table 3). Two-point linkage analysis placed 1,229 loci in 29 linkage groups at LOD ≥8.75. An addi- tional 192 markers with two-point LOD <8.75 were added to linkage groups manually, of which only six markers had a two-point LOD <3.0 (2.90, 2.89, 2.64, 2.12, 2.10 and 1.80). The specific best of two-point LOD score for each marker is provided in Additional file 3, Worksheet 1. The total combined sex averaged map dis- tance was 3,346.3 cM (Kosambi). A sample map repre- senting chromosome 2 is presented in Figure 1, and maps representing all chromosomes are presented in Additional file 4. Multipoint linkage analysis was con- ducted on individual linkage groups to assign LOD scores for the specific position of each marker within the linkage group. The number of markers included in a framework map created at LOD ≥4 for the specific posi- tion of the marker in the linkage group was 460. The only chromosome that did not contain any framework markers at LOD ≥4 was OMY21, for which a framework map was created at LOD ≥3. Additional loci were added at LOD ≥3 (77), ≥2 (80) ≥1 (56), and ≥0 (748) (Table 3). Immune response genes The integrated map Anchoring of 203 BAC contigs from the physical map to linkage groups was accomplished through mapping of 266 loci onto the NCCCWA genetic map. The marker loci were derived from the PCR screening of the BAC superpools, BES microsatellites (OMY4000), microsatel- lites isolated from BACs that harbor genes of interest (OMM3000) and one SNP marker matched with BES (OMS00144). A schematic illustration of a BAC finger- printing contig anchored to a linkage group is presented in Figure 2. Markers from 12 of the anchored contigs were mapped to two different linkage groups as a result of PFC assembly errors or linkage mapping errors as we have previously discussed [8]. The fraction of contigs that are in disagreement between the physical map and genetic map is used to estimate the error rate in the FPC assembly. This error rate of 6% (12/203) is similar to the 5% estimated for the catfish physical map of Qui- niou et al. [45] or the 4% rate detected in the 3-color HICF physical map of the maize genome [46]. The number of contigs anchored per chromosome ranged from 3 to 17 with an average of 7.4. Chromosomes OMY18, 24 and 28 had the lowest number of 3 anchored contigs each, and OMY12 had the highest number with 17 anchored contigs. In this version of the map, we have added to the map of Rexroad et al. [16] through multipoint linkage analy- sis 159 RRL SNPs, 126 microsatellites from BES and 9 microsatellites isolated from BACs that harbor immune response genes (Additional file 2, Table S2). The SNPs were distributed in all the chromosomes (2-10 per chro- mosome; Additional file 3, worksheet 3) and the BES microsatellites were mapped to all but chromosome 24 (1-10 per chromosome; Additional file 3, worksheet 4). Twenty seven loci that were previously mapped to expand the length of linkage groups [16] were not mapped in this version, and 29 loci that were previously genotyped but were not linked, were assigned to linkage groups in the current version. A high frequency of The combined physical length of the 203 anchored contigs was 138,525 consensus bands (CB) which is equal to 235,493 Kb based on a conversion ratio of 1 CB = 1.7 Kb [8]. Immune response genes The BAC library was also screened with PCR primers from 12 immune response genes that were not pre- viously mapped to the rainbow trout genome (Addi- tional file 2, Table S1). Positive clones were verified by Table 2 Summary of BAC library screening results with previously mapped microsatellites using PCR super-pools INRAa USDAb Combined No. of markers tested 265 137 396 Localized to a single FPC contig 98 41 135 Localized to two FPC contigs 7 5 11 Singletons or failed DNA fingerprinting 21 15 35 Not validated by single clone PCR 4 12 16 Not positive by PCR screening of superpools 135 64 199 No. of chromosomes covered 29 22 29 aMarkers previously mapped on the INRA genetic map [13]. bMarkers previously mapped on the USDA-NCCCWA genetic map [16]. Table 2 Summary of BAC library screening results with previously mapped microsatellites using PCR super-pools INRAa USDAb Combined Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 4 of 9 Table 3 Genetic loci sources and linkage mapping statistics Marker Source Inputa Mapped LOD4 LOD3 LOD2 LOD1 LOD0 % of Input Rexroad et al. 2008 [16] 1180 1126b 396 62 57 43 568 95% SNPs (OMS) 167 159 21 5 13 8 112 95% OMY4000 (BES) 128 127 40 10 10 5 62 98% Immune Genesc 10 9 3 0 0 0 6 90% Total 1485 1421d 460 77 80 56 748 96% Percent 100% 32% 5% 6% 4% 53% aLoci that were genotyped and were informative for linkage analysis in the NCCCWA mapping panel. bTwenty seven loci that were previously mapped to linkage groups in the genetic map version of Rexroad et al. (2008) were not linked to other loci in this version, and 29 loci that were previously genotyped but not linked were assigned to linkage groups in the current version. cMicrosatellites or SNPs isolated from immune response genes (Additional file 2, Table S2). dEighty eight duplicated loci were mapped to linkage groups in the current version (total number of markers was 1,333). Table 3 Genetic loci sources and linkage mapping statistics Additional file 3, worksheet 1 contains this information and can be used to recreate maps using MapChart soft- ware [44]. The average resolution of the genetic map was 2.35 cM with inter-marker distances ranging from 1.31 to 3.59 cM for individual chromosomes (Additional file 3, Worksheet 2). Immune response genes duplicated microsatellite loci was observed as previously reported [16], but in many cases only one locus was successfully ordered on the map. Overall, 88 duplicated markers were successfully mapped to two loci (176 loci), which means that the total number of markers mapped was 1,333. The female:male recombination ratio was 1.65:1, with the female having a map length of 4,775.7 cM and the male map 2,897.8 cM. This ratio varied by chromosome, ranging from 0.53:1 to 11.87:1 (Additional file 3, Work- sheet 2). It is noteworthy that this type of sex recombi- nation ratio estimates do not take into account the larger differences in recombination rate that exist between males and females throughout most of the length of the linkage groups. It is likely that female:male ratios will be elevated throughout most of the length of the chromosome arms, while they will be much lower in the more contracted telomeric ends of the linkage groups because of elevated male recombination rates in these regions [15]. It should be pointed out that overall estimates of recombination rate may not be accurately depicted in the current study, because recombination estimates were not obtained by direct comparisons of adjacent intervals. Therefore, the reported recombina- tion distances given in this study are likely an underesti- mate of the real recombination ratio values. Conclusions The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chro- mosomes. This map provides a frame work for a robust composite genome map. The availability of an integrated physical and genetic map will enable detailed compara- tive genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for eco- nomically important traits and the incorporation of MAS into rainbow trout breeding programs. A compre- hensive integrated map will also provide a minimal tiling path for genome sequencing and a framework for whole genome sequence assembly. Figure 1 Chromosome 2 from the new NCCCWA linkage map is shown as an example. Annotation of genes linked to the marker or BAC contig from the 1st generation physical map are connected to the marker name by underscore (e.g. OMM3080_TAP1_ctg260). Annotation of “or_?” means that the marker is duplicated and only one of two BAC contig was identified for the marker. Blue, Green, Red, Black and Italicized font markers were mapped to their specific location on the linkage group at LOD scores of 4, 3, 2, 1 and 0, respectively. Sex average distances between markers are shown in cM. The integrated map Therefore, we estimate that the inte- grated map covers ~12% of the physical map, or ~10% of the rainbow trout genome, assuming haploid genome size of 2.4 × 109 bp. The length of anchored contigs ranged from 119 Kb to 4,590 Kb with an average length Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 5 of 9 Page 5 of 9 average of 3,033. In addition, 35 contigs were anchored to linkage groups of the INRA map through markers that were not informative for linkage analysis in the NCCCWA mapping panel (Additional file 3, worksheet 7), bringing the total number of anchored contigs to 238. Rainbow Trout Chr. 2 Figure 1 Chromosome 2 from the new NCCCWA linkage map is shown as an example. Annotation of genes linked to the marker or BAC contig from the 1st generation physical map are connected to the marker name by underscore (e.g. OMM3080_TAP1_ctg260). Annotation of “or_?” means that the marker is duplicated and only one of two BAC contig was identified An FPC map with all the genetic markers that we have assigned to BAC contigs can be viewed and searched online through: http://www.genome.clemson. edu/activities/projects/rainbowTrout The integrated map we developed for the rainbow trout genome will facilitate comparative genomics stu- dies with other salmonids and with model fish species. Many microsatellite markers can be used for genetic mapping across salmonid species which is very useful for comparative genome mapping [23,48] and can bene- fit research in species with less developed genome maps. In addition, the rainbow trout BAC end sequences can be used to infer conserved synteny with other fish gen- omes as we have previously shown (Genet et al.: Analy- sis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes, submitted), and this inte- grated map provides a larger frame-work expanding the size of the syntenic blocks that can be identified between fish genomes. BAC end sequencing and markers development ŽŶƚŝŐϮϲϬ;ĂŶĐŚŽƌĞĚƚŽŚƌ͘ϮͿ Figure 2 A schematic illustration of a BAC fingerprinting contig anchored to the rainbow trout Chr. 2 using microsatellites isolated from BACs. The four microsatellite markers from Ctg260 (224 clones; 1,584 CB or approximately 2.7 Mb) were mapped to Chr. 2 and the TAP1 positive BACs (highlighted in green) were previously identified by probe hybridization and confirmed by PCR and direct sequencing. The microsatellites order shown is based on the FPC map (not the genetic map). Markers in bold blue (OMY4005 and 4006) were localized on the linkage group at LOD4 and markers in regular font at LOD0. The genetic distance between the LOD4 markers is marked by a solid-line arrow and between markers that were localized at lower confidence by broken-line arrows. concentrations. PCR reactions (12 μl total volume) included 50 ng DNA, 1.5-2.5 mM MgCl2, 2 pmol of for- ward primer, 6 pmol of reverse primer, 1 pmol of fluores- cent labeled primer, 200 μM dNTPs, 1X manufacturer’s reaction buffer, and 0.5 unit Taq Polymerase (ABI, Foster City, CA, USA). Amplifications were conducted in an MJ Research DNA Engine thermal cycler model PTC 200 (MJ Research, Waltham, MA) as follows: an initial dena- turation at 95°C for 10 min, 30 cycles consisting of 94°C for 60 sec, annealing temperature for 45 sec, 72°C exten- sion for 45 sec; followed by a final extension of 72°C for 10 min. PCR products were visualized on agarose gels after staining with ethidium bromide. Three μl of each PCR product was added to 20 μl of water, 1 μl of the diluted sample was added to 12.5 μl of loading mixture made up with 12 μl of HiDi formamide and 0.5 μl of Genscan 400 ROX internal size standard. Samples were denatured at 95°C for 5 min and kept on ice until loading on an ABI 3730 DNA Analyzer (ABI, Foster City, CA, USA). Output files were analyzed using GeneMapper version 3.7 (ABI, Foster City, CA, USA), formatted using Microsoft Excel and stored in a Microsoft Access database. software [49]. The PHRED quality score cut-off value was set at 20 for the acquisition of Q20 values. The BESs were trimmed of vector sequences (pBeloBAC11 vector [50]) and filtered of E. coli sequences. Microsatel- lites and other simple sequence repeats (SSR) were ana- lyzed using Tandem repeat Finder software [51]. BAC end sequencing and markers development BAC end sequencing and markers development The 10X HindIII Rainbow trout BAC library [6] was used for BAC-end sequencing (BES) as previously described (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assess- ment of synteny between trout and other fish genomes, submitted). Briefly, BAC culture was conducted using standard protocols and end sequencing with SP6 and T7 primers was done using standard Sanger technique. The raw, untrimmed files were processed by PHRED of 1,160 Kb (Additional file 3, worksheet 5). The ratio of physical to genetic linkage distances varied substantially among the 33 anchored contigs that contained spaced markers, which is similar to other vertebrate genomes [45,47]. The 33 contigs represent segments from 21 of the 29 chromosomes (Additional file 3, worksheet 6). The Kb/cM ratio ranged from 37 to 17,000 with an Palti et al. BMC Genomics 2011, 12:180 Page 6 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Ϭ͘ϬDď Ϭ͘ϱDď ϭ͘ϬDď ϭ͘ϱDď Ϯ͘ϬDď Ϯ͘ϱDď ϯ͘ϬDď ŽŶƚŝŐϮϲϬ;ĂŶĐŚŽƌĞĚƚŽŚƌ͘ϮͿ RT453K16 RT401D05 RT011D19 RT411P21 RT332L07 RT146D09 RT116G20 RT360D03 RT287N06 RT247H11 RT256I12 RT241K09 RT431F21 RT162L19 RT521M20 RT463B12 RT017G19 RT476N09 RT459K05 RT440E04 RT370E22 RT102I21 RT462G03 RT410F12 RT182L04 RT186B04 RT535K23 RT383D08 RT121D07 RT433E15 RT012N07 RT293M21 RT484C04 RT333C01 RT406J04 RT154E12 RT501K19 RT353G05 RT433P02 RT177O05 RT470J20 RT495P06 RT165A05 RT358G09 RT183N23 RT325O11 RT119M15 RT374F19 RT190G04 RT399E06 RT177L08 RT020B13 RT265E05 RT338C12 RT197D04 RT001E01 RT534K13 RT456H04 RT227B03 RT221N22 RT516J20 RT251J04 RT375L08 RT542H15 RT350C10 RT230M18 RT416P19 RT452F06 RT215A10 RT372O14 RT292E17 RT162K21 RT188H22 RT381F24 RT101E21 RT019L08 RT495G23 RT344F20 RT107B20 RT279D06 RT170F24 RT170F20 RT438N02 RT181E06 RT168M24 OMY4005 OMY4007 TAP1 OMM3080 OMY4006 0.5 cM 6.5 cM 5.8 cM Figure 2 A schematic illustration of a BAC fingerprinting contig anchored to the rainbow trout Chr. 2 using microsatellites isolated from BACs. The four microsatellite markers from Ctg260 (224 clones; 1,584 CB or approximately 2.7 Mb) were mapped to Chr. 2 and the TAP1 positive BACs (highlighted in green) were previously identified by probe hybridization and confirmed by PCR and direct sequencing. The microsatellites order shown is based on the FPC map (not the genetic map). Markers in bold blue (OMY4005 and 4006) were localized on the linkage group at LOD4 and markers in regular font at LOD0. The genetic distance between the LOD4 markers is marked by a solid-line arrow and between markers that were localized at lower confidence by broken-line arrows. BAC end sequencing and markers development We examined ten classes of SSRs by using a maximum per- iod size of 10. BES reads harboring at least 50 base pairs (bp) flanking sequences on either side of the SSRs were selected for PCR primer design. Primers for BESs con- taining microsatellites were designed using Primer3 soft- ware [52]. The primer product size range was chosen between 150 and 450 nucleotides. The optimum size of primers was set to 20 nucleotides (range from 18 to 27 nucleotides) with an optimum melting temperature of 60.0°C (range from 57 to 63°C). Microsatellites Genotyping The NCCCWA mapping panel of 5 families was geno- typed with microsatellites as previously described [16]. A total of 205 microsatellite markers isolated from BAC end sequences (Additional file 1, sheet 1) were genotyped using the tailed protocol of Boutin-Ganache et al. [53]. Primers were obtained from commercial sources (Alpha DNA, Montreal, Quebec, Canada). Three oligonucleotide primers were used in each DNA amplification reaction (Forward: 5’ GAGTTTTCCCAGTCACGAC-primer sequence 3’; reverse: 5’ GTTT-primer sequence 3’; fluor- escent labeled primer with FAM: 5’ GAGTTTTCCCA GTCACGAC 3’). Primers were optimized for amplifica- tion by varying annealing temperatures and MgCl2 Author details 1 The microsatellites and SNPs were placed on the rain- bow trout genetic map using the genetic linkage map- ping programs MULTIMAP [59] and CRI-MAP [60]. First, genotype data combined for both sexes were for- matted into the standard LINKAGE [61] file format and checked for Mendelian inheritance using PEDCHECK [62]. RECODE [63] was then used to convert the allele sizes into number-coded alleles. Using an in-house Perl script, make_gen, the genotype data and the locus names were assembled into CRI-MAP input format. The 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA. 2INRA, UMR1313, Génétique Animale et Biologie Intégrative, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. 3Department of Plant Sciences, University of California, One Shields Ave., Davis, CA 95616, USA. 4West Virginia University, Animal and Nutritional Sciences, Morgantown, WV, 26506, USA. 5INRA, UMR 444 ENVT Génétique Cellulaire, 31326 Castanet-Tolosan, France. SNPs discovery using reduced representation libraries (RRL) Protocols developed and used for SNPs discovery in cat- tle and swine [56-58] were adapted for rainbow trout using RRL libraries and high throughput parallel 454 GS FLX pyrosequencing. The experimental design and results of the rainbow trout work were recently pub- lished [42]. Briefly, DNA from 96 unrelated individuals representing the families of the NCCCWA broodstock was pooled into one sample. The reduced representation library consisted of 440 bp fragments resulting from complete digestion of the pooled DNA with the restric- tion enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the esti- mated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent computa- tional data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually repre- sent paralogous loci in the semi-tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Alignments between SNPs and BES To find matches we aligned the HaeIII RRL SNP discov- ery database of Castaño-Sánchez et al. [42] with the BES database (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assess- ment of synteny between trout and other fish genomes, submitted). Matches were found using SSAHA2 [43] (http://www.sanger.ac.uk/Software/analysis/SSAHA2/) for pairwise sequence alignment with a threshold Smith- Waterman score of 160 (very restrictive and conserved to avoid matches between paralogous loci). Acknowledgements h g This project was supported by National Research Initiative Competitive Grant no. 2007-35616-17875 from the USDA National Institute of Food and Agriculture and by internal base funds provided by the Agricultural Research Service project no. 1930-31000-009. The INRA BAC library pooling was granted by the French program AGENOP. The BAC end sequencing was made possible by the financial support of Genoscope. We thank Brian Smith, Kristy Shewbridge, Renee Fincham and Roseanna Long for their technical support in the microsatellites genotyping, and Amandine Launay for technical support in the INRA PCR superpools screening. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Library screening with PCR superpools The 10x Swanson BAC library was screened using the NCCCWA or the INRA PCR superpools with microsa- tellites that were mapped to the NCCCWA or INRA genetic maps [13,16] as previously described [54,55]. The screening results were cross-referenced with the physical map to localize the positive clones onto contigs. Page 7 of 9 Page 7 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 resulting file was then added to that of Rexroad et al. [16] using another in-house Perl script, join_gens, and MULTIMAP was used to conduct two-point linkage analyses to identify the closest markers with LOD ≥8.75 and recombination fraction r ≤0.2. An additional 192 markers with two-point LOD <8.75 were added to link- age groups manually, of which only six markers had a two-point LOD <3.0 (2.90, 2.89, 2.64, 2.12, 2.10 and 1.80). The specific best of two-point LOD score for each marker is provided in Additional File 3, Worksheet 1. Multipoint linkage analysis was conducted on individual linkage groups to assign LOD scores for the specific position of each marker within the linkage group. Fra- mework maps were constructed at LOD ≥4 for all link- age groups but OMY21, for which the framework map was created at LOD ≥3. Markers were added to compre- hensive maps by lowering the LOD threshold one inte- ger at a time and starting with the previous order. Resulting maps are consensus maps, accounting for co- informative meiosis across the five families. Chromo- some numbers were assigned to linkage groups using the integrated cytogenetic/linkage map of Phillips et al. [24]. For microsatellite markers that did not have at least two positive clones from the same FPC BAC contig, the individual positive clones were picked from glycerol stock and confirmed by PCR as previously described [6]. Additional material Additional file 1: BES microsatellites. Additional file 2: Table S1. Additional file 3: Additional material. Additional file 4: chromosome maps. References Palti Y, Gahr SA, Hansen JD, Rexroad CE: Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication. Anim Genet 2004, 35(2):130-133. 6. Palti Y, Gahr SA, Hansen JD, Rexroad CE: Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication. Anim Genet 2004, 35(2):130-133. 27. Rodriguez MF, LaPatra S, Williams S, Famula T, May B: Genetic markers associated with resistance to infectious hematopoietic necrosis in rainbow and steelhead trout. Aquaculture 2004, 241(1):93-115. 7. Katagiri T, Asakawa S, Minagawa S, Shimizu N, Hirono I, Aoki T: Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia. Anim Genet 2001, 32(4):200-204. 28. Ozaki A, Sakamoto T, Khoo S, Nakamura K, Coimbra MR, Akutsu T, Okamoto N: Quantitative trait loci (QTLs) associated with resistance/ susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss). Mol Genet Genomics 2001, 265(1):23-31. 8. Palti Y, Luo MC, Hu Y, Genet C, You F, Vallejo R, Thorgaard G, Wheeler P, Rexroad C: A first generation BAC-based physical map of the rainbow trout genome. BMC Genomics 2009, 10(1):462. 29. Baerwald MR, Petersen JL, Hedrick RP, Schisler GJ, May B: A major effect quantitative trait locus for whirling disease resistance identified in rainbow trout (Oncorhynchus mykiss). Heredity 2010. 9. Robison BD, Wheeler PA, Sundin K, Sikka P, Thorgaard GH: Composite interval mapping reveals a major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss). J Hered 2001, 92(1):16-22. 30. Zimmerman A, Evenhuis J, Thorgaard G, Ristow S: A single major chromosomal region controls natural killer cell-like activity in rainbow trout. Immunogenetics 2004, 55(12):825-835. 10. Robison BD, Wheeler PA, Thorgaard GH: Variation in development rate among clonal lines of rainbow trout (Oncorhynchus mykiss). Aquaculture 1999, 173(1-4):131-141. 31. Perry GM, Danzmann RG, Ferguson MM, Gibson JP: Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss). Heredity 2001, 86(Pt 3):333-341. 11. Young WP, Wheeler PA, Fields RD, Thorgaard GH: DNA fingerprinting confirms isogenicity of androgenetically derived rainbow trout lines. J Hered 1996, 87(1):77-80. 32. Perry GM, Ferguson MM, Sakamoto T, Danzmann RG: Sex-linked quantitative trait loci for thermotolerance and length in the rainbow trout. J Hered 2005, 96(2):97-107. 12. Quillet E, Dorson M, Le Guillou S, Benmansour A, Boudinot P: Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout. Fish & Shellfish Immunology 2007, 22(5):510-519. 33. Received: 22 December 2010 Accepted: 7 April 2011 Published: 7 April 2011 Received: 22 December 2010 Accepted: 7 April 2011 Published: 7 April 2011 References 22. 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Genetics 1998, 148(2):839-850. 25. Nichols KM, Bartholomew J, Thorgaard GH: Mapping multiple genetic loci associated with Ceratomyxa shasta resistance in Oncorhynchus mykiss. Dis Aquat Organ 2003, 56(2):145-154. 5. Allendorf FW, Thorgaard GH: Tetraploidy and the evolution of salmonid fishes. In Evolutionary Genetics of Fishes. Edited by: Turner BJ. New York: Plenum Press; 1984:1-46. 5. Allendorf FW, Thorgaard GH: Tetraploidy and the evolution of salmonid fishes. In Evolutionary Genetics of Fishes. Edited by: Turner BJ. New York: Plenum Press; 1984:1-46. 26. Barroso RM, Wheeler PA, LaPatra SE, Drew RE, Thorgaard GH: QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) xYellowstone cutthroat (Oncorhynchus. Aquaculture 2008, 277(3):156-163. 6. Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. 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Genetics 2000, 155(3):1331-1345. physical map and wrote the manuscript draft; CG participated in the study design, obtained BAC end sequences, identified microsatellites in BAC end sequences and designed PCR primers for microsatellites genotyping and supervised the INRA BAC library screening; MCL participated in the study design, supervised DNA extractions and BAC fingerprinting and assembled the physical map; AC screened the INRA PCR superpools; GG improved the genetic linkage analysis pipeline, conducted the linkage analysis and assembled the genetic map, and conducted the SSAHA2 alignment between the SNP and BES databases; YH conducted DNA extractions and BAC fingerprinting; KTA contributed to the INRA BAC library pooling and screening; FK contributed to the INRA BAC library screening; CCS conducted the SNPs discovery and validation experiments; JY co-supervised the SNPs discovery and validation experiments; RLV participated in the study design and developed the genetic linkage analysis pipeline; CER participated in the study design and in the genetic linkage analysis and supervised the SNPs discovery and validation experiments. All authors reviewed and contributed to the manuscript. 16. Rexroad CE, Palti Y, Gahr SA, Vallejo RL: A second generation genetic map for rainbow trout (Oncorhynchus mykiss). BMC Genet 2008, 9:74. rainbow trout (Oncorhynchus mykiss). BMC Genet 2008, 9:74. 17. Rexroad CE, Lee Y, Keele JW, Karamycheva S, Brown G, Koop B, Gahr SA, Palti Y, Quackenbush J: Sequence analysis of a rainbow trout cDNA library and creation of a gene index. Cytogenet Genome Res 2003, 102(1- 4):347-354. 18. Govoroun M, Le Gac F, Guiguen Y: Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries. BMC Genomics 2006, 7(1):196. 19. Salem M, Rexroad C, Wang J, Thorgaard G, Yao J: Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches. BMC Genomics 2010, 11(1):564. 20. Salem M, Xiao C, Womack J, Rexroad CE, Yao J: MicroRNA repertoire for functional genome research in rainbow trout (Oncorhynchus mykiss). Marine Biotech 2009, 12(4):410-429. 21. Rise ML, von Schalburg KR, Brown GD, Mawer MA, Devlin RH, Kuipers N, Busby M, Beetz-Sargent M, Alberto R, Gibbs AR, et al: Development and Application of a Salmonid EST Database and cDNA Microarray: Data Mining and Interspecific Hybridization Characteristics. Genome Res 2004, 14(3):478-490. Authors’ contributions conceived and designed the study, supervised and analyzed microsatellites genotyping, supervised the USDA BAC library screening, integrated the markers information from the genetic and physical maps, updated the Page 8 of 9 Page 8 of 9 References Am J Hum Genet 1998, 63(1):259-266. 39. Nichols KM, Edo AF, Wheeler PA, Thorgaard GH: The Genetic Basis of Smoltification-Related Traits in Oncorhynchus mykiss. Genetics 2008, 179(3):1559-1575. 63. O’Connell JR, Weeks DE: The VITESSE algorithm for rapid exact multilocus linkage analysis via genotype set-recoding and fuzzy inheritance. Nat Genet 1995, 11(4):402-408. 63. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 56. Van Tassell CP, Smith TP, Matukumalli LK, Taylor JF, Schnabel RD, Lawley CT Haudenschild CD, Moore SS, Warren WC, Sonstegard TS: SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat Methods 2008, 5(3):247-252. 57. Wiedmann RT, Smith TP, Nonneman DJ: SNP discovery in swine by reduced representation and high throughput pyrosequencing. BMC Genet 2008, 9:81. 58. 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https://openalex.org/W2078766378	https://periodicos.uem.br/ojs/index.php/ActaSciTechnol/article/download/1196/708	Portuguese	null	Influência da aplicação de esgoto sanitário tratado no desempenho de um sistema de irrigação por gotejamento montado em campo	Acta Scientiarum. Technology/Acta scientiarum. Technology	2,006	cc-by	3,684	Rafael Oliveira Batista, Antônio Alves Soares, Antonio Teixeira de Matos e Everardo Chartuni Mantovani Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Av. Ph Rolfs, s/n, 36570-000, Viçosa, Minas Gerais, Brasil. Autor para correspondência. e-mail: ms36384@zipmail.com.br RESUMO. Este trabalho verificou o efeito da aplicação de esgoto sanitário tratado no desempenho de um sistema de irrigação por gotejamento montado em campo. Para a realização do experimento, utilizou-se um sistema de irrigação por gotejamento montado na Estação-Piloto de Tratamento de Esgoto do Departamento de Engenharia Agrícola da Universidade Federal de Viçosa, Estado de Minas Gerais. Tal sistema constava de uma unidade de controle (composta por motobomba de 3 cv e filtro de 550 mesh), de linhas principal e de derivação e de linhas laterais com gotejadores do modelo G1. Os resultados mostraram que o efluente apresentou considerável potencial de obstrução de gotejadores, tendo como conseqüência direta o decréscimo tanto da uniformidade de aplicação do efluente quando da vazão dos gotejadores. Palavras-chave: gotejadores, água residuária, obstrução. ABSTRACT. Influence of treated sanitary wastewater application on performance of a drip irrigation system built in field. This work verified the effect of the application of treated sanitary wastewater on performance of a drip irrigation system built in field. For the accomplishment of the experiment, a drip irrigation system was built in Sewer Treatment Pilot Plant of the Federal University of Viçosa’s, State of Minas Gerais, Agricultural Engineering Department. The system consisted of a control unit (composed of a 3 cv pump and a 550-mesh filtration system), lateral lines, and derivation and main lines with model dripper G1. Results showed that the effluent presented considerable potential of clogging drippers, having as direct consequence as much decrease of the uniformity application of effluent as flow rate of drippers. Key words: drippers wastewater clogging Acta Sci. Technol. Influência da aplicação de esgoto sanitário tratado no desempenho Influência da aplicação de esgoto sanitário tratado no desempenho Influência da aplicação de esgoto sanitário tratado no desempenho Influência da aplicação de esgoto sanitário tratado no desempenho de um sistema de irrigação por gotejamento montado em campo de um sistema de irrigação por gotejamento montado em campo de um sistema de irrigação por gotejamento montado em campo de um sistema de irrigação por gotejamento montado em campo Rafael Oliveira Batista*, Antônio Alves Soares, Antonio Teixeira de Matos e Everardo Chartuni Mantovani IIIIntrodução ntrodução ntrodução ntrodução passagem para o líquido em escoamento e a velocidade da água por essa passagem. Geralmente, gotejadores com tamanho de seção transversal inferior a 1,5 mm apresentam alguma sensibilidade à obstrução. Porém, velocidades que oscilam entre 4,0 e 6,0 m s-1 reduzem o entupimento nas estreitas passagens dos gotejadores (Keller e Bliesner, 1990). Por meio de avaliações experimentais, Resende et al. (2000) verificaram que o entupimento por causa biológica estava mais relacionado à arquitetura interna dos gotejadores do que a parâmetros de vazão e diâmetro do orifício. A utilização de águas residuárias na agricultura é uma alternativa para o controle da poluição dos corpos hídrico receptores e disponibilização de água e fertilizantes para as culturas. Entretanto, para que isso possa se tornar uma prática viável, é preciso que sejam desenvolvidas técnicas de tratamento, aplicação e manejo de águas residuárias. O grande problema associado à utilização de águas residuárias em sistemas de irrigação por gotejamento consiste na alteração da vazão pelo entupimento parcial ou total dos gotejadores e como esta afeta a uniformidade de distribuição de água. Existem muitos fatores físicos, químicos e biológicos nas águas residuárias com elevado potencial de entupimento de gotejadores. A formação de depósitos gelatinosos, resultantes da interação entre mucilagens bacterianas, algas e A sensibilidade ao entupimento é uma consideração muito importante quando da seleção do gotejador. Dois parâmetros críticos relacionados à sensibilidade de obstrução são o tamanho da Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006 214 Batista et al. zooplâncton, tem sido o fator central no processo de entupimento de gotejadores que aplicam esgotos sanitários tratados (Ravina et al., 1992; Ravina et al., 1997). Estudos realizados por Taylor et al. (1995) evidenciaram que as interações entre fatores físicos, químicos e biológicos foram responsáveis por 90% dos gotejadores entupidos. entanto, aquelas plantas que recebiam a lâmina adequada passam a ter problema de irrigação excessiva, aumentando a perda por percolação (López et. al., 1992). A uniformidade de aplicação de água em sistemas de irrigação localizada pode ser expressa por meio de vários coeficientes. O coeficiente de uniformidade de Christiansen (CUC), apresentado na equação 1, foi adaptado da irrigação por aspersão. Mantovani (2002) apresentou o seguinte critério geral para a interpretação dos valores do CUC para sistemas de irrigação por gotejamento: entre 90 e 100%, excelente, entre 80 e 90%, bom, entre 70 e 80%, razoável, entre 60 e 70% ruim e menor que 60%, inaceitável. Acta Sci. Technol. IIIIntrodução ntrodução ntrodução ntrodução Colônias de protozoários do gênero Ciliatea e colônias de Bryozoa plumatella foram identificadas nos gotejadores entupidos e ao longo das linhas laterais de sistemas de irrigação por gotejamento abastecidos com esgoto sanitário tratado (Ravina et al., 1992). Problema idêntico foi relatado por Sagi et al. (1995), os quais porém, constataram, nos gotejadores obstruídos, apenas colônias de protozoários (Epystilus balanarum). Os protozoários aderiram-se às paredes do equipamento de irrigação somente quando a velocidade do escoamento do efluente era inferior a 2 m s-1. Adin e Sacks (1991) constataram ovos de Dafnia (efípios) e larvas (com 1 cm de comprimento) dentro de gotejadores autocompensantes que aplicavam esgoto sanitário não-filtrado.             − − = ∑ = q n q q CUC e n i i 1 1 100 (1)             − − = ∑ = q n q q CUC e n i i 1 1 100 (1) (1) em que: em que: qi = vazão de cada gotejador, L h-1; qi = vazão de cada gotejador, L h-1; Rav-Acha et al. (1995) verificaram diminuição de 68% na vazão nominal de gotejadores abastecidos com esgoto sanitário tratado, após 60 horas do início do experimento. Fato similar foi descrito por Sagi et al. (1995), que identificaram colônias de protozoário ocupando 57% da área dos gotejadores, o que acarretou queda de 38% na vazão nominal. Ravina et al. (1992) observaram que os gotejadores autocompensantes foram mais propensos ao aumento de vazão do que os demais gotejadores, devido à deterioração da membrana de autocompensação pela atividade microbiana. q = vazão média dos gotejadores, L h-1; e ne = número de gotejadores. Material e métod Material e métod Material e métod Material e métodos os os os O trabalho foi realizado na Estação-Piloto de Tratamento de Esgoto (EPTE), uma das áreas experimentais do Departamento de Engenharia Agrícola da Universidade Federal de Viçosa (DEA/UFV), localizada em Viçosa, Estado de Minas Gerais. Foi montada uma Estação Elevatória de Esgoto Experimental (EEEe), com a finalidade de abastecer a EPTE com esgoto sanitário bruto proveniente do conjunto residencial Condomínio Bosque Acamari, situado em Viçosa. O esgoto sanitário bruto recalcado para a EPTE recebeu tratamento em três etapas distintas. Inicialmente, o esgoto bruto era bombeado para o tratamento preliminar, em que um desarenador removia os sólidos de elevada massa específica. A diminuição na carga orgânica (DBO5) foi obtida pela disposição do esgoto sanitário sobre faixas com 1,0 m de largura, 25 m de comprimento e declividade de 2%, cultivadas com capim Tifton 85 do gênero Cynodon. Após o tratamento secundário, o esgoto sanitário era lançado em uma lagoa de maturação com capacidade armazenadora de 300 m3, com as dimensões de 50 m de comprimento x 6 m de largura e 1 m de profundidade, para remoção de organismos patogênicos. Os dados de campo foram coletados até o dia 12/12/2003, somente nas oito linhas laterais situadas no lado direito da subárea, conforme apresentado na Figura 1. Nessa parcela, o comprimento das linhas laterais variou de 31 a 33,8 m, tendo como valor médio 32,5 m. No início de cada linha lateral, foi instalada uma válvula para tomada de pressão, devido à variação da declividade ao longo das linhas de derivação. A pressão média de serviço mantida no início das linhas laterais foi de 133 kPa. O esgoto sanitário proveniente da lagoa de maturação foi utilizado na fertirrigação de cafeeiros da variedade Catuaí IAC 99, com dois anos de idade, cultivados, no espaçamento de 2,50 m entre linhas e 0,75 m entre plantas, em uma subárea de 1.400 m2, situada dentro da própria EPTE. A aplicação do esgoto sanitário foi realizada via sistema de irrigação por gotejamento. Figura 1. Esquema do experimento montado em campo para avaliação do desempenho do sistema de irrigação por gotejamento aplicando esgoto sanitário da lagoa de maturação, e apresentação da parcela escolhida para a coleta dos dados. Aplicação de esgoto sanitário em um sistema de irrigação por gotejamento solar e precipitação pluviométrica. efluente e na vazão de gotejadores de um sistema de irrigação por gotejamento montado em campo. solar e precipitação pluviométrica. Para a realização do experimento, utilizou-se o sistema de irrigação por gotejamento montado na EPTE. Tal sistema constava de uma unidade de controle (composta por conjunto motobomba de 3 cv, sistema de filtração automatizado e injetor de fertilizantes, tipo Venturi, com 70 L h-1 de capacidade de injeção), de linhas principal e de derivação, ambas em PVC, e de linhas laterais com gotejadores do modelo G1. Este modelo de gotejador apresentava as seguintes características técnicas: não-autocompensante, vazão nominal de 2,3 L h-1, faixa de pressão de serviço de 50 a 400 kPa, espaçamento entre gotejadores de 0,40 m, comprimento do labirinto de 298 mm, largura do labirinto de 2 mm e um único filtro secundário por gotejador. Esse modelo de gotejador é comumente utilizado em sistemas de irrigação por gotejamento, implantados no Brasil, para irrigação de cafeeiros. A escolha do modelo foi realizada com base na arquitetura interna de seu labirinto, que segundo o fabricante torna o emissor resistente ao entupimento. O labirinto do gotejador era de percurso tortuoso com protuberâncias salientes, ocasionando um fluxo turbulento, que minimizava a deposição de partículas em seu interior. ne = número de gotejadores. Keller e Karmeli (1975) sugerem a utilização da equação 2, que compara a média de 25% dos menores valores de vazões observadas com a média total das vazões para a determinação da uniformidade de aplicação de água de sistemas de irrigação por gotejamento. Merriam e Keller (1978) apresentaram o seguinte critério geral para interpretação dos valores de CUD para sistemas que estejam em operação por um ou mais anos: maior que 90%, excelente; entre 80 e 90%, bom; 70 e 80%, regular; e menor que 70%, ruim. Nakayama e Bucks (1981) concluíram que reduções consideráveis na uniformidade de distribuição de água podem ocorrer mesmo quando existem poucos gotejadores entupidos no sistema de irrigação. Hills e El-Ebaby (1990) verificaram que o acúmulo de material orgânico dentro de gotejadores ocasionou redução de 48,3% no coeficiente de uniformidade estatística de aplicação de água (Us), após 1.000 horas de funcionamento do sistema de irrigação. q q CUD % 25 100 = (2) (2) CUD = coeficiente de uniformidade de distribuição, %; e CUD = coeficiente de uniformidade de distribuição, %; e CUD = coeficiente de uniformidade de distribuição, %; e q25% = valor médio dos 25% menores valores de vazões observadas, L h-1. No que se refere ao manejo da irrigação, outra conseqüência direta da baixa uniformidade de aplicação de água consiste no aumento do volume aplicado, isto é, o irrigante, ao constatar a diminuição da vazão média dos gotejadores, pelo efeito do entupimento, aumenta o tempo de irrigação; logo, as plantas que receberam menor lâmina de irrigação passam a receber maior quantidade de água, de modo a atender às suas exigências hídricas. No Estudos realizados por vários pesquisadores, no mundo, mostram que a aplicação de águas residuárias via sistema de irrigação por gotejamento acarretam sérios problemas de obstrução dos gotejadores. Por esta razão, o presente trabalho objetivou avaliar o efeito da aplicação de esgoto sanitário tratado na uniformidade de aplicação de Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006 215 Acta Sci. Technol. Material e métod Material e métod Material e métod Material e métodos os os os A aplicação do esgoto sanitário da lagoa de maturação ao cafeeiro teve início no dia 9/5/2003, porém, a partir do dia 8/6/2003, tais aplicações foram realizadas sob turno de rega variável, com a finalidade de atender à variação da demanda evapotranspiratória e parte das exigências nutricionais da cultura. O manejo da aplicação do esgoto sanitário tratado foi realizado por meio do balanço de água no solo com o suporte do programa computacional Irriga, levando-se em consideração as características física e hídrica do solo, as características fenológicas da cultura e os dados climáticos da região. Os dados climáticos foram obtidos de uma estação meteorológica automática instalada na EPTE, com a finalidade de fornecer dados de temperaturas máxima, média e mínima, umidade relativa do ar, velocidade do vento, radiação Figura 1. Esquema do experimento montado em campo para avaliação do desempenho do sistema de irrigação por gotejamento aplicando esgoto sanitário da lagoa de maturação, e apresentação da parcela escolhida para a coleta dos dados. O sistema de irrigação operou por um período de 120 horas, porém, com o manejo da fertirrigação, foi Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006 216 Batista et al. possível a aplicação do esgoto sanitário tratado de forma intermitente para a realização dos estudos com relação à uniformidade de aplicação de água. funcionamento de 0 e 120 horas. No entanto, observou-se, também, que todos os valores de CUC foram superiores a 90%, sendo assim, classificados como excelentes por Mantovani (2002). Nos tempos de funcionamento de 0, 50 e 100 horas, os valores de CUD foram classificados como excelentes, segundo o critério proposto por Merriam e Keller (1978). Porém, quando se analisou o valor do CUD no tempo de funcionamento de 120 horas, esse foi classificado como bom. Na unidade de controle foi instalado um filtro tipo membrana, autolimpante, automático, de 550 mesh e com capacidade para 5 m3 h-1. A retrolavagem era acionada por um sistema temporizador, a cada 30 minutos de funcionamento, durante 20 segundos. Sendo a pressão mínima requerida pela retrolavagem de 304 kPa. Em cada uma das oito linhas laterais foram selecionados 24 gotejadores, igualmente espaçados, totalizando 192 emissores a serem avaliados no sistema. Para a determinação da vazão foi necessário obter o volume aplicado pelo emissor em um intervalo de tempo de três minutos. Em seguida, tal volume era medido em proveta graduada de 250 mL. Acta Sci. Technol. Material e métod Material e métod Material e métod Material e métodos os os os Foram realizadas quatro avaliações para determinação da uniformidade de aplicação de água, nos tempos de operação de 0, 50, 100 e 120 horas, respectivamente. 80 84 88 92 96 100 0 50 100 120 CUC (%) (a) 80 84 88 92 96 100 0 50 100 120 Tempo de funcionamento (horas) CUD (%) (b) Figura 2. Valores do CUC (a) e do CUD (b) do modelo de gotejador G1, nos tempos de funcionamento de 0, 50, 100 e 120 horas. 80 84 88 92 96 100 0 50 100 120 CUC (%) 80 84 88 92 96 100 0 50 100 120 CUC (%) Os níveis da uniformidade de aplicação de água foram obtidos por meio dos coeficientes CUC e CUD, descritos pelas equações 1 e 2, respectivamente. As medições de pressão foram realizadas no início e final das linhas laterais, utilizando-se um manômetro. (a) 80 84 88 92 96 100 0 50 100 120 Tempo de funcionamento (horas) CUD (%) (b) Após as 560 horas de aplicação do esgoto sanitário pelo sistema, retiraram-se amostras de gotejadores entupidos para identificação do material de obstrução nos laboratórios específicos dos Departamentos de Biologia Vegetal e de Microbiologia da Universidade Federal Viçosa, Estado de Minas Gerais. (b) Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006 RRRResultados e discussão esultados e discussão esultados e discussão esultados e discussão verificou-se que o material de obstrução resultou da interação entre bactérias e algas presentes na água residuária, que formaram aglomerados na forma de cocos e de pequenos bastonetes. Os gêneros de bactérias Clostridium, Bacillus, Pseudomonas e Enterobacter, juntamente com a ferrobactéria da espécie Cremothix sp., formaram um muco microbiano, no qual se aderiram partículas, principalmente de origem orgânica, representadas por células de algas vivas ou em decomposição. As algas predominantes pertenciam aos filos Cyanophyta (gênero Chlorococcus), Euglenophyta (gêneros Euglena e Phacus) e Chlorophyta (gêneros Selenastrum, Scenedesmus e Sphaerocystis). filtração com filtro de membrana de 550 mesh e abertura dos finais de linha a cada duas semanas não previnem a obstrução de gotejadores. Referências Referências Referências Referências ADIN, A.; SACKS, M. Dripper-clogging factors in wastewater irrigation. J. Irrig. Drain. Eng., New York, v. 117, n. 6, p. 813-826, 1991. HILLS, D.J.; EL-EBABY, F.G. Evaluation of microirrigation self-cleaning emitters. Appl. Eng. Agric., St. Joseph, v. 6, n. 4, p. 441-445, 1990. KELLER, J.; BLIESNER, R.D. Sprinkle and trickle irrigation. New York: Avibook, 1990. O biofilme se desenvolveu no interior dos gotejadores, acarretando mundanças no regime de escoamento, e, conseqüentemente, favorecendo a deposição de partículas sobre o mesmo. Com o aumento da massa do biofilme no interior dos gotejadores, ocorreu ao longo do tempo o entupimento parcial e total dos mesmos. KELLER, J.; KARMELI, D. Trickle irrigation desing. Glendora: Rain Bird Sprinkler Manufacturing, 1975. LÓPEZ, J.R. et al. Riego localizado. Madrid: Mundi-Prensa, 1992. MANTOVANI, E.C. AVALIA. Manual do usuário. Viçosa: DEA/UFV–PNP&D/café Embrapa, 2002. MERRIAM, J.L.; KELLER, J. Farm irrigation system evaluation: a guide for management. Logan: Utah State University, 1978. 2,2 2,3 2,4 2,5 2,6 2,7 0 50 100 120 Tempo de funcionamento (horas) Vazão (L h-1) Figura 3. Valores da vazão do modelo de gotejador G1, nos tempos de funcionamento de 0, 50, 100 e 120 horas. NAKAYAMA, F.S.; BUCKS, D.A. Emitter clogging effects on trickle irrigation uniformity. Trans. ASAE, St. Joseph, v. 24, n. 4, p. 77-80, 1981. RAV-ACHA, C. et al. The effect of chemical oxidants on effluent constituents for drip irrigation. Water Res., London, v. 29, n. 1, p. 119-129, 1995. RAVINA, I. et al. Control of clogging in drip irrigation with stored reclaimed wastewater. Irrig. Sci., New York, v. 13, p. 129-139, 1992. Tempo de funcionamento (horas) RAVINA, I. et al. Control of clogging in drip irrigation with stored treated municipal sewage effluent. Agric. Water Manag., Amsterdam, v. 33, p. 127-137, 1997. Figura 3. Valores da vazão do modelo de gotejador G1, nos tempos de funcionamento de 0, 50, 100 e 120 horas. RESENDE, R.S. et al. Suscetibilidade de gotejadores ao entupimento de causa biológica. Rev. Bras. Eng. Agric. Amb., Campina Grande, v. 4, n. 3, p. 368-375, 2000. Acta Sci. Technol. Received on April 07, 2006. Accepted on December 06, 2006. RRRResultados e discussão esultados e discussão esultados e discussão esultados e discussão Figura 2. Valores do CUC (a) e do CUD (b) do modelo de gotejador G1, nos tempos de funcionamento de 0, 50, 100 e 120 horas. No sistema de irrigação por gotejamento montado em campo, foram realizadas quatro avaliações e, com os dados obtidos, calculados o coeficiente de uniformidade de Christiansen (CUC) e o coeficiente de uniformidade de distribuição (CUD), que são apresentados a seguir. As medidas preventivas com relação ao entupimento de gotejadores utilizadas neste experimento foram a abertura do final das linhas laterais a cada duas semanas e um sistema de filtração automático autolimpante de 550 mesh. O entupimento dos gotejadores, além de diminuir a uniformidade de aplicação de água, propiciou decréscimo em sua vazão, como pode ser visto na Figura 3. Comparando os tempos de funcionamento de 0 e 120 horas, constatou-se redução de 4,56% na vazão média do sistema de irrigação. Por meio das avaliações da uniformidade de aplicação de água, verificou-se que o entupimento de gotejadores foi mais acentuado no final das linhas laterais. Durante a abertura do final dessas linhas, observou-se a saída de um esgoto sanitário tratado com coloração verde-escura, devido à presença de matéria orgânica. De acordo com a Figura 2a e b, os valores do CUC e do CUD do sistema de irrigação decresceram ao longo do tempo, devido ao entupimento dos gotejadores. Verificaram-se, nessas figuras, reduções de 4,49% e 10,58%, nos valores do CUC e do CUD, respectivamente, quando se estabeleceu comparação entre os tempos de Por meio de análises microbiológicas e identificação visual com auxílio de microscópio, Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006 217 Aplicação de esgoto sanitário em um sistema de irrigação por gotejamento com o modelo de gotejador G1 de 4,49%, 10,58% e 4,56%, respectivamente; com o modelo de gotejador G1 de 4,49%, 10,58% e 4,56%, respectivamente; verificou-se que o material de obstrução resultou da interação entre bactérias e algas presentes na água residuária, que formaram aglomerados na forma de cocos e de pequenos bastonetes. Os gêneros de bactérias Clostridium, Bacillus, Pseudomonas e Enterobacter, juntamente com a ferrobactéria da espécie Cremothix sp., formaram um muco microbiano, no qual se aderiram partículas, principalmente de origem orgânica, representadas por células de algas vivas ou em decomposição. As algas predominantes pertenciam aos filos Cyanophyta (gênero Chlorococcus), Euglenophyta (gêneros Euglena e Phacus) e Chlorophyta (gêneros Selenastrum, Scenedesmus e Sphaerocystis). Conclusão Conclusão Conclusão Conclusão Diante dos resultados apresentados concluiu-se que: SAGI, G. et al. Clogging of drip irrigation systems by colonial protozoa and sulfur bacteria. INTERNATIONAL MICROIRRIGATION CONGRESS, 5., 1995. Orlando. Proceedings… St. Joseph: ASAE, 1995. p. 250-254. o esgoto sanitário tratado apresentou um considerável potencial de obstrução de gotejadores. A formação de um biofilme, de coloração verde, resultante da interação entre colônias de bactérias e algas, propiciou entupimento parcial e total dos gotejadores; TAYLOR, H.D. et al. Drip irrigation with waste stabilisation pond effluents: Solving the problem of emitter fouling. Water Sci. Technol., London, v. 31, n. 12, p. 417-424, 1995. g j ocorreu decréscimo do CUC, do CUD e da vazão do gotejador testado, devido à aplicação do esgoto sanitário tratado. Após 120 horas de funcionamento ocorreram reduções nos valores do CUC, do CUD e da vazão do sistema de irrigação Maringá, v. 28, n. 2, p. 213-217, July/Dec., 2006
https://openalex.org/W2280887465	https://europepmc.org/articles/pmc4757889?pdf=render	English	null	An approach to quantifying 3D responses of cells to extreme strain	Scientific reports	2,016	cc-by	9,242	An approach to quantifying 3D responses of cells to extreme strain Yuhui Li1,2,, Guoyou Huang1,2,, Moxiao Li2, Lin Wang1,2,3, Elliot L. Elson2,3,5, Tian Jian Lu2, Guy M. Genin1,2,4,5 & Feng Xu1,2 OPEN received: 26 June 2015 accepted: 18 November 2015 Published: 18 February 2016 The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells. In vivo tissues commonly experience large deformation in both physiological and pathological conditions. For instance, the solid tissues of hollow organs (e.g., bladder) can routinely stretch up to 2.5 times their length, or a nominal strain of 150%1. The alveolar can expand to 1.5 times their volume in normal mice and sustainably keep at 3 times in emphysematous mice2. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells (e.g., fibroblasts) are not known at these levels of sustained strain3–5. The responses of cells to sustained stretches in this upper range has not yet been established definitively for cells in three-dimensional (3D) culture, in part due to challenges with presenting cells a realistic and well-defined culture environment that can sustain such strains6,7. The need for effective 3D culture systems is particularly pressing because the well-established responses to mechanical stimuli of cells in two-dimensional (2D) culture appear to differ substantially from those of cells in 3D8–11. Even the toolbox of transmembrane molecules such as integrins that cells use to connect with their environment might be different in 3D environments8. Further, established tools for mechanobiology in 2D do not extend easily to 3D mechanobiology12–15. Observations acquired using tools such as micropost arrays16, 2D traction microscopy17, and stretchable substrata18 are difficult to relate to 3D due to fundamental differences in the morphologies of cells in 2D and 3D19–21. f p g A number of systems have been developed to probe cell mechanobiology in 3D tissue constructs, but these cannot accommodate the high strains of interest. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results O i i Optimized μMASTs were stable at high strains. μ MASTs each contained (i) a stiff, strong “magnet- ically-actuated” layer of poly(ethylene glycol) dimethacrylate (PEGDMA) encapsulating an iron microsphere, and (ii) a tunable-stiffness “synthetic tissue” hydrogel layer of gelatin methacrylate (GelMA) encapsulating a population of NIH/3T3 cells (Fig. 1a,b, Supplementary Figs S1 and S2). The interface between the two layers was toughened using the double-network principle39 (Fig. 1c,d), and differential swelling of the two layers was over- come by tuning the GelMA concentration (Supplementary Fig. S3). To stretch the synthetic tissues within μ MASTs, the iron microspheres were actuated through a magnetic field that was applied focally by a custom-designed array of magnetic field focusers (Supplementary Figs S4 and S5). Each magnetic field focuser was composed of a cylindrical NdFeB rare earth magnet (2 mm in diameter and 2 mm in height) and an iron wire (1 mm in diameter and 8 mm in length). Simulations showed that the magnetic field focusers, held at fixed spacing in a polytetrafluoroethene (PTFE) mold, amplified the field from the NdFeB magnets on the order of tenfold in the direction of the focusing wires to generate a strong and focal magnetic field with minimal crosstalk (Supplementary Fig. S5). When iron microspheres were actuated through focused magnetic fields, μ MASTs responded linearly and uniformly up to exceptionally high levels of strain (Fig. 1e,f). The stress-strain curves and ultimate strains were repeatable and tunable by varying GelMA concentration. The focused, high magnetic fields showed no statistically significant effects on cellular responses independent of the mechanical stretches they actuated (Supplementary Fig. S6). μ MASTs were synthesized in high-throughput arrays that enabled performance of experiments with high statistical significance. Cell responses were dependent upon combined effects of stiffness and strain. Several cell responses, including cell spreading, polarization and proliferation, relate to important observations in develop- ment and in granulation tissue. To clarify whether the μ MASTs remain stiff with increasing culturing time, we firstly tested the elastic modulus of μ MASTs (6 kPa) at day 1, 3 and 5 of culturing, respectively (Supplementary Fig. S7). The results suggested that there is no significantly decrease even after 5 days culturing, indicating that the proliferated cells and their secreted matrix will not have an obvious effect on remodeling the local microen- vironment in μ MASTs. The effects of matrix stiffness on spreading were diametrically opposite those observed in 2D culture. Results O i i NIH/3T3 cells in 2D culture spread more on stiffer substrata40; however, the opposite was observed for NIH/3T3 cells in 3D, with cells in the 10 kPa synthetic tissues spreading much less on average than those in the 2 and 6 kPa synthetic tissues (Fig. 2a,b and Supplementary Fig. S8). In all cases, cell spreading increased non- linearly with increasing strain, rising rapidly to an asymptotic saturation level at a critical strain in the range of 20–30%. In 10 kPa synthetic tissues, the mean cell spreading area increased almost 5 times when strained up to 30%. These phenomena can be explained only through active cellular processes: spreading area was greater that could be explained by passive stretching of cells, and the lower spreading area in stiffer synthetic tissues would not be expected if passive strain simply opened more volume for cells to occupy. The ~72 hour time interval that cell populations required to spread to steady state mean size did not depend upon strain and matrix stiffness. A possi- ble reason for stronger cell activity in softer μ MASTs is that μ MASTs with lower modulus and monomer concen- tration may be easier to be degraded by encapsulated cells. It is well-known that matrix metalloproteinase (MMP) is a kind of proteolytic enzymes and can degrade virtually any component of the ECM and remodel their local microenvironment, whose expression and activation is increased in almost all human cancers compared with normal tissue41. To clarify if the MMP activity of cells will significantly degrade their local matrix and regulate cell behaviors in μ MASTs, we tested the cell spreading and proliferation in strained group immersed in culture medium with a broad-spectrum MMP inhibitor (Batimastat, BB94). The results indicated that BB94 significantly inhibited the activity of cell behaviors, including mean cell spreading area and proliferated cell numbers, in all three kinds of μ MASTs (2 kPa, 6 kPa and 10 kPa, respectively) during 5 days culturing (Supplementary Fig. S9). In 50% strained 6 kPa group with BB94, the maximum mean cell spreading area is 1850 ± 200 μ m2 and the cell numbers per μ MAST is 620 ± 120 after 5 days of culturing, which is lower than group without BB94 (mean cell spreading area is 2550 ± 100 μ m2 and cell numbers per μ MAST is 750 ± 50). An approach to quantifying 3D responses of cells to extreme strain Yuhui Li1,2,, Guoyou Huang1,2,, Moxiao Li2, Lin Wang1,2,3, Elliot L. Elson2,3,5, Tian Jian Lu2, Guy M. Genin1,2,4,5 & Feng Xu1,2 OPEN Although the strains experienced by cells in a tissue stretched 150% can be lower than 150% due to structural features such as crimping22 and due to the details of load-sharing between cells and extracellular matrix (ECM)23,24, existing systems can sustain stretches that are only a small fraction of this, typically on the order of 30%. One of the earliest such systems is slab-like10 or ring-like tissue constructs25 consisting of cells seeded in a collagen tissue construct. These have provided much insight into issues of tissue remodeling that involve large deformations of the tissue constructs themselves, but only small strains at the level of cells26–29. Like these specimens, tissue constructs synthesized between flexible posts30–32 and tissue constructs adhered to flexible substrates33 cannot attain the high strain levels of interest. Another attractive 3D system is based on fibrous protein hydrogels, including collagen, gelatin, elastin and fibrin, which exhibit sim- ilar structural and mechanical properties with the native ECM34,35. A variety of fibrous hydrogel systems have been developed to help understand how ECM mechanics regulate cell behaviors36,37. For instance, alignment of vascular-derived cells was successfully engineered in collagen hydrogels with 10% cyclic strain33. Differentiation 1The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China. 2Bioinspired Engineering and Biomechanics Center, Xi’an Jiaotong University, Xi’an 710049, China. 3Department of Biochemistry and Molecular Biophysics, Saint Louis, Missouri 63110, USA. 4Department of Neurological Surgery, Washington University School of Medicine, Saint Louis, Missouri 63110, USA. 5Department of Mechanical Engineering and Materials Science, Washington University, Saint Louis, Missouri 63130, USA. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.M.G. (email: genin@wustl.edu) or F.X. (email: fengxu@mail.xjtu.edu.cn) Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 1 www.nature.com/scientificreports/ of cardiac fibroblasts into myofibroblasts was observed in 5% strained collagen hydrogel, which is an important mechanoregulation of fibrosis38. However, existing fibrous hydrogel systems can hardly achieve high strain level, which always happens in hollow tissues (e.g., lung and bladder). Another challenge is that these materials systems do not always allow one to perform 3D cell culture where matrix stiffness is decoupled from external mechanical stimulation (e.g., stress/strain). An approach to quantifying 3D responses of cells to extreme strain Yuhui Li1,2,, Guoyou Huang1,2,, Moxiao Li2, Lin Wang1,2,3, Elliot L. Elson2,3,5, Tian Jian Lu2, Guy M. Genin1,2,4,5 & Feng Xu1,2 OPEN Therefore, it is important to develop a platform that can decouple the effect of matrix stiffness and high strain and help to answer the question how mechanical strain affects cell behaviors.i ff Herein, we presented defined 3D cellular microenvironments called microscale magnetically actuated syn- thetic tissues (micro-MASTs) which could undergo reversible, relatively homogeneous deformation up to 160% following non-contact, focused magnetic actuation. μ MASTs were stable under sustained, accurate, and large (up to 160%) stretches. We describe here the μ MAST system and its application to the study how stretch and ECM stiffness affect proliferation, spreading, polarization, differentiation and apoptosis of several cell types in 3D culture. Results O i i These phenomena can be also found in 2 kPa and 10 kPa strained groups. However, the increasing trend of mean cell spreading area and proliferated cell numbers keep consistent with the group without BB94, suggesting that mechanical strains still made signifi- cant contributions to enhance cell activity in μ MASTs.i Polarization of cells and stress fibers were also functions of both strain and modulus. In unstrained synthetic tissues with an elastic modulus of 6 kPa, NIH/3T3 cells showed random orientation (Fig. 2c and Supplementary Fig. S10). However, cells polarized perpendicular to the stretch direction when the synthetic tissues in which they were cultured under 20% strain for 3 days. This response is consistent with cellular responses at the periphery of a wound but inconsistent with observations of cells cultured on flexible 2D substrata, which always align with the Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ entificreports/ Figure 1. Characterization of microscale, magnetically-actuated synthetic tissues (μMASTs) and interdependence of strain and modulus on cell mechanosensitivity. (a) Each μ MAST contained a stiff, str “magnetically-actuated” PEGDMA layer encapsulating an iron microsphere and a tunable-stiffness “synthet tissue” GelMA layer encapsulating a population of cells. Strains were controlled magnetically using a magne field focusing device. (b) Top view (upper) of the fabricated μ MASTs and side view (bottom) of one stretche μ MAST under different straining. Synthetic tissue modulus: 6 kPa. (c) The failure strain that μ MASTs could withstand prior to failure was maximized by modulating the “soaking time,” the interval between the pourin of the GelMA precursor/cell mixture into the rectangular cut-out in the mold “cover” and the initiation of U photo-crosslinking. Here, UV photo-crosslinking time was fixed at 20 s and UV light power density was fixe at 2.9 mW cm−2. (d) UV photo-crosslinking time was modulated to maximize the failure strain. Here, soakin time was fixed at the optimum of 10 min, and UV light power density at 2.9 mW cm−2. (e) Representative str strain curves for synthetic tissue layers composed of different GelMA fraction were highly linear (symbols: experiment; lines: simulation) (f) Simulated strain (left) and stress (right) distributions in synthetic tissue la Figure 1. Characterization of microscale, magnetically-actuated synthetic tissues (μMASTs) and interdependence of strain and modulus on cell mechanosensitivity (a) Each μMAST contained a sti Figure 1. Characterization of microscale, magnetically-actuated synthetic tissues (μMASTs) and interdependence of strain and modulus on cell mechanosensitivity. Results O i i (a) Each μ MAST contained a stiff, strong “magnetically-actuated” PEGDMA layer encapsulating an iron microsphere and a tunable-stiffness “synthetic tissue” GelMA layer encapsulating a population of cells. Strains were controlled magnetically using a magnetic field focusing device. (b) Top view (upper) of the fabricated μ MASTs and side view (bottom) of one stretched μ MAST under different straining. Synthetic tissue modulus: 6 kPa. (c) The failure strain that μ MASTs could withstand prior to failure was maximized by modulating the “soaking time,” the interval between the pouring of the GelMA precursor/cell mixture into the rectangular cut-out in the mold “cover” and the initiation of UV photo-crosslinking. Here, UV photo-crosslinking time was fixed at 20 s and UV light power density was fixed at 2.9 mW cm−2. (d) UV photo-crosslinking time was modulated to maximize the failure strain. Here, soaking time was fixed at the optimum of 10 min, and UV light power density at 2.9 mW cm−2. (e) Representative stress- strain curves for synthetic tissue layers composed of different GelMA fraction were highly linear (symbols: experiment; lines: simulation). (f) Simulated strain (left) and stress (right) distributions in synthetic tissue layers under magnetic actuation showed highly uniform mechanical fields. Contours were normalized to the peak values. Scale bars: 800 μ m. Figure 1. Characterization of microscale, magnetically-actuated synthetic tissues (μMASTs) and interdependence of strain and modulus on cell mechanosensitivity. (a) Each μ MAST contained a stiff, strong “magnetically-actuated” PEGDMA layer encapsulating an iron microsphere and a tunable-stiffness “synthetic tissue” GelMA layer encapsulating a population of cells. Strains were controlled magnetically using a magnetic field focusing device. (b) Top view (upper) of the fabricated μ MASTs and side view (bottom) of one stretched μ MAST under different straining. Synthetic tissue modulus: 6 kPa. (c) The failure strain that μ MASTs could withstand prior to failure was maximized by modulating the “soaking time,” the interval between the pouring of the GelMA precursor/cell mixture into the rectangular cut-out in the mold “cover” and the initiation of UV photo-crosslinking. Here, UV photo-crosslinking time was fixed at 20 s and UV light power density was fixed at 2.9 mW cm−2. (d) UV photo-crosslinking time was modulated to maximize the failure strain. Here, soaking time was fixed at the optimum of 10 min, and UV light power density at 2.9 mW cm−2. Results O i i (e) Representative stress- strain curves for synthetic tissue layers composed of different GelMA fraction were highly linear (symbols: experiment; lines: simulation). (f) Simulated strain (left) and stress (right) distributions in synthetic tissue layers under magnetic actuation showed highly uniform mechanical fields. Contours were normalized to the peak values. Scale bars: 800 μ m. Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 3 www.nature.com/scientificreports/ Figure 2. Cellular responses in strained μMASTs. (a) Confocal fluorescence images of cells in μ MASTs after 3 days of straining to different levels (Green:F-actin (phalloidin); blue: nuclei (DAPI)). (b) Mean cell spreading area increased with culture time and with strain level, up to a threshold. In all cases, cell spreading increased nonlinearly with increasing strain, rising rapidly to an asymptotic saturation level at a critical strain in the range of 20–30%. In 10 kPa synthetic tissues, the rise in mean cell spreading area was nearly an order of magnitude, 5 times greater than for the more compliant synthetic tissues. (c) Polarization of NIH/3T3 cells in strained μ MASTs. For 6 kPa μ MASTs, cells oriented perpendicular to the applied stretch at lower strain levels and parallel to the applied stretch (90°) at higher strain levels. At the lowest and highest levels of stretch, no dominant polarization was observed. For 2 and 10 kPa μ MASTs, cell orientation with the direction of stretch was evident at intermediate levels of strain. (d) The number of cells per μ MAST increased with time, and up to a threshold (10% strain for 2 kPa μ MASTs, 20% strain for 6 kPa μ MASTs and 30% strain for 10 kPa μ MASTs, respectively), with different strain levels. (e,f) Quantification of live, apoptotic and late apoptotic (dead) cells in μ MASTs as a function of straining showed a substantial drop in cell viability in the range of 100–150% strain. Cells in μ MASTs were analyzed for apoptosis using Annexin V/FITC and PI staining and flow cytometry after 5 days of straining to prescribed levels. Live cells: Q3 (Annexin V(− )/PI(− )); early apoptotic cells: Q4 (Annexin V(+ )/PI(− )); late apoptotic cells: Q2 (Annexin V(+ )/PI(+ )). Control group: cells cultured in μ MASTs without straining. Synthetic tissue modulus: 6 kPa. Error bars, s.d. Results O i i Cellular responses in strained μMASTs. (a) Confocal fluorescence images of cells in μ MASTs after 3 days of straining to different levels (Green:F-actin (phalloidin); blue: nuclei (DAPI)). (b) Mean cell spreading area increased with culture time and with strain level, up to a threshold. In all cases, cell spreading increased nonlinearly with increasing strain, rising rapidly to an asymptotic saturation level at a critical strain in the range of 20–30%. In 10 kPa synthetic tissues, the rise in mean cell spreading area was nearly an order of magnitude, 5 times greater than for the more compliant synthetic tissues. (c) Polarization of NIH/3T3 cells in strained μ MASTs. For 6 kPa μ MASTs, cells oriented perpendicular to the applied stretch at lower strain levels and parallel to the applied stretch (90°) at higher strain levels. At the lowest and highest levels of stretch, no dominant polarization was observed. For 2 and 10 kPa μ MASTs, cell orientation with the direction of stretch was evident at intermediate levels of strain. (d) The number of cells per μ MAST increased with time, and up to a threshold (10% strain for 2 kPa μ MASTs, 20% strain for 6 kPa μ MASTs and 30% strain for 10 kPa μ MASTs, respectively), with different strain levels. (e,f) Quantification of live, apoptotic and late apoptotic (dead) cells in μ MASTs as a function of straining showed a substantial drop in cell viability in the range of 100–150% strain. Cells in μ MASTs were analyzed for apoptosis using Annexin V/FITC and PI staining and flow cytometry after 5 days of straining to prescribed levels. Live cells: Q3 (Annexin V(− )/PI(− )); early apoptotic cells: Q4 (Annexin V(+ )/PI(− )); late apoptotic cells: Q2 (Annexin V(+ )/PI(+ )). Control group: cells cultured in μ MASTs without straining. Synthetic tissue modulus: 6 kPa. Error bars, s.d. (b–d: 10 ≤ n ≤ 15 μ MASTs for each strain level; e,f: 60 ≤ n ≤ 80 μ MASTs for each strain level, p < 0.01, p < 0.001). Scale bars: 50 μ m. direction of a sustained stretch, and cells cultured on isometrically stretched substrate, which orient randomly42. The dominant strain-energy theories of polarization predict the latter43, suggesting that the principles underlying cell polarization in 3D differ fundamentally from those in 2D. The significance of biphasic alignment for 6 kPa stiffness μ MASTs occurred at 60% strain level. Results O i i (b–d: 10 ≤ n ≤ 15 μ MASTs for each strain level; e,f: 60 ≤ n ≤ 80 μ MASTs for each strain level, p < 0.01, *p < 0.001). Scale bars: 50 μ m. Figure 2. Cellular responses in strained μMASTs. (a) Confocal fluorescence images of cells in μ MASTs tf Figure 2. Cellular responses in strained μMASTs. (a) Confocal fluorescence images of cells in μ MASTs after 3 days of straining to different levels (Green:F-actin (phalloidin); blue: nuclei (DAPI)). (b) Mean cell spreading area increased with culture time and with strain level, up to a threshold. In all cases, cell spreading increased nonlinearly with increasing strain, rising rapidly to an asymptotic saturation level at a critical strain in the range of 20–30%. In 10 kPa synthetic tissues, the rise in mean cell spreading area was nearly an order of magnitude, 5 times greater than for the more compliant synthetic tissues. (c) Polarization of NIH/3T3 cells in strained μ MASTs. For 6 kPa μ MASTs, cells oriented perpendicular to the applied stretch at lower strain levels and parallel to the applied stretch (90°) at higher strain levels. At the lowest and highest levels of stretch, no dominant polarization was observed. For 2 and 10 kPa μ MASTs, cell orientation with the direction of stretch was evident at intermediate levels of strain. (d) The number of cells per μ MAST increased with time, and up to a threshold (10% strain for 2 kPa μ MASTs, 20% strain for 6 kPa μ MASTs and 30% strain for 10 kPa μ MASTs, respectively), with different strain levels. (e,f) Quantification of live, apoptotic and late apoptotic (dead) cells in μ MASTs as a function of straining showed a substantial drop in cell viability in the range of 100–150% strain. Cells in μ MASTs were analyzed for apoptosis using Annexin V/FITC and PI staining and flow cytometry after 5 days of straining to prescribed levels. Live cells: Q3 (Annexin V(− )/PI(− )); early apoptotic cells: Q4 (Annexin V(+ )/PI(− )); late apoptotic cells: Q2 (Annexin V(+ )/PI(+ )). Control group: cells cultured in μ MASTs withou straining. Synthetic tissue modulus: 6 kPa. Error bars, s.d. (b–d: 10 ≤ n ≤ 15 μ MASTs for each strain level; e,f: 60 ≤ n ≤ 80 μ MASTs for each strain level, p < 0.01, ***p < 0.001). Scale bars: 50 μ m. Figure 2. Results O i i Theoretical modeling also suggests that the interaction of the contractile force generated by the cellular stress fibers and FAs with the external mechanical strain triggers cell repolariza- tions46. However, most of these studies still stagnate in the interactions between cells and 2D substrata, which can hardly match with in vivo situation because of its complexity. Our system provides a potential to move this from 2D to 3D and can be used to study the mechanism that links mechanical force to cellular responses, which is important during tissue development and disease. Further challenges still need to be addressed. For instance, cells in hydrogels may dynamically adhere to matrix during the stretching process. Cell may release their adhesions (e.g., FAs) from the matrix and reattach. There may be no memory of strain when this happens. Therefore, further studies should focus on the dynamic changing of cellular FAs when applied extreme strain on μ MASTs.fi y g g pp μ Another surprise was that cells adhered to the matrix at strain levels sufficient to cause apoptosis, rather than release from it. In 6 kPa synthetic tissues, cell viability decreased gradually from 90% viability at no strain to 70% viability at 100% strain (Fig. 2f). Early apoptosis was evident in the 100% and 120% strain groups, and viability plummeted to 10% at 130% strain, the latter with widespread late apoptosis and cell death. Cells were viable to μ MAST failure in the 2 kPa and 10 kPa specimens.l μ p Another key factor that may influence cell behaviors is the change of μ MAST’s microarchitectures when applied external strains. To study the deformation of pore structures due to strain alone in three kinds of μ MASTs (2 kPa, 6 kPa and 10 kPa), we characterized the pore size, aspect ratio of interconnected pores and porosity of hydrogel samples (Supplementary Figs S14). Our results showed that pore size decreased by increasing the stiff- ness of μ MASTs, which is caused by the increased monomer fraction (Supplementary Figs S14a). For 2 kPa and 6 kPa groups, the average pore size is 180 ± 20 μ m and 110 ± 15 μ m, respectively, which is larger than 10 kPa group (55 ± 10 μ m). Similarly, the porosity of μ MASTs decreased with increasing monomer fractions. Results O i i To check if the MMP activity affects this behavior, we added MMP inhibitor (BB94) in culture medium of 6 kPa μ MASTs with 60% strain (Supplementary Fig. S11). Both fluores- cent images and alignment quantification showed that the biphasic alignment disappeared and cell reoriented perpendicular to the strain direction. These results indicated that cell polarization in 6 kPa μ MASTs at 60% strain could be dependent on the concentration of ligand and MMP-susceptible sites. Further studies should focus on testing the distribution of adhesion ligands and MMP-susceptible sites under mechanical strain. Cells in 75–90% strained synthetic tissues showed increasing polarization parallel to the stretch direction, but coordinated polar- ization diminished in synthetic tissues strained above 100%.hf y The applied strain will affect cell polarization in all three kinds of μ MASTs (2 kPa, 6 kPa and 10 kPa). In syn- thetic tissues with a modulus of 10 kPa, cells orientated perpendicular to the stretch direction at strain levels up to 30%, and parallel to the stretch direction for strains above 40% (Fig. 2c and Supplementary Fig. S10). In synthetic tissues with a modulus of 2 kPa, however, orientation perpendicular to the stretch direction was never observed; cells aligned parallel to the stretch direction with peak parallel alignment at a strain of 60%. These observations are again consistent with variations in cellular orientation seen in granulation tissue, with cells least aligned in the most compliant areas. Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 4 www.nature.com/scientificreports/ Strain-activation was required for substantial proliferation of cells in all μ MASTs tested (Fig. 2d and Supplementary Fig. S12). Straining of 10–20% was required, with the threshold increasing with matrix modulus. A saturation value was reached in all cases except at the highest strain levels (> 100% in the 6 kPa synthetic tis- sues), in which proliferation ceased and gave way to apoptosis (Fig. 2e,f and Supplementary Figs S13). ) p g y p p ( g pp y g ) Cellular cytoskeleton is a dynamic, adaptive and functional entity that can connect the cell physically and biochemically to the external microenvironment and generate coordinate force enabling the cell to change shape and polarization44. A key bridge to cytoskeleton and ECM is focal adhesions (FAs). For instance, existing stud- ies demonstrated that myosin II and other crosslinkers can generate traction forces on FAs by which they are anchored to ECM surrounding the cell45. Results O i i All these results suggested that the cell activity in μ MASTs with larger modulus may be also inhibited due to the less growth space supported by the hydrogel. To clarify the relationship between the hydrogel architecture applied strains, we characterized the aspect ratio of pores in 6 kPa μ MASTs under different strain levels (Supplementary Figs S14b-f). The results showed that aspect ratio of pores increased with increasing applied strains and the pore significantly aligned parallel to the stretch direction, which may be a factor to affect cell repolarization in strained μ MASTs. The deformation of pore structure may also apply off-axis compressive stresses on encapsulated cells and narrow down the region for cell growth when applied uniaxial strain, which may induce the cell apoptosis. To clarify this, we have compared the area and aspect ratio of cell nucleus in 6 kPa synthetic tissues between 120% and 60% group (Supplementary Fig. S15). The results showed that the nuclear area significantly decreased when applied 120% strain and aspect ratio of cell nuclei is larger than 60% group, indicating that the hydrogel may collapse off-axis and compress the cell, including the nucleus and lead to apoptosis. Discussionh The mechanical environment is known to affect cell morphology, proliferation, polarization, and differentiation in both 2D and 3D culture33,47. The stiffness and strain of the mechanical environment are intertwined in 3D, with fibroblast cells in 3D both stiffening the ECM and proliferating/dying to reach the point at which their modulus match; in cell-seeded collagen ECM, this corresponds to the steric percolation threshold23,48. Because the stiffness and strain are definable independently with μ MASTs, we were able for the first time to probe their effects on cells independently in a 3D culture environment. In addition, μ MASTs provided a platform for applying simultaneous, non-contact, and tunable mechanical stimulation to parallel arrays of cells in 3D culture. This platform overcomes challenges faced by other 3D culture systems associated with sample handling, diffusion barriers, specimen varia- bility, and non-uniformity of mechanical loading. The system enabled the first-ever extreme strain testing of cells in a uniform 3D environment.hi The μ MAST system has limitations that bear mention. For instance, non-fibrous matrix that enabled strain uniformity is not fully representative of the cell environment in a real tissue. Another challenge is that ECM porosity and stiffness cannot be prescribed independently. This presents a challenge to interpreting surprising results about the cellular apoptosis, as we cannot be certain that cells in real tissues will “hold on” at strains associated with apoptosis like those in μ MASTs do. However, we believe that μ MASTs provide useful and well-calibrated information about cell mechanobiology that can be applied to understand future work in more complicated fibrous mechanical environments. We are also hopeful that the system will continue to be useful for dynamic characterization of mechanobiological responses of cells in 3D. Methods Following a 5× dilution with additional warm (40 °C) DPBS to stop the reaction, the mixture was dialyzed against distilled water using 12–14 kDa cutoff dialysis tubing (Spectrum Labs, Inc.) at 40 °C for 1 week. Water was changed every day to removed salts and methacrylic acid. After that, the GelMA solution was lyophilized for 1 week to generate a porous foam and stored at –80 °C for further use.i NIH/3T3 fibroblasts (Cell Bank of the Chinese Academy of Sciences) were cultured in Dulbecco’s modi- fied Eagle’s medium (DMEM; high glucose, Gibco BRL Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco BRL Life Technologies, Inc.) and 1% penicillin–streptomycin mixture (Gibco BRL Life Technologies, Inc.) at 37 °C, 95% humidity, and 5% CO2. Cells were passaged approximately 2 times per week with the media changed every 2 days. Fabrication of μMASTs. A two-step lithography approach was developed to fabricate μ MASTs (Supplementary Fig. S1). In the first step, one iron microsphere (350 μ m diameter, nickel-coated) (K&J Magnetics) was placed in each of the 15 through-thickness holes in the PMMA interlayer, and positioned and held in place using the magnetic field focusing array. The through-thickness holes were then filled with 12.5% (w/v) PEGDMA precur- sor solution (MW = 1000, Polysciences, Inc.), which was then polymerized by exposing to 365 nm UV light at a power of 2.9 mW cm–2 (model XLE-1000 A/F, Spectroline) for 25–30 s. p ( / , p ) In the second step, NIH/3T3 fibroblasts and GelMA precursor mixture were first mixed gently at the final concentration of 1.5 × 105 cells mL–1. 500 μ L mixture was then pipetted onto the crosslinked PEGDMA within each of the 15 holes, and the PEGDMA was soaked in GelMA solution for 10 min. A glass cover slide, modified with 3-(trimethoxysilyl) propyl methacrylate (TMSPMA; Sigma-Aldrich) to conjugate with GelMA, was placed atop the molds to secure the fabricated μ MASTs. Photopolymerization then proceeded using a custom-designed, commercially manufactured photomask (Shenzhen Qingyi Precision Mask Making Co., Ltd.). This photo- mask (Supplementary Fig. S16c) contained an array of 15 holes (0.8 mm diameter), and was placed atop the glass cover slide (Fig. S1 D) between the mold and the UV light source described above. The UV light source photo-crosslinked the GelMA layer for 15–25 s. Methods Preparation and fabrication of μMASTs. We developed a two-step lithography approach to fabricate μ MASTs composed of (i) a stiff, strong “magnetically-actuated” layer of PEGDMA and (ii) a tunable-stiffness “synthetic tissue” layer of cells encapsulated in GelMA. The magnetically actuated PEGDMA layer was created from a poly(methyl methacrylate) (PMMA) mold, and the GelMA synthetic tissue later was created by masked photolithography. Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 5 www.nature.com/scientificreports/ Preparation of PMMA mold and magnetic field focusers. A PMMA (Shuguang Plexiglas Co., Ltd.) mold was designed using CAD software (AutoCAD 2010, Autodesk Inc.) and fabricated using a CO2 laser engraving system (Universal VLS2.30, Universal Laser Systems, Inc.). The rectangular PMMA mold was composed of three parts, each 50 mm × 36 mm (Supplementary Fig.S16): (i) a 1 mm thick cover containing a 20 mm × 24 mm rectangular cut-out; (ii) a 1 mm thick interlayer containing 15 circular holes of 1 mm diameter, situated beneath the cover’s rectangular cut-out; and (iii) a 2 mm thick base plate.i g p A custom-designed array of magnetic field focusers was used for both positioning of iron microspheres during fabrication of the magnetically-actuated PEGDMA layers and for mechanical straining of μ MASTs. This array was constructed by assembling 15 magnetic field focusing elements on a PTFE (DuPontTM, Inc.) base (Supplementary Fig. S5a). Each magnetic field focusing element was composed of a cylindrical NdFeB rare earth magnet (2 mm in diameter and 2 mm in height) (K&J Magnetics) and an iron wire (1 mm in diameter and 8 mm in length). Preparation of GelMA and cells. GelMA, a biocompatible and photocrosslinkable hydrogel that is effective for 3D cell encapsulation, was synthesized as described previously47. Briefly, type A porcine skin gelatin powder (Sigma-Aldrich) was added into Dulbecco’s phosphate buffered saline (DPBS; Gibco BRL Life Technologies, Inc.) at a concentration of 10% (w/v) and a temperature of 65 °C, and stirred until fully dissolved. Methacrylate (Sigma-Aldrich) was added into this solution at a rate of 0.5 mL min–1 under stirring at 50 °C until the target volume was reached. The solution was then allowed to react for 2 hours. The fraction of lysine groups that reacted could be controlled by varying the amount of added methacrylate. Methods The exerted on the iron microsphere was then estimated using:  π ν = + ( ) F R U ma 6 2 magnetic ( ) 2  π ν = + F R U ma 6 magnetic where the first term represents viscous drag, in which v is the dynamic viscosity of the PEO solution, R is the radius of the iron microsphere, and  U is the speed of the iron microsphere; and the second term represents an inertial force, in which m is the mass of the iron microsphere and a is the acceleration. A relationship between force and separation (Supplementary Fig. S2b) was calibrated from these experiments; as expected, the relation- ship was nearly inverse cubic. The dynamic viscosity v was estimated similarly from downward motions of iron microspheres release into a beaker of PEO solution in the absence of magnetic fields. h f h l d h d l d d During stretching of μ MASTs, microspheres were encapsulated in hydrogels and strain was estimated opti- cally from images recorded using a high-resolution inverted fluorescence microscopy (IX-81, Olympus, Inc.) and analyzed using Image-Pro Plus (IPP; version 6.0, Media Cybernetics)46. Nominal strain, ε, was calculated in each layer as ε = λ − 1, λ is the ratio of the current to initial length of the layer. The average strain over the synthetic tissue layers was calculated by averaging the strain of 15 samples. The relationship between the Cauchy stress (force divided by current cross-sectional area) and this strain was found to be linear over a very broad range of strains for all GelMA concentrations tested. The elastic moduli of the synthetic tissues were derived from these relationships. Structural characterization of μMASTs. For sample preparation, the hydrogel (control and strained) was firstly frozen at − 80 °C for 24 h and then lyophilized by using a freeze-dryer (Heto PowerDry LL1500, Thermo Scientific) at room temperature for 12 h. The freeze-dried specimens were submerged into liquid nitrogen for about 5 minutes, and then fractured with a scalpel blade. The pore structure of hydrogels were sputter coated with platinum and then examined using a S-3000N, HITACHI scanning electron microscope (SEM). To test the porosity of hydrogels, hydrogel scaffolds were first thawed and hydrated for 24 hours. Methods Hydrated scaffolds were weighed on a scale, and a Kimwipe was lightly applied to the scaffold surface for 40 s to wick away loosely held water, and the mass was again recorded. The interconnected volume was calculated as the mass of water wicked away divided by the total hydrated mass. Cell viability and proliferation characterization. To measure cell viability and proliferation, cells encapsulated in synthetic tissues were stained with using a live/dead assay (Molecular Probes) following manufacturer’s instructions. Briefly, synthetic tissues were cut into several ~300 μ m thickness circular cross-sectional slices using razor blades and incubated in a solution of 2 μ g mL–1 calcein AM and 5 μ g mL–1 propidium iodide at 37 °C for 20–30 min. Fluorescence microscopy was performed to identify cells that were living (stained green by calcein AM) and dead (stained red by propidium iodide). Cell nucleus were stained for cell number counting using IPP. Briefly, each florescent image of hydrogel slice was imported in IPP software. The “Area” option in “count/ size-measure-select measurement” menu was then chosen. The cell number and was calculated through “count/ size-count” option (Supplementary Fig. S17). Cell spreading area characterization. To measure cell spreading area, F-actin stress fibers and the nuclei of cells were stained by fluorescein isothiocyanate (FITC) conjugated phalloidin (Acti-stain 488 phalloidin, Cytoskeleton, Inc.) and 4′ , 6-diamidino-2-Phenylindole (DAPI; InvitrogenTM, Life Technologies, Inc.), respectively. Briefly, for stress fiber staining, cells in synthetic tissue slices were fixed by 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min, and then incubated with 200 μ L of 100 nM FITC phalloidin solution in the dark at room temperature for 30–40 min. For nuclear stain- ing, cells were counterstained with 200 μ L of 100 nM DAPI in DPBS for 30 s. The stained synthetic tissue slices were placed in a drop of antifade mounting medium (Invitrogen) on a glass slide and stored in the dark at 4 °C until imaging. Confocal fluorescence images were captured using a Zeiss LSM 700 microscope (Carl Zeiss). Cell spreading area was analyzed from these images using IPP. Cell orientation analysis. The images acquired for cell spreading analysis were also analyzed to extract the ori- entation distribution and mean orientation of cells using the semi-automated “binarization-based extraction of alignment score” method described elsewhere49. Methods 2-hydroxy-2-methylpropiophenone (TCI, Shanghai Development Co., Ltd.) was used as photoinitiator at the concentration of 0.05% (w/v) for crosslinking of both PEGDMA and GelMA. Both PEDMA and GelMA precursor solution were sterilized by filtration (0.22 μ m pore size, Thermo Scientific Co., Ltd.) before using. Mechanical stretching of μMASTs. To design the magnetic field focusers and characterize the mechanical fields within μ MASTs, we performed a series of numerical simulations using commercially available software (COMSOL Multiphysics 4.0a, Comsol Inc.). First, we predicted the magnetic field gradients experienced by the iron microspheres within the magnetically-actuated layers as a function of the separations between the ends of magnetic field focusers and the centers of iron microspheres. The magnetic force vector (F ) applied to each iron microsphere was estimated according to: ∫µ = ∇ ( ) ( ) ( ) − F M B x n x dA 1 A 0 1 sat     ( ) 1 where A is the surface of the iron microsphere, n  is the outward normal of dA, μ0 is the magnetic permeability of a vacuum, Msat is the saturation moment of an individual iron microsphere, and  ∇ ( ) B x is the external magnetic field gradient tensor at position x.h i This force was subsequently applied to a synthetic tissue as a uniform pressure on the upper surface; in-plane displacements were constrained to be zero on the upper surface, and all displacements were constrained to be zero on the lower surface. These boundary conditions are appropriate given the difference in stiffness between the PEGDMA and GelMA layers. GelMA were treated as an incompressible, isotropic Neo-Hookean with elastic modulus derived from experimental work described below. Experimental characterization of μMASTs. Mechanical characterization of μMASTs. To experimen- tally characterize magnetic force applied to iron microspheres in magnetically-actuated layers, the Stokes drag Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 6 www.nature.com/scientificreports/ method was used. An iron microsphere was placed in a poly (ethylene oxide) solution of defined viscosity (PEO; Sigma-Aldrich), then subjected to a magnetic field. 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To suppress the enzymatic activity of the matrix metalloproteinases (MMPs) secreted by NIH/3T3 fibroblasts, culture media was supplemented with a broad-spectrum MMP inhibitor BB94, (Selleck Chemicals, S7155) at a concentration of 400 μ M, which has been previously shown to inhibit MMP expression and activation without significantly affecting cell function or viability50. The culture medium was exchanged per day. The samples were then stained for studying the effect of MMP inhibition on cell spreading and proliferation in μ MASTs. Cell apoptosis experiment. Cells in synthetic tissues were dissociated with 2.5% trypsin (Gibco BRL Life echnologies, Inc.), collected and suspended in DMEM. 5 μ L annexin-V-FITC (InvitrogenTM, Life Technologies, Inc.) Scientific Reports \| 6:19550 \| DOI: 10.1038/srep19550 7 www.nature.com/scientificreports/ was then added into the cell suspension and incubated for 15 min at room temperature, followed by addition of 10 μ L of propidium iodide (InvitrogenTM, Life Technologies, Inc.) and incubation for 5 min. Immediately after staining, a quantitative analysis was performed by using a flow cytometer (Becton, Dickinson and Company). Data were analyzed with FlowJo software (Tomy Digital Biology). was then added into the cell suspension and incubated for 15 min at room temperature, followed by addition of 10 μ L of propidium iodide (InvitrogenTM, Life Technologies, Inc.) and incubation for 5 min. Immediately after staining, a quantitative analysis was performed by using a flow cytometer (Becton, Dickinson and Company). Data were analyzed with FlowJo software (Tomy Digital Biology). References 1. Sherwood, L. & Pysiology, H. From Cells to Systems. (Human Physiology, 2010).h 1. 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Bio. 17, 397–430 (1988). h 11. Elson, E. L. Cellular Mechanics as an Indicator of Cytoskeletal Structure and Function. Annu. Rev. Biophys. Bio. 17, 397–430 (1 2. Eschenhagen, T. et al. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscl model system. Faseb. J. Acknowledgements g This work was supported by the National Natural Science Foundation of China (11372243, 11522219 and 11532009), the International Cooperation and Exchange Program of the National Natural Science Foundation of China (11120101002), the International Science and Technology Cooperation Program of China (2013DFG02930), the China Postdoctoral Science Foundation (2013M540742), and the Natural Science Basic Research Plan in Shaanxi Province of China (2014JQ1004). F.X. was partially supported by the China Young 1000-Talent Program. G.M.G. supported in part by the Changjiang (Yangtze River) Scholars Program of the Chinese Ministry of Education. G.M.G. and E.L.E. also acknowledge funding from the National Institutes of Health (R01HL109505). References Part C-Me. 17, 641–649 (2011). 50 Aubin H et al Directed 3D cell alignment and elongation in microengineered hydrogels Biomaterials 31 6941 6951 (2010) J , ( ) 49. Feng, X., Beyazoglu, T., Hefner, E., Gurkan, U. A. & Demirci, U. Automated and Adaptable Quantification of Cellular Alignment from Microscopic Images for Tissue Engineering Applications. Tissue Eng. Part C-Me. 17, 641–649 (2011). 49. Feng, X., Beyazoglu, T., Hefner, E., Gurkan, U. A. & Demirci, U. Automated and Adaptable Quantification of Cellular Align from Microscopic Images for Tissue Engineering Applications. Tissue Eng. Part C-Me. 17, 641–649 (2011). g, , y g , , , , k , , p Qi g from Microscopic Images for Tissue Engineering Applications. Tissue Eng. 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Additional Information upplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Li, Y. et al. An approach to quantifying 3D responses of cells to extreme strain. Sci. Rep. 6, 19550; doi: 10.1038/srep19550 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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https://openalex.org/W3169647735	https://www.researchsquare.com/article/rs-561153/latest.pdf	English	null	Evaluation of Anti-diabetic Effects of Eryngium Billardieri on Streptozotocin-induced Diabetes in Male Mice	Research Square (Research Square)	2,021	cc-by	6,508	Kamran Hosseini Research Keywords: Eryngium billardieri, Maceration, Mice, STZ, OGTT, Insulin, MDA Posted Date: June 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-561153/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Evaluation of Anti-diabetic Effects of Eryngium Billardieri on Streptozotocin-induced Diabetes in Male Mice Kamran Hosseini Shiraz University of Medical Sciences Elaheh Touri Tabriz University of Medical Sciences Faculty of Pharmacy Haleh Vaez Tabriz University of Medical Sciences Faculty of Pharmacy Alireza Garjani Tabriz University of Medical Sciences Faculty of Pharmacy Parina Asgharian Tabriz University of Medical Sciences Drug Applied Research Center Vahideh Tarhriz (  t.tarhriz@yahoo.com ) Tabriz University of Medical Sciences Research Keywords: Eryngium billardieri, Maceration, Mice, STZ, OGTT, Insulin, MDA Posted Date: June 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-561153/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Evaluation of Anti-diabetic Effects of Eryngium Billardieri on Streptozotocin-induced Diabetes in Male Mice Kamran Hosseini Shiraz University of Medical Sciences Elaheh Touri Tabriz University of Medical Sciences Faculty of Pharmacy Haleh Vaez Tabriz University of Medical Sciences Faculty of Pharmacy Alireza Garjani Tabriz University of Medical Sciences Faculty of Pharmacy Parina Asgharian Tabriz University of Medical Sciences Drug Applied Research Center Vahideh Tarhriz (  t.tarhriz@yahoo.com ) Tabriz University of Medical Sciences Research Keywords: Eryngium billardieri, Maceration, Mice, STZ, OGTT, Insulin, MDA Posted Date: June 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-561153/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/17 Page 1/17 Page 1/17 Abstract Background: One of the largest plant genera of the Umbelliferae family is Eryngium including 274 species, of which only 9 species are native to Iran and one of the most dominant species is E. billardieri. Numerous pharmacological effects of E. billardieri such as anti-inflammatory, antimicrobial, antioxidant have been reported in several studies. Objective: The present study aimed to investigate the anti-diabetic effects of E. billardieri on streptozotocin-induced diabetes in male mice. Methods: The extraction was performed by maceration with 70% ethanol solvent. Forty eight mice, weighing 32 g, were randomly divided into 8 groups (6 mice per group): healthy control, diabetic control (151 mg/kg STZ; IP), healthy extract control group (receiving the high dose of the extract orally), diabetic groups (receiving low dose and high dose of extract in the form of IP and Oral), the treatment group which received STZ (IP) and high dose of the extract group (Oral) for five days. To induce diabetes, a single dose of STZ (151 mg/kg) was injected intra-peritoneally. After diabetes, 100 mg/kg and 300 mg/kg of extract were injected into diabetic animals for 18 days as IP and Oral were given, and finally, serum samples were isolated and stored at -71 °C. Three days before surgery, OGTT test was performed. Results: IP injection of 300 mg/kg of hydroalcoholic extract of E. billardieri reduced the weight of diabetic rats and significantly reduced glucose tolerance. Furthermore, gavage at a dose of 300 mg/kg of extract caused significantly the increase in serum insulin while the decrease in blood sugar, glucose tolerance and malondialdehyde. Conclusion: E. billardieri extract, due to its components, probably increases serum insulin and decreases serum malondialdehyde by two mechanisms including protects pancreatic beta cells from further damage by streptozotocin and stimulation of insulin secretion from the remaining healthy pancreatic beta cells. In other words, part of the beneficial effects of the extract is associated with inhibition of oxidative stress and increased insulin secretion. Introduction Diabetes mellitus (DM) is a group of metabolic disorders that cause high blood sugar levels in a long time [1]. The cause of the disease depends on the amount of insulin production or insufficient production of insulin by pancreatic beta cells which are generally divided into three main types: type I diabetes, type II diabetes and gestational diabetes [2]. Common clinical manifestations of this disease include: weight loss, polyuria, polydipsia, polyphagia, headache, fatigue, slow wound healing, and if left untreated, can result in significant consequences such as neuropathy, retinopathy, and nephropathy [3, 4]. The global prevalence of adult diabetes was 6.4% in 2010, equivalent to 285 million people, and in 2012 was about 371 million people, estimated to reach 552 million by 2030 [5]. It has been estimated that the prevalence of diabetes in the Middle East will increase significantly and it is prognosticated that the annual growth rate of diabetes by 2030 in Iran will be second only to Pakistan [6]. Overall, the incidence of DM increased Page 2/17 Page 2/17 from 11.3 million in 1990 to 22.9 million in 2017 [7]. Important risk factors for the disease include obesity physical inactivity, family history, race, age, gender, high blood pressure and so on [8]. Exercise, weight control, proper diet, blood sugar lowering drugs and insulin injections are common treatments for this disease [9]. The study of natural compounds is very important in the treatment of various diseases, including diabetes. Due to the side effects of insulin and hypoglycemic drugs such as sulfonylureas, metformin, alpha-glucosidase inhibitors, troglitazone, etc., patients tend to use natural hypoglycemic products. One of the plant compounds with pharmacological properties is a plant of the Umbelliferae family called Eryngium. The distribution of this plant has been observed in the northern, northwestern, central and northeastern regions of Iran [10]. The anti-diabetic effects of some Eryngium species have been reported in previous studies [11, 12]. Considering the increasing prevalence of diabetes and the importance of medicinal plants in the treatment of diabetes and also taking into account that no study has been done to investigate the anti-diabetic effects of E. billardieri, this study aimed to evaluate the anti-diabetic effects of the extract of E. billardieri on diabetic mice. Materials And Methods In general, mice were randomly divided into 8 groups (n = 6): Healthy control group, diabetic control group Page 3/17 (receiving STZ), healthy extract control group (gavage dose of 300 mg/kg extract), diabetic group (receiving 100 mg/kg extract from the third day as IP), diabetic group (receiving 300 mg/kg extract from the third day as IP), diabetic group (gavage 100 mg/kg extract from the third day), diabetic group (gavage 300 mg/kg extract from the third day), STZ receiving group and dose 300 mg/kg of extract (Oral) for 5 days. (receiving STZ), healthy extract control group (gavage dose of 300 mg/kg extract), diabetic group (receiving 100 mg/kg extract from the third day as IP), diabetic group (receiving 300 mg/kg extract from the third day as IP), diabetic group (gavage 100 mg/kg extract from the third day), diabetic group (gavage 300 mg/kg extract from the third day), STZ receiving group and dose 300 mg/kg of extract (Oral) for 5 days. Streptozotocin preparation. Since STZ is a powder and cannot be injected directly into the peritoneum, the powder was dissolved in a 0.1 M sodium citrate dihydrate buffer. Due to the sensitivity of STZ to light, all steps of STZ preparation were performed in the dark [13, 14]. Induction of diabetes in male mice by streptozotocin. First, the weight and blood sugar of each mouse was measured and recorded. Blood was taken from the tail vein of a mice to measure blood sugar; in this way, first the mice tail was disinfected with alcohol and then the tail picking method was used for blood sampling. The dose of streptozotocin (150 mg/kg) for each mice was calculated based on mouse weight [13, 14]. The injection site was disinfected with 70% ethanol and then streptozotocin was injected intraperitoneally. The mice were monitored for three days, during which time they had access to water and food. After three days, the rats' weight and blood sugar were measured again. Mice with blood glucose above 200 mg/dl were selected as diabetic groups and this procedure was repeated every three days. Receiving the extract by diabetic mice as IP and Oral and their surgery. From the third day, diabetic mice (groups 4, 5, 6 and 7) received the extract for 18 days. After 18 days of receiving the extract, mice were anesthetized by intraperitoneal injection of ketamine (100–100 mg/kg) and xylazine (3–10 mg/kg) [15– 17]. Materials And Methods Chemicals. Streptozotocin (Merck Germany), Ethanol 70% (Caledon Canada), Serum Normal Saline and Distilled Water, Sodium Citrate Dihydrate Buffer (Merck Germany), Citric Acid Monohydrate (Merck Germany), STAR Glucometer for Blood Glucose (Made in Taiwan), Insulin Kit (manufactured by Shibayagi Company), and Malone Dealdehyde Kit (Jiancheng Institute of Bioengineering Company, Nanjing, China). Preparation of samples and plant powder. E. billardieri plant was collected in August 2016 from Urmia region, Iran. After identification, its herbarium specimen with number 1249 was kept in the herbarium of Tabriz University of Medical Sciences. At first, the aerial parts of the mentioned plant were dried naturally in the open air, and turned into a very fine powder. The powder obtained from this step was very light with a low density. Extraction of the Plant Material. Extraction was performed by maceration method; first, pour 200 g of plant powder into a wide-mouthed Erlenmeyer flask and add 70% ethanol ratio. Then, the Erlenmeyer was placed in a quiet place away from sunlight and shook the Erlenmeyer flask once a day to mix its contents. We did this until the color of the solvent did not change. The supernatant is passed through a filter and the solvent of the extract is dried via an evaporator at low pressure and temperature. The dried extract was placed in a sealed container and stored at 4°C for further procedure. Experimental Animals. This study was conducted in four stages including streptozotocin injection, injection and gavage of the extract, collection of blood and tissue samples, measurement of serum insulin and malondialdehyde. Forty eight male NMRI mice race purchased from Pasteur Institute of Iran with an average age of 12 weeks and a weight range of 32 g in standard clear polyethylene cages under standard conditions of 21°C, and humidity ± 12 hours light-dark, and in normal temperature of laboratory (10 ± 60%) were maintained. They had free access to sufficient water and food until the experiment. Results Weight changes in healthy and diabetic groups. The weight of the healthy control group increased during the experiment. The weight of the healthy control group of the extract did not change much compared to the healthy control group. The diabetic control group lost weight until the ninth day, which is not significant compared to the weight of the healthy control group. In the diabetic group receiving 100 mg/kg of extract (IP) compared to the diabetic control group, weight loss was observed which was significant on the last day of the experiment (day 21) (P < 0.05). The weight of the diabetic group receiving 300 mg/kg of extract (IP) had a decreasing trend during the experiment, which was not significant compared to the weight of the diabetic control group. No weight loss was observed in the diabetic groups receiving 100 mg/kg and 300 mg/kg of the extract (Oral) compared to the weight of the diabetic control group (Fig. 1). To better evaluate the results of weight changes during the experiment, a graph of weight changes compared to the first day is drawn. As illustrated in the chart (Fig. 2), streptozotocin caused significant weight loss in the diabetic control group compared to the healthy control group (P < 0.05 and P < 0.01). Weight changes in simultaneous administration of STZ and 300 mg/kg dose (Oral). The weight of the samples in the simultaneous administration of STZ and the dose of 300 mg/kg of the extract did not change much compared to the weight of the diabetic control group (Fig. 3). Change of blood sugar in healthy and diabetic groups. The blood sugar of the healthy control group did not show much change during the experiment and had a steady trend. Blood sugar in the healthy control group from extract did not change compared to the blood sugar in the healthy control group and had a steady trend as in the healthy control group. In the diabetic control group compared to the healthy control group, on the third day, an increase in blood sugar was observed and from the sixth day to the last day of the experiment (day 21), the increase in blood sugar was significant (P < 0.001). The diabetic groups receiving 100 mg/kg and 300 mg/kg extract (IP) did not have regular hypoglycemia compared to the diabetic control group. Materials And Methods After deep anesthesia, cervical dislocation was performed to ensure the cessation of vital signs in mice. Blood samples were centrifuged at 4500 rpm and 4 ° C for 30 minutes and then isolated serum samples were stored at -70 ° C. OGTT test. Three days before surgery, mice were given 2 g/kg glucose by gavage after 12 hours of fasting. At times, zero (before glucose consumption), 30, 60, 90, and 120 minutes after glucose consumption, blood samples were collected from the venous vein to measure blood glucose levels [18]. Measurement of serum insulin levels. Determination of insulin levels in serum samples was performed by ELISA method. For this purpose the company kit Shibayagi was used. 10 µl of serum samples were incubated in wells coated with biotin conjugated to insulin antimonoclonal for 2 hours at room temperature. After incubation, the wash buffer was added to the wells and incubated with streptavidin- conjugated HRP (Horseradish Peroxidase) for 30 minutes at room temperature. In the next step, the streptavidin-conjugated HRP residue reacted with the chromogenic reagent at room temperature for 30 minutes. The reaction was stopped by adding the solution. The absorbance was read with a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm [19]. Measuring the amount of malondialdehyde (MDA). The amount of malondialdehyde was measured using colorimetry method and thiobarbituric acid reagent (TBARS) according to the instructions of the Page 4/17 Page 4/17 manufacturer (Jiancheng Institute of Bioengineering Company, Nanjing, China) and its absorption was determined at 500 nm [20]. manufacturer (Jiancheng Institute of Bioengineering Company, Nanjing, China) and its absorption was determined at 500 nm [20]. Statistical analysis of data. Data were represented as Mean ± SEM. One-way ANOVA and post-hoc Tukey test were used as statistical methods. P < 0.05 is considered as a significant difference. hange of blood sugar in concomitant administration of STZ and 300 mg/kg Change of blood sugar in concomitant administration of STZ and 300 mg/kg extract (Oral). As shown in Fig. 6, the blood glucose of the samples in the simultaneous administration of STZ and the dose of 300 mg/kg of extract (Oral), compared to the blood sugar of the diabetic control group, on the fifth day, was significantly reduced. Change of blood sugar on the OGTT test. The healthy control group and the healthy control group of the extract had an increase in blood sugar at 30 minutes after glucose administration and in the following minutes, their blood sugar gradually decreased. In the diabetic control group compared to the healthy control group, after glucose administration, at all times, blood sugar was significantly high (P < 0.001). The diabetic groups receiving 100 mg/kg and 300 mg/kg of extract (IP), compared with the diabetic control group, after glucose administration, showed a decreasing trend in blood sugar, which decreased for the group receiving 100 mg/kg of the extract was significant at 120 min time. In the group receiving 300 mg/kg of the extract, it was significant in 120 − 60 minutes (P < 0.001). The diabetic group receiving 100 mg/kg extract (IP) had hyperglycemia at 30 minutes after glucose administration and no significant decrease in blood glucose was observed at subsequent times compared to the diabetic control group. The diabetic group receiving 300 mg/kg extract (Oral), compared with the diabetic control group, had a significant reduction in blood sugar at 90 and 120 minutes (P < 0.001) (Fig. 7). In order to better evaluate the results obtained from changes of blood sugar in the OGTT test, a graph of changes in blood sugar compared to zero minutes (before glucose administration) has been drawn (Fig. 8). As can be seen, the diabetic group receiving 300 mg/kg extract (IP) had a significant reduction in blood sugar compared to the diabetic control group (P < 0.05 and P < 0.001). Evaluation of insulin levels in the serum of mice. As shown in Fig. 9, the healthy control group of the extract did not change much in insulin levels compared to the healthy control group. In the diabetic control group, compared to the healthy control group, a significant decrease in insulin levels was observed (P < 0.001). Results In the diabetic group receiving 100 mg/kg of extract (Oral), compared with the diabetic control group, no reduction in blood sugar was observed. From the ninth day to the last day of the experiment (day 21), the blood sugar of the diabetic group receiving 300 mg/kg of the extract (Oral) had a significant decrease compared to the diabetic control group (P < 0.05 for the ninth day, P < 0.001 for days 12–21) (Fig. 4). To better evaluate the results obtained from changes in blood sugar during the test, a graph of changes in blood sugar compared to the first day is drawn (Fig. 5). Streptozotocin caused a significant increase in Page 5/17 blood sugar in the diabetic control group compared to the healthy control group. In addition, oral administration of 300 mg/kg of the extract significantly reduced the blood sugar of diabetic mice compared to the diabetic control group (P < 0.05, P < 0.001). Discussion One of the chronic metabolic disorders and the leading causes of death in the world is diabetes mellitus (DM). Among the most common symptoms of this disease is high blood sugar levels, which can be caused by a defect in insulin secretion (type 1 diabetes) or insulin resistance (type 2 diabetes) [21]. Common treatments include exercise, weight control, proper diet, blood sugar-lowering drugs, and insulin injections. The reason why patients are more inclined to use natural products (herbs) to lower blood sugar is the side effects of blood sugar lowering drugs such as insulin, sulfonylureas, metformin, alpha- glucosidase inhibitors, troglitazone, etc. [22]. Eryngium species are cultivated as ornamental, herbal and medicinal products. However, the phytochemical and medicinal properties of most 251 species of this genus are not yet known. 23 species of the genus Eryngium have been identified, of which at least 127 compounds have been isolated and identified, most of which are compounds such as terpenoid and triterpenoid saponins, flavonoids, coumarins, polystylenes and steroids. According to in vivo and in vitro studies on extracts of some species of the genus Eryngium, they were found to have anti-cancer, anti- inflammatory, anti-snake and anti-scorpion, anti-bacterial, anti-fungal, anti-malarial anti-oxidants and anti- hyperglycemic effects [23]. Considering the increasing prevalence of diabetes and the importance of herbs in the treatment of diabetes and also considering that no study has been done to investigate the anti-diabetic effects of E. billardieri, this study was investigated to evaluate the effect of E. billardieri extract on glucose, insulin and malondialdehyde in diabetic mice. For this purpose, diabetes was caused by streptozotocin. Considering the increasing prevalence of diabetes and the importance of herbs in the treatment of diabetes and also considering that no study has been done to investigate the anti-diabetic effects of E. billardieri, this study was investigated to evaluate the effect of E. billardieri extract on glucose, insulin and malondialdehyde in diabetic mice. For this purpose, diabetes was caused by streptozotocin. Streptozotocin enters the pancreatic beta cells through the glucose transporter (GLUT2) and alkylates the cell DNA. DNA damage induces activation of Poly ADP-ribosylation which reduces NAD + and cellular ATP; As a result, streptozotocin has toxic effect on the insulin-producing beta cells of the pancreas [24]. Based on the results of previous studies with STZ, this combination reduces the weight of mice [25]. hange of blood sugar in concomitant administration of STZ and 300 mg/kg The diabetic groups receiving 100 mg/kg and 300 mg/kg extract (IP) did not have a significant increase in insulin levels compared to the diabetic control group. No significant increase in insulin was observed in the diabetic group receiving 100 mg/kg extract (Oral) compared to the diabetic control group. Insulin levels in the diabetic group receiving 300 mg/kg of extract (Oral) had a significant increase compared to the diabetic control group (P < 0.001). Evaluation of MDA in mice. As shown in Fig. 10, the amount of MDA in the healthy control group of the extract did not change significantly compared to the healthy control group. In the diabetic control group, compared to the healthy control group, a significant increase in MDA was observed (P < 0.001). The diabetic groups receiving 100 mg/kg and 300 mg/kg extract (IP) did not have a significant reduction in MDA compared to the diabetic control group. In the diabetic groups receiving the dose of 100 mg/kg and Page 6/17 Page 6/17 the dose of 300 mg/kg of the extract (Oral), compared to the diabetic control group, a significant decrease in the amount of MDA was observed (P < 0.01 and P < 0.001, respectively). Discussion In this study, it was observed that IP injection of 300 mg/kg of E. billardieri hydroalcoholic extract caused a decrease in the weight of diabetic mice, while gavage at 100 mg/kg and 300 mg/kg, as well as IP injection at a dose 100 mg/kg of extract did not cause significant change in weight of diabetic mice. In a study by Jagabir et al., on the antidiabetic effects of E. campestre leaves has been shown that oral use of the aqueous extract of this plant reduced the blood glucose concentration of hyperglycemic rats [11]. In another similar study of E. foetidum, Rauter et al., found that the plant's flavonoid compounds had anti- diabetic effects [26]. Afshari et al., extracted the hydroalcoholic extract of E. caucasisum and found that the extract of this plant reduces blood glucose concentration in a dose-dependent manner (doses of 200 and 300 mg/kg) and has anti-diabetic properties [12]. The results of the present study also showed that the oral dose of 300 mg/kg of extract significantly reduced blood sugar in diabetic mice (P < 0.001) and increased insulin secretion (P < 0.001). To evaluate the effect of E. billardieri hydroalcoholic extract on glucose homeostasis in mice, OGTT test was Page 7/17 Page 7/17 performed as an indicator to evaluate the function of Langerhans islands. OGTT results showed that diabetic control group had high glucose tolerance, while IP injection of 300 mg/kg and gavage of 300 mg/kg hydroalcoholic extract remarkably improved glucose tolerance in diabetic mice (P < 0.001). To investigate the protective effect of E. billardieri extract on the development of diabetes by streptozotocin, concomitant administration of streptozotocin (IP) and 300 mg/kg extract (Oral) was performed in mice and developed on the third day of diabetes. However, on the fifth day, blood sugar dropped dramatically. It seems that the gavage at a dose of 300 mg/kg of the extract could not completely protect against damage to pancreatic beta cells by streptozotocin, so mice became diabetic on the third day. Significant reduction of blood sugar in diabetic mice on the fifth day can have two possible mechanisms including preventing further damage to pancreatic beta cells caused by streptozotocin and stimulating insulin secretion in the remaining healthy pancreatic beta cells. Conclusion Based on our findings, E. billardieri extract, increases serum insulin and decreases serum MDA. It is likely that E. billardieri, with two mechanisms protects the pancreatic beta cells from further damage by streptozotocin and stimulates insulin secretion by remaining healthy pancreatic beta cells which causes to increase serum insulin levels, lower blood sugar, and decrease serum MDA levels. Therefore, it seems that in oral administration of the extract, the substances in the extract are metabolized by chemical or enzymatic reactions in the gastrointestinal tract and become substances that have the ability to significantly inhibit oxidative stress and increase insulin secretion, which needs more studies. Moreover, according to the present study, it can be found that weight change in mice is often observed in diabetic groups and with increasing the dose of the extract, a decreasing trend was observed in the weight of mice. Discussion In addition, our experimental results showed that the use of 100 mg/kg and 300 mg/kg of extract orally significantly reduced MDA levels (P < 0.01 and P < 0.001, respectively) compared with diabetic control group, which in general shows the antioxidant effects of the extract which confirmed the anti-oxidant effects of E. billardieri in previous studies [23, 27] Ethics approval and consent to participate The experimental procedures were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals and were approved by the Animal Ethical Committee of the Tabriz University of Medical Sciences (Permit Number: IR.TBZMED.VCR.RFR.1398.039). In addition, there is no involvement of human in this study. Funding This work was supported by Tabriz University of Medical Sciences. Authors' contributions ET and KH performed the experiments; PA, HV, AG and VT analyzed of data; KH, VT, HV and PA prepared the manuscript; KH and VT wrote and edited the manuscript; PA designed the experiments; PA led and supervised the project. Acknowledgements We would like to thank Dr. Baradaran for his supportive help. We would like to thank Dr. Baradaran for his supportive help. Consent for publication All other authors declare no conflict of interest. Page 8/17 Page 8/17 Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article. The authors confirm that the data supporting the findings of this study are available within the article. Competing interests We declare that we have no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript. References 1. Piero M, Nzaro G, Njagi J. 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Antidiabetic effect of the scorpion Scorpio maurus palmatus body extract using alloxan-induced diabetic mice model. Journal of Taibah University for Science. 2019;13(1):504-13. 22. Luna B, Feinglos MN. Oral agents in the management of type 2 diabetes mellitus. American family physician. 2001;63(9):1747. 23. Wang P, Su Z, Yuan W, Deng G, Li S. Phytochemical constituents and pharmacological activities of Eryngium L.(Apiaceae). 2012. 24. Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiological research. 2001;50(6):537-46. 25. Babby A, Elanchezhiyan C, Suhasini S, Chandirasegaran G. 26. Rauter AP, Martins A, Borges C, Mota‐Filipe H, Pinto R, Sepodes B, et al. Antihyperglycaemic and protective effects of flavonoids on streptozotocin–induced diabetic rats. Phytotherapy Research. 2010;24(S2):S133-S8. References Antihyperglycemic effect of tannic acid in streptozotocin induced diabetic rats. Int J Curr Res. 2014;6(3):5396-8. Page 10/17 Page 10/17 26. Rauter AP, Martins A, Borges C, Mota‐Filipe H, Pinto R, Sepodes B, et al. Antihyperglycaemic and protective effects of flavonoids on streptozotocin–induced diabetic rats. Phytotherapy Research. 2010;24(S2):S133-S8. 27. Zarei A, Ashtiyani SC, Hamidizadeh S, Rezaei A. The study of the effects hydro-alcoholic extract of Eryngium billardieri on lipid profiles levels and liver and renal functions tests in hypercholesterolemic rats Global J Pharmacol 2015;9(1):21 7 26. Rauter AP, Martins A, Borges C, Mota‐Filipe H, Pinto R, Sepodes B, et al. Antihyperglycaemic and protective effects of flavonoids on streptozotocin–induced diabetic rats. Phytotherapy Research. 2010;24(S2):S133-S8. 27. Zarei A, Ashtiyani SC, Hamidizadeh S, Rezaei A. The study of the effects hydro-alcoholic extract of Eryngium billardieri on lipid profiles levels and liver and renal functions tests in hypercholesterolemic rats. Global J Pharmacol. 2015;9(1):21-7. 27. Zarei A, Ashtiyani SC, Hamidizadeh S, Rezaei A. The study of the effects hydro-alcoholic extract of Eryngium billardieri on lipid profiles levels and liver and renal functions tests in hypercholesterolemic rats. Global J Pharmacol. 2015;9(1):21-7. Figures Fi 1 Figures Figure 1 Weight changes in healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal) Figure 1 Weight changes in healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal) Page 11/17 Page 11/17 Page 11/17 Figure 2 Weight changes in healthy and diabetic groups compared to the first day. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and ** P < 0.01 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP Intraperitoneal). Figure 2 Weight changes in healthy and diabetic groups compared to the first day. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and ** P < 0.01 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Page 12/17 Figure 3 Figure 3 Page 12/17 Weight changes in the group receiving concomitant STZ and 300 mg/kg of extract (Oral). Data is reported as Mean ± SEM (STZ: Streptozotocin ، E.b: Eryngium billardieri) Figure 4 Changes in blood sugar of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and *** P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 4 Changes in blood sugar of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and * P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Page 13/17 Page 13/17 Figure 5 Changes in blood sugar of healthy and diabetic groups compared to the first day. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. P < 0.01 and * P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Changes in blood sugar of healthy and diabetic groups compared to the first day. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. P < 0.01 and * P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 6 Changes in blood glucose in the group receiving concomitant STZ and dose of 300 mg/kg of extract (Oral). Data are reported as Mean ± SEM. * P < 0.001 compared with the diabetic control group (STZ: Streptozotocin, E.b: Eryngium billardieri). Figure 6 Figure 6 Changes in blood glucose in the group receiving concomitant STZ and dose of 300 mg/kg of extract (Oral). Data are reported as Mean ± SEM. * P < 0.001 compared with the diabetic control group (STZ: Streptozotocin, E.b: Eryngium billardieri). Changes in blood glucose in the group receiving concomitant STZ and dose of 300 mg/kg of extract (Oral). Data are reported as Mean ± SEM. * P < 0.001 compared with the diabetic control group (STZ: Streptozotocin, E.b: Eryngium billardieri). Page 14/17 Page 14/17 Page 14/17 Figure 7 Changes of blood glucose of different groups in the OGTT test. Data are reported as Mean ± SEM. One- way ANOVA and post hoc tukey statistical methods were used. * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 7 Changes of blood glucose of different groups in the OGTT test. Data are reported as Mean ± SEM. One- way ANOVA and post hoc tukey statistical methods were used. * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Page 15/17 Figure 8 Figure 8 Page 15/17 Changes in blood glucose of different groups in the OGTT test compared to zero minutes (before glucose administration). Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and *** P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Changes in blood glucose of different groups in the OGTT test compared to zero minutes (before glucose administration). Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.05 and * P < 0.001 compared with the diabetic control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 9 Serum insulin levels of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 9 Figure 9 Serum insulin levels of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Serum insulin levels of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Page 16/17 Figure 10 Serum MDA levels of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. P < 0.01 and * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Figure 10 Figure 10 Serum MDA levels of healthy and diabetic groups during the experiment. Data are reported as Mean ± SEM. One-way ANOVA and post hoc tukey statistical methods were used. P < 0.01 and * P < 0.001 compared with the diabetic control group and ♦♦♦ P < 0.001 compared with the healthy control group (Dia: Diabetes, STZ: Streptozotocin, E.b: Eryngium billardieri, IP: Intraperitoneal). Page 17/17 Page 17/17
https://openalex.org/W3098479819	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09260136.pdf	English	null	Unmanned Surface Vehicle Collision Avoidance Trajectory Planning in an Uncertain Environment	IEEE access	2,020	cc-by	10,747	Received October 21, 2020, accepted November 11, 2020, date of publication November 16, 2020, date of current version November 30, 2020. Received October 21, 2020, accepted November 11, 2020, date of publication November 16, 2020, date of current version November 30, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3038187 Digital Object Identifier 10.1109/ACCESS.2020.3038187 INDEX TERMS Collision avoidance, COLREGs, trajectory planning, trajectory prediction, Gaussian process regression, Gaussian mixture model, velocity obstacle method. INDEX TERMS Collision avoidance, COLREGs, trajectory planning, trajectory prediction, Gaussian process regression, Gaussian mixture model, velocity obstacle method. under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 8, 20 Unmanned Surface Vehicle Collision Avoidance Trajectory Planning in an Uncertain Environment This work was supported in part by the 7th Generation Ultra Deep Water Drilling Platform (Ship) Innovation Project, and in part by the National Natural Science Foundation of China under Grant 51879049. ABSTRACT Unmanned surface vehicles (USVs) can encounter undetected moving obstacles while sailing along a planned global path. USVs need to plan collision avoidance trajectories for moving obstacles. In this paper, an algorithm based on the Gaussian mixture model (GMM) and Gaussian process regression (GPR) is proposed to predict the motion of moving obstacles and estimate the uncertainty of the prediction. A nonlinear ﬁnite-time velocity obstacle (NLFVO) method is developed for obstacle avoidance. The NLFVO method analyzes the velocity of the USV and the predicted uncertain velocity vectors of the moving obstacles and selects a collision-free velocity for the USV and minimizes the objective function. To enable the actual navigation of USVs, the International Regulations for Preventing Collisions at Sea (COLREGs) are considered in addition to the NLFVO method. The simulation results show that the prediction algorithm can effectively predict the trajectory of moving obstacles, and the NLFVO method can obtain a collision-free trajectory for a USV. A. BASIC VELOCITY OBSTACLE METHOD In this section, we brieﬂy review the basic VO method. A schematic of the basic VO method is shown in Figure 1. Assuming that at time t that the motion state of the USV is deﬁned as Susv(t) = (Pusv(t), vusv(t)) in the coordinate system {X, Y}, where Pusv = (xusv, yusv) is the position of the USV, and vusv = (vx usv, vy usv) is the velocity of the USV. The motion state of the moving obstacle (mo) is deﬁned as Smo(t) = (Pmo(t), vmo(t)). The motion state of the moving obstacle can be calculated according to the contents described in Section III. Kalman ﬁltering, the Markov model, and the support vec- tor machine (SVM) are popular prediction algorithms. How- ever, their prediction accuracy degrades signiﬁcantly in the case of large changes in motion state [11]. The development of theory and practice in recent years has made Gaussian pro- cess regression (GPR) an important algorithm for supervised learning applications [12]. The advantage of the GPR model is that it seamlessly integrates many machine learning tasks, including model training, hyperparameters and uncertainty estimation [13]. GPR has been applied in many ﬁelds, such as wear prediction [14], wind speed prediction [15], and trajectory prediction [16]. In [17], GPR has been adopted for representing vehicular motion patterns, because it is robust against noise in measured data. FIGURE 1. Geometric expression of the VO method. Because a moving obstacle in the sea is affected by many factors, its motion patterns are complex. Therefore, it is necessary to analyze the motion patterns of obstacles by clustering and select the motion pattern with the maximum probability in the future. According to motion coherence, the trajectory was clustered into a few groups by the Gaussian mixture model (GMM) in [18] and [19]. This paper proposes a trajectory prediction algorithm based on GMM-GPR and a USV collision avoidance algo- rithm based on a nonlinear ﬁnite-time velocity obsta- cle (NLFVO) method. The main contributions are as follows: FIGURE 1. Geometric expression of the VO method. D(p, r) is a disk with p as the center and r as the radius: D(p, r) = q\|∥q −p∥2 < r . (1) (1) (1) The trajectory prediction algorithm based on GMM-GPR is proposed to predict the trajectory of a moving obstacle where the velocity may experience sudden changes. I. INTRODUCTION VO methods were applied to avoid collisions with moving obstacles whose trajectories were nonlinear and predictable. Compared to traditional approaches, the algorithms in [3] could detect collision dangers with moving obstacles sailing nonlinearly and predictably and ﬁnd collision-free velocities in multi-ship scenarios. In [4], COLREGs were considered as an additional constraint in the VO method. Due to the COL- REGs, VO methods speciﬁed on which side of an obstacle the vehicle should pass during an avoidance maneuver. In [5], a reciprocal VO (RVO) method for n-body collision avoid- ance was presented, where multiple mobile vehicles needed to avoid collisions with each other while moving in a com- mon workspace. In [6], a ﬁnite-time VO (FVO) method was introduced by considering the collision avoidance duration. A mobile robot that completed an unknown environment crossing using the FVO method was reported in [7]. An unmanned surface vehicle (USV), as a kind of autonomous marine vehicle, is suitable for tasks that are dan- gerous or unsuitable for manned vehicles. A USV typically plans a global path connecting the starting point and the target point according to the existing information before performing the task. Due to limited information, a USV can encounter moving obstacles that were not previously known. There- fore, the global path of a USV should be adjusted locally to avoid collisions [1]. As a part of local trajectory planning, USV collision avoidance for moving obstacles is carried out online based on real-time information from shipborne sensors. Many studies on USV collision avoidance have been con- ducted. The velocity obstacle (VO) method is a collision avoidance method that was ﬁrst proposed for robots to avoid collisions with obstacles in [2], and the potential of colli- sions between robots and obstacles was analyzed by their velocities. In [3], linear VO, nonlinear VO, and probabilistic The dynamic window approach (DWA) calculates the velocity of a robot can reach within a given time window [8]. The reachable velocities constitute a set in the velocity space, known as a dynamic window. The DWA is widely used for robot collision avoidance in a dynamic environment. In [9], the DWA and VO methods were combined to calculate the The associate editor coordinating the review of this manuscript and approving it for publication was Heng Wang . 207844 207844 VOLUME 8, 2020 G. I. INTRODUCTION Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment reachable velocity and course in a given time window for a USV to avoid obstacles. GMM-GPR is provided. In Section IV, a trajectory plan- ning algorithm considering the uncertainty of the obstacle’s motion is presented. Simulations for testing the performance of the proposed trajectory prediction algorithm and colli- sion avoidance trajectory planning algorithm are presented in Section V. In Section VI, conclusions are provided. In practice, it is necessary to also consider environmental uncertainty, model uncertainty, and information uncertainty in collision avoidance. The velocity and course of a moving obstacle are uncertain. According to historical motion infor- mation, a USV can predict a moving obstacle’s trajectory in the future, and then collision avoidance operations can be performed based on the predicted trajectory. It is difﬁcult for shipborne sensors to explicitly show motion information such as the velocity and course of a moving obstacle. The motion information is implicit in the obstacle’s position at the next timeframe, so the motion information of the obstacle is estimated by a prediction algorithm [10]. A. BASIC VELOCITY OBSTACLE METHOD The formula for determining whether the USV will collide with a moving obstacle is: Geometrically, if (9) and (12) are true, that is, the USV will collide with the moving obstacle in the future, the angle vusv ∈ Pmo(tcol) −Pusv(t0) tcol −t0 ⊕D P, rsum tcol −t0 , (9) where vusv represents that if the USV sails at this velocity during the time interval [t0, tcol], it will collide with the moving obstacle at the time tcol [3]. g Hence, (5) is equivalently expressed as (θuo −α) ≤χuo ≤(θuo + α) . (16) (16) VOuo = ∞ [ tcol Pmo(tcol) −Pusv(t0) tcol −t0 ⊕D P, rsum tcol −t0 , (10 A. BASIC VELOCITY OBSTACLE METHOD The Gaussian distributions that represent the uncertainty of the future velocity vectors of the moving obstacle are obtained. In this paper, the USV is simpliﬁed as a point, and the moving obstacle is expanded into a circle with rsum = rusv + rmo as the radius and any point P as the center, where rusv and rmo are the radii of the affected area of the USV and the moving obstacle, respectively. Therefore, D(P, rsum) is represented as the area affected by the moving obstacle, that is, the prohibited zone of the USV. (2) According to the motion uncertainty of the moving obsta- cle, a collision avoidance trajectory planning algorithm based on the NLFVO method and the DWA is proposed for collision avoidance considering the changes in the velocities of obstacles, collision time, and kinematic con- straints. The relative velocity vector vuo between the USV and the moving obstacle is vuo = vusv −vmo. (2) (2) The remainder of this paper is organized as follows. In Section II, the collision avoidance trajectory plan- ning algorithm based on the NLFVO method is proposed. In Section III, the trajectory prediction algorithm based on The ray emitted from the point Pusv in the direction of the velocity vector vuo is λ(Pusv, vuo) = {Pusv + vuot\|t ≥0} . (3) λ(Pusv, vuo) = {Pusv + vuot\|t ≥0} . (3) 207845 207845 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment FIGURE 2. Schematic diagram of collision avoidance judgment. When ray λ(Pusv, vuo) intersects the expanded obstacle, that is, ray λ(Pusv, vuo) is within the angle formed by the two rays tangent to D(P, rsum) from Pusv, the USV will collide with the moving obstacle in the future. The relative collision cone of the USV and D(P, rsum) is deﬁned as RCCuo: RCCuo = n vuo\|λ (Pusv, vuo) \ D (P, rsum) ̸= ∅ o , (4) (4) where RCCuo is an area located between the left tangent λL and the right tangent λR of D(P, rsum) emitted from Pusv. In other words, RCCuo is the set of all the relative velocities vuo that cause the USV to collide with the moving obstacle. A. BASIC VELOCITY OBSTACLE METHOD The velocity obstacle for the USV induced by the moving obstacle, denoted as VOuo, is a set of the USV velocities that result in a collision with the moving obstacle in the future: where RCCuo is an area located between the left tangent λL and the right tangent λR of D(P, rsum) emitted from Pusv. In other words, RCCuo is the set of all the relative velocities vuo that cause the USV to collide with the moving obstacle. The velocity obstacle for the USV induced by the moving obstacle, denoted as VOuo, is a set of the USV velocities that result in a collision with the moving obstacle in the future: FIGURE 2. Schematic diagram of collision avoidance judgment. VOuo = RCCuo ⊕vmo, (5) (5) where Pmo(t0), Pusv(t0), t0, and tcol are known. Therefore, item Pmo(t0)−Pusv(t0) tcol−t0 can be obtained as a constant. where ⊕is the Minkowski sum operation. tcol t0 As shown in Figure 2, the angle between the velocity vecto col 0 As shown in Figure 2, the angle between the velocity vector vuo and the X-axis is deﬁned as χuo, the angle between the vector (Pmo −Pusv) and the X-axis is θuo, and the angle between the vector (Pmo −Pusv) and the tangent λL is α. The angles χuo, θuo, and α are respectively calculated as follows: If the USV detects a moving obstacle at t0 and it will collide with it at time tcol, Pusv(tcol) is in area Pmo(tcol)⊕D(P, rsum), that is Pusv(tcol) ∈Pmo(tcol) ⊕D(P, rsum). (6) (6) If vusv is not changed within [t0, tcol], the position of the USV at tcol is χuo = arctan vy uo vxuo = arctan vy usv −vy mo vxusv −vxmo . (13) θuo = arctan yusv −ymo xusv −xmo . (14) α = arcsin rsum ∥Puo∥2 , (15) (13) Pusv(tcol) = Pusv(t0) + vusv(tcol −t0). (7) (7) (14) Then, (6) is rewritten as (15) Pusv(t0) + vusv(tcol −t0) ∈Pmo(tcol) ⊕D(P, rsum). (8) where Puo = Pusv −Pmo. The following formula is obtained by transforming (8): Geometrically, if (9) and (12) are true, that is, the USV will collide with the moving obstacle in the future, the angle χuo of the velocity vector vuo is within the angle between the left tangent λL and the right tangent λR of D(P, rsum) emitted from Pusv. B. NONLINEAR VELOCITY OBSTACLE METHOD According to the above two parts, a series of prohibited zones for the USV are obtained in the velocity space. The envelope of the prohibited zones is the set of NLVOuo, whose shape is similar to a curved cone in the velocity space, as shown in Figure 3. ∞ [ tcol Pmo(t0) −Pusv(t0) + PN i=1 (vmo(i)1t) tcol −t0 ! represents a curve in velocity space. The second part ∞ S tcol D P, rsum tcol−t0 represents the shape of the area affected by the moving obstacle, whose radius is adjusted by (tcol −t0)−1, and its center P lies on the curve represented by the ﬁrst part [3]. According to the above two parts, a series of prohibited zones for the USV are obtained in the velocity space. The envelope of the prohibited zones is the set of NLVOuo, whose shape is similar to a curved cone in the velocity space, as shown in Figure 3. ∞ [ tcol Pmo(t0) −Pusv(t0) + PN i=1 (vmo(i)1t) tcol −t0 ! where τ is the minimum allowable collision time; if the USV sails at vusv, the collision will occur at least after the time interval (τ −t0). Before time τ, the USV can sail at the current velocity vusv so that the USV has a larger range of optional collision avoidance velocity vectors. As shown in Figure 4, FVOuo is a new polygon formed by VOuo being truncated by D(PF, rF), where the position of PF is (Pusv−Pmo)/τ, and the radius is rF = r/τ. Geometrically, if vusv is in the area of FVOuo, it will cause the USV and the moving obstacle to collide at least after the time interval (τ −t0); otherwise, it will not. In this way, FVOuo has a larger velocity space than VOuo to obtain the velocity that can avoid collisions, which is signiﬁcant for the situation of multi-obstacle collision avoidance. col by the moving obstacle, whose radius is adjusted by (tcol −t0)−1, and its center P lies on the curve represented by the ﬁrst part [3]. According to the above two parts, a series of prohibited zones for the USV are obtained in the velocity space. The envelope of the prohibited zones is the set of NLVOuo, whose shape is similar to a curved cone in the velocity space, as shown in Figure 3. FIGURE 3. Geometric expression of NLVO in the velocity space. B. NONLINEAR VELOCITY OBSTACLE METHOD FIGURE 4. Geometric expression of FVO. FIGURE 3. Geometric expression of NLVO in the velocity space. FIGURE 3. Geometric expression of NLVO in the velocity space. B. NONLINEAR VELOCITY OBSTACLE METHOD (10) In the basic VO algorithm, the obstacle is regarded as moving uniformly in a straight line. In the nonlinear VO (NLVO) method, it is assumed that the motion of the moving obstacle is nonlinear, that is, its velocity vector vmo is time-varying, and vmo(t) within t ∈[t0, tcol] can be detected by the USV. Thus, the position of the moving obstacle at tcol is obtained by where tcol ∈(t0, ∞). If the USV continues to sail at the velocity vector in VOuo, it will deﬁnitely collide with the moving obstacle at a time within (t0, ∞). In other words, the USV can avoid collision with the moving obstacle by adjusting the current velocity vector vusv out of VOuo within a given time interval. Pmo(tcol) = Pmo(t0) + N X i=1 (vmo(i)1t), (17) If vmo is not changed within [t0, tcol], the position of the moving obstacle at tcol is (17) Pmo(tcol) = Pmo(t0) + vmo(tcol −t0). (11) (11) where N = tcol−t0 1t and 1t is the length of the detection cycle of the shipborne sensors. Then, (9) can be rewritten as vuo ∈ Pmo(t0) −Pusv(t0) tcol −t0 ⊕D P, rsum tcol −t0 , (12) By substituting (17) into (9), the velocity vector vusv that may cause a collision between the USV and the moving 207846 VOLUME 8, 2020 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment 1) FINITE-TIME VELOCITY OBSTACLE METHOD The ﬁnite-time velocity obstacle (FVOuo) formed by the USV and the moving obstacle at time t0 is deﬁned as: FVOuo = {vusv\|∃0 < τ ≤t, Pusv(t0) + vusv(t −t0) ∈Pmo(t) ⊕D(P, rsum)}, (20) obstacle with nonlinear motion is obtained as (18), as shown at the bottom of the page [8], [20]. Therefore, the NLVO for the USV is obtained as (19), as shown at the bottom of the page [3]. Two parts on the right side of (19) perform the Minkowski sum operation. The ﬁrst part (20) ∞ [ tcol Pmo(t0) −Pusv(t0) + PN i=1 (vmo(i)1t) tcol −t0 ! represents a curve in velocity space. The second part ∞ S tcol D P, rsum tcol−t0 represents the shape of the area affected by the moving obstacle, whose radius is adjusted by (tcol −t0)−1, and its center P lies on the curve represented by the ﬁrst part [3]. C. NONLINEAR FINITE-TIME VELOCITY OBSTACLE METHOD C. NONLINEAR FINITE-TIME VELOCITY OBSTACLE METHOD If there are numerous moving obstacles near the USV, the USV will have very few velocity candidates [9]. To solve the above problem, the collision time between the USV and the moving obstacle is considered in the VO method. Assuming the USV sails at the velocity vector in VOuo, it will collide with the moving obstacle after a long time. Thus, a ﬁnite-time velocity obstacle (FVO) method is proposed in [6], [7]. FIGURE 4. Geometric expression of FVO. However, the previous references on the FVO method are only for the linear motion of obstacles, and the nonlinear motion of obstacles and the minimum allowable collision time are rarely considered. Therefore, in the following con- tent, FVO will be applied to the nonlinear motion of obstacles. vusv ∈   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) tcol −t0 ⊕D P, rsum tcol −t0  . (18) NLVOuo = ∞ [ tcol   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) tcol −t0 ⊕D P, rsum tcol −t0  . (19) vusv ∈   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) tcol −t0 ⊕D P, rsum tcol −t0  . (18) (18) NLVOuo = ∞ [ tcol   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) tcol −t0 ⊕D P, rsum tcol −t0  . (19) NLVOuo = ∞ [ tcol   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) tcol −t0 ⊕D P, rsum tcol −t0  . (19) (19) VOLUME 8, 2020 207847 VOLUME 8, 2020 207847 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment 2) NONLINEAR FINITE-TIME VELOCITY OBSTACLE METHOD Combining (19) and (20), the nonlinear ﬁnite-time velocity obstacle (NLFVO) caused by the USV and the moving obsta- cle with the nonlinear motion is obtained as (21), as shown at the bottom of the next page where 0 < t ≤τ, and N = τ−t0 1t . the USV measure the m + 1 historical trajectory points {Pmo(tc −m), · · · , Pmo(tc)} of the obstacle before the current time tc. The purpose of trajectory prediction is to obtain the n future trajectory points {Pmo(tc + 1), · · · , Pmo(tc + n)} of the obstacle after the current time tc. III. TRAJECTORY PREDICTION ALGORITHM m(T) = E[vx mo(T)], (23) k(T, T′) = E vx mo(T) −m(T) vx mo(T′)) −m(T′) = σ 2 f exp −(T −T′)(T −T′)T 2σ 2 l ! , (24) (23) Trajectory prediction is important for collision detection and trajectory planning. In this section, we ﬁrst introduce the motion pattern model based on GPR. In some complex envi- ronments, the velocity vectors of the moving obstacle will change randomly, and the GMM is used to cluster the motion patterns and calculate the probability distribution of different motion patterns. Finally, the method to predict the trajectory of the moving obstacle is proposed. (24) where σf and σl are hyperparameters. At the current time tc, t∗ j is deﬁned as the future time, i.e., t∗ j = tc + j, where j = 1, · · · , n. vx mo ∗ = vx mo(t∗ 1), · · · , vx mo(t∗ n) represents the component of the n future velocity vectors of the moving obstacle in the X-axis direction. T∗= {t∗ 1, · · · , t∗ n} represents n future time after the current time tc and is used as the input of the prediction data set. C. NONLINEAR FINITE-TIME VELOCITY OBSTACLE METHOD Deﬁnition 1: The motion pattern is deﬁned as a map- ping from the location (x, y) to a distribution over trajectory derivatives (vx, vy) = 1x 1t , 1y 1t indicating the agent’s future motion [17]. 1t The geometric expression of the NLFVO in velocity space is shown in Figure 5. FIGURE 5. Geometric expression of NLFVO in the velocity space. The m historical velocities of the obstacle are obtained by the m + 1 historical trajectory points of the obstacle. The two elements of the moving obstacle’s velocity vector vmo = (vx mo, vy mo)T are regarded as two independent ran- dom variables. At the current time tc, ti is deﬁned as the historical time, i.e., ti = tc −m −1 + i, where i = 1, · · · , m. T = {t1, · · · , tm} represents m historical time and is used as the input of the observation data set. vx mo = vx mo(t1), · · · , vx mo(tm) is the component of the m historical velocity vectors of the moving obstacle in the X-axis direc- tion, and vy mo = vy mo(t1), · · · , vy mo(tm) is the component of the m historical velocity vectors of the moving obstacle in the Y-axis direction. Since the expression of vy mo is similar to that of vx mo, vx mo is discussed only in the following content. vx mo is regarded as the output of the observation data set. Therefore, an observation data set Dx = ti, vx mo(ti) m i=1 is obtained. According to the known input data, a random variable set vx mo(T) is formed. A GPR model is determined by its mean function m(T) and covariance function k(T, T′) [12]: FIGURE 5. Geometric expression of NLFVO in the velocity space. In (21), the minimum collision time τ is signiﬁcant for the determination of NLFVOuo. Meanwhile, the minimum collision time τ is determined by the current position and velocity of the USV and the moving obstacle, respectively. However, the motion uncertainty of the moving obstacles sailing in the sea leads to an inaccurate minimum collision time. Therefore, to obtain the minimum collision time in an uncertain environment, the trajectory prediction algorithm is used to predict the future trajectories of the moving obstacle, and then the range of the minimum collision time is calcu- lated. C. NONLINEAR FINITE-TIME VELOCITY OBSTACLE METHOD vx mo(T) ∼GP m(T), k(T, T′) (22) (22) where III. TRAJECTORY PREDICTION ALGORITHM B. SELECTION OF MOTION PATTERNS BASED ON GMM Since vehicles are affected by many factors, their motion patterns are complex. In a complex motion pattern scenario, a trajectory may belong to multiple motion patterns, which is difﬁcult to describe by a Gaussian process. If a trajectory needs to be accurately described, a mixture model is required. Assuming that a moving obstacle has K possible motion patterns, which are denoted as {b1, b2, · · · , bK}, the proba- bilities that the moving obstacle adopts each motion pattern to move are deﬁned as {p(b1), p(b2), · · · , p(bK)}. The GMM of vx mo(ti) in all motion patterns is expressed as follows: (37) A. MODELING OF MOTION PATTERNS USING GPR Assume that there is no communication between the USV and the moving obstacle. Some parameters of the moving obstacle, such as position, velocity, and course, are difﬁcult to measure in real time. Thus, the trajectory prediction algo- rithm is used to obtain the future positions of the obstacle, and then the velocities of the obstacle are calculated. Due to the kinematic and kinetic constraints of the moving obstacles in the sea, their motion state will not change signiﬁcantly. Therefore, we can assume that the motion state of the moving obstacles will not change signiﬁcantly in a short time. The output vx mo of the observation data set and the out- put vx mo ∗of the prediction data set follow a joint Gaussian distribution: vx mo vx mo ∗ ∼N 0, K(T, T) K(T∗, T)T K(T∗, T) K(T∗, T∗) , (25) (25) where the covariances matrices K(T, T), K(T∗, T), and K(T∗, T∗) are considered to be zero mean [12]. The position of the moving obstacle at time t is set as Pmo(t) = (xmo(t), ymo(t)). The shipborne sensors on VOLUME 8, 2020 207848 VOLUME 8, 2020 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment K(T, T) is an m×m kernel matrix, called the Gram matrix, whose expression is shown as: where wk is the weight, which satisﬁes 0 ≤wk ≤1, and PK k=1 wk = 1. GP vx mo(ti)\|µk, σk is the Gaussian distribu- tion of vx mo(ti) in motion pattern bk. µk and σk represent the mean and covariance of the kth motion pattern, respectively. Since the expression of the GMM of vy mo(ti) is similar to that of vx mo(ti), vx mo(ti) is discussed only in the following section. K(T, T) =   k(T1, T1) · · · k(T1, Tm) ... ... ... k(Tm, T1) · · · k(Tm, Tm)  . (26) (26) o o For the entire observation data set vx mo = {vx mo(t1), · · · , vx mo(tm)}, the likelihood function of its GMM is The expressions of K(T∗, T) and K(T∗, T∗) are shown as follows: P(vx mo) = m Y i=1 K X k=1 wkGP vx mo(ti)\|µk, σk . (34) K(T∗, T) = k(T∗, T1), · · · , k(T∗, Tm) , (27) K(T∗, T∗) = k(T∗, T∗). ˆvx mo = K(T∗, T)K(T, T)−1vx mo (32) p bk\|vx mo(ti) = wkp bk\|vx mo(ti) KP k=1 wkp bk\|vxmo(ti) . (36) (36) At time t + 1, the new m velocity vectors of the moving obstacle are remeasured. In other words, the ﬁrst of the predicted values is taken as the observation data for the next prediction cycle. This process is repeated, the velocities of the moving obstacle are obtained online, and the prediction errors are continuously corrected. M-step: Recalculate the parameters of the Gaussian model according to the updated probability. Nk = N X i=1 p bk\|vx mo(ti) , wk = Nk N , µk = 1 NK N X i=1 p bk\|vx mo(ti) vx mo(ti), σk = v u u t 1 NK N X i=1 p bk\|vxmo(ti) vxmo(ti) −µk 2. (37) Nk = N X i=1 p bk\|vx mo(ti) , wk = Nk N , µk = 1 NK N X i=1 p bk\|vx mo(ti) vx mo(ti), σk = v u u t 1 NK N X i=1 p bk\|vxmo(ti) vxmo(ti) −µk 2. (37) A. MODELING OF MOTION PATTERNS USING GPR (28) (34) (28) The log-likelihood function is The log-likelihood function is After calculating the output vx mo of the observation data set, the conditional distribution of the output vx mo ∗of the prediction data set is obtained as [15]: ln P(vx mo) = m X i=1 ln K X k=1 wkGP vx mo(ti)\|µk, σk ! . (35) vx mo ∗\|T, vx mo, T∗∼N(mvxmo∗, σ 2 vxmo∗) (29) (29) The expectation-maximization (EM) algorithm is used to train the model. First, the model parameters are initialized. The expectation-maximization (EM) algorithm is used to train the model. First, the model parameters are initialized. Then, the E-step and M-step are iterated to update the param- eters until the likelihood function value no longer changes signiﬁcantly or the maximum number of iterations is reached [19]. where where mvxmo∗= K(T∗, T)K(T, T)−1vx mo (30) σ 2 vxmo∗= K(T∗, T∗) −K(T∗, T)K(T, T)−1K(T∗, T)T (31) (30) Then, the E-step and M-step are iterated to update the param- eters until the likelihood function value no longer changes signiﬁcantly or the maximum number of iterations is reached [19]. The mean value of the conditional distribution in (29) is used as the estimated value of ˆvx mo: E-step: Calculate the probability that the ith sample vx mo(ti) belongs to the motion pattern bk as ˆvx mo = K(T∗, T)K(T, T)−1vx mo (32) By analogy, the predicted position at time (t + n) is By analogy, the predicted position at time (t + n) is C. METHOD FOR OBTAINING USV COLLISION AVOIDANCE VELOCITY Pmo(t + n) = xmo(t + n −1) + ˆvx mo(t + n −1)1t ymo(t + n −1) + ˆvy mo(t + n −1)1t T . (40) According to the above contents, the velocity vusv needs to be adjusted out of the area of VOuo to avoid a collision between the USV and the moving obstacle. However, due to the kinematics of the USV, the adjustment range of the USV’s velocity is limited. However, a USV at sea is subject to the rules of COLREGs. Therefore, we should choose the USV’s velocity vusv to avoid collision and minimize the cost function value under the USV’s kinematic constraints and the rules of COLREGs. (40) A. NLFVO METHOD WITH CONSIDERING UNCERTAINTY As described in Section III, the two elements of vmo, vx mo and vy mo, follow Gaussian distributions. Hence, according to the 3σ rule of thumb, the velocity ranges of the moving obstacle can be obtained: vx mo ∈[µx −3σx, µx + 3σx], (41) vy mo ∈[µy −3σy, µy + 3σy]. (42) (41) (42) According to the historical trajectory points of the moving obstacle from t1 to tk, the uncertainty of the moving obstacle’s velocity at tk+1 is described by Gaussian distributions. There- fore, the uncertainties of the moving obstacle’s velocity rep- resented by (41) and (42) are added to item PN i=1 (vmo(i)1t) in (21). Considering the uncertainty, the curve represented by the item ∞ S tcol Pmo(t0)−Pusv(t0)+PN i=1 (vmo(i)1t) tcol−t0 in (21) will be replaced by a set of curves. The area indicated by NLFVOuo in the velocity space will be expanded accordingly. According to the historical trajectory points of the moving obstacle from t1 to tk, the uncertainty of the moving obstacle’s velocity at tk+1 is described by Gaussian distributions. There- fore, the uncertainties of the moving obstacle’s velocity rep- resented by (41) and (42) are added to item PN i=1 (vmo(i)1t) in (21). Considering the uncertainty, the curve represented by (1) Overtaking rule: when the relative course angle 1χ between the USV and the moving obstacle is within [112.5◦, 247.5◦), the USV and the moving obstacle are in the situation of overtaking. The USV will sail along the port side of the moving obstacle, as shown in Figure 7 (a). ( ) g y, p y the item ∞ S tcol Pmo(t0)−Pusv(t0)+PN i=1 (vmo(i)1t) tcol−t0 in (21) will be item ∞ S tcol Pmo(t0)−Pusv(t0)+PN i=1 (vmo(i)1t) tcol−t0 in (21) will be tcol replaced by a set of curves. The area indicated by NLFVOuo in the velocity space will be expanded accordingly. (2) Head-on rule: when the relative course angle 1χ between the USV and the moving obstacle is within [345◦, 360◦) S[0◦, 15◦), the USV and the moving obsta- cle are in the situation of head-on. The USV will sail along the starboard side of the moving obstacle, as shown in Figure 7 (b). 1) COLLISION AVOIDANCE RULES BASED ON COLREGs According to the relative course angle between the USV and the moving obstacle, the angle ranges of the four encounters of overtaking, head-on, left crossing, and right crossing are determined by [21] and [22], as shown in Figure 6. Because the USV is unmanned, it cannot assume the responsibility of being avoided by other vehicles. Therefore, in the above four encounter situations, the USV should actively avoid the moving obstacle, and its collision avoidance trajectories are shown in Figure 7 (a), (b), (c), and (d), respectively. Based on the characteristics of USVs and the rules of COLREGs in [21] and [23], the rules for USV collision avoidance based on COLREGs are designed as follows: IV. TRAJECTORY PLANNING ALGORITHM CONSIDERING UNCERTAINTY The trajectory prediction algorithm based on GMM-GPR has been introduced in Section III, and the Gaussian distributions representing the uncertainty of the moving obstacle’s veloc- ities are obtained. In the following, we will combine the tra- jectory planning algorithm based on the NLFVO method by considering the motion uncertainty of the obstacle to obtain a USV collision-free trajectory in an uncertain environment. C. EXPRESSIONS OF THE PREDICTED VELOCITY AND POSITION OF THE OBSTACLE p vx mo(ti) = K X k=1 p(bk)p vx mo(ti)\|bk = K X k=1 wkGP vx mo(ti)\|µk, σk , (33) After determining the motion pattern bi of the moving obsta- cle and the corresponding GPR model, the predicted velocity of the moving obstacle at time t is obtained by (32), that is, (33) ˆvmo(t) = ˆvx mo(t), ˆvy mo(t) . (38) (38) NLFVOuo = ∞ [ t   Pmo(t0) −Pusv(t0) + NP i=1 (vmo(i)1t) t −t0 ⊕D P, rsum tcol −t0  , (21) (21) VOLUME 8, 2020 207849 VOLUME 8, 2020 207849 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment Comparing the Euclidean distance between the USV and the moving obstacle, if the following exists If the current position Pmo(t) = (xmo(t), ymo(t)) of the moving obstacle is known, the predicted position of the mov- ing obstacle at time (t + 1) is calculated as: ∥Pusv(tk) −Pmo(tk)∥2 ≤rsum, (44) (44) Pmo(t + 1) = xmo(t) + ˆvx mo(t)1t, ymo(t) + ˆvy mo(t)1t . (39 then it is considered that the two will collide at time tk, where t1 ≤tk ≤tn. The time tk is the minimum collision time, that is τ = tk. (39) By analogy, the predicted position at time (t + n) is 3) COST FUNCTION FIGURE 6. The angle range of possible encounter situations between a USV and a moving obstacle. FIGURE 6. The angle range of possible encounter situations between a USV and a moving obstacle. There are several USV velocities outside VOuo in the velocity space. To select an optimized velocity vector vusv to avoid a collision, a cost function is established: min J = w1J1 + w2J2, (47) (47) where J1 is the course variation function, and J2 is the USV’s energy consumption function. w1 and w2 are the weights of J1 and J2, respectively. The course variation function is expressed as: J1 = θgoal(t) −χ(t) /π, (48) (48) where J1 ∈[0, 1]. As shown in Figure 8, χ(t) is the angle between the direction of vusv(t) and the X-axis direction, and θgoal(t) is the direction of the vector Pgoal −Pusv(t) . Thus, θgoal(t) −χ(t) is the angle between the course of the USV at time t and the line connecting the USV’s current position Pusv(t) with the goal position Pgoal. where J1 ∈[0, 1]. As shown in Figure 8, χ(t) is the angle between the direction of vusv(t) and the X-axis direction, and θgoal(t) is the direction of the vector Pgoal −Pusv(t) . Thus, θgoal(t) −χ(t) is the angle between the course of the USV at time t and the line connecting the USV’s current position Pusv(t) with the goal position Pgoal. FIGURE 7. The collision trajectories in the situations of overtaking, head-on, left crossing, and right crossing are shown in (a), (b), (c), and (d), respectively. g The USV’s energy consumption function is shown as fol- lows [25], [26]: course to its starboard side and sail along the tail of the moving obstacle, as shown in Figure 7 (d). J2 = 0.5ρ\|U\|3CDA1t, (49) (49) 2) DYNAMIC WINDOW APPROACH where ρ is the density of the water, CD is the drag coefﬁ- cient, A is the reference area, and U is the relative velocity of the USV. Due to the constraints of USV kinematics, the adjustment range of the velocity vector vusv is limited in a given dura- tion. The dynamic window approach (DWA) is introduced to handle the kinematic constraints [24]. The reachable velocity of the USV in a given time window 1t, which is called the velocity window, is B. MINIMUM ALLOWABLE COLLISION TIME If the USV does not change its original velocity, its trajectory point sequence within the time interval [t1, tn] is (3) Left crossing rule: when the relative course angle 1χ between the USV and the moving obstacle is within [15◦, 112.5◦), the USV and the moving obstacle are in the situation of left crossing. The USV will adjust the course to its port side and sail along the tail of the moving obstacle, as shown in Figure 7 (c). {Pusv(t1), · · · , Pusv(tn)} = {Pusv(t1), · · · , Pusv(t1) + (n −1)vusv(t1)1t} (43) (43) According to Section III, the predicted trajectory point sequence of the moving obstacle within the time interval [t1, tn] is {Pmo(t1), · · · , Pmo(tn)}. (4) Right crossing rule: when the relative course angle 1χ between the USV and the moving obstacle is within [247.5◦, 345◦), the USV and the moving obstacle are in the situation of right crossing. The USV will adjust the In the time interval [t1, tn], the Euclidean distance sequence between the USV and the moving obstacle is {∥Pusv(t1) −Pmo(t1)∥2, · · · , ∥Pusv(tn) −Pmo(tn)∥2}. 207850 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment FIGURE 6. The angle range of possible encounter situations between a USV and a moving obstacle. FIGURE 7. The collision trajectories in the situations of overtaking, head-on, left crossing, and right crossing are shown in (a), (b), (c), and (d) respectively. FIGURE 8. Schematic of the course variation in cost function J1. FIGURE 8. Schematic of the course variation in cost function J1. FIGURE 6. The angle range of possible encounter situations between a USV and a moving obstacle. FIGURE 7. The collision trajectories in the situations of overtaking, head-on, left crossing, and right crossing are shown in (a), (b), (c), and (d), respectively. VOLUME 8, 2020 V. SIMULATION STUDIES By using the GMM clustering algorithm, the moving obsta- cle’s velocity data in the observation dataset are partitioned into multiple clusters. We choose different cluster numbers to perform cluster analysis on historical velocity data in this paper. The analysis results, such as RMSEx and RMSEy between the predicted positions and the actual positions of the obstacle, and calculation time are listed in Table 1. 4) OBTAINING USV COLLISION AVOIDANCE VELOCITY The velocity window vd and course window χd of the USV are obtained. However, the elements in vd and χd are contin- uous. Due to the large amount of calculation data, it takes a long time to obtain a velocity vector that satisﬁes (45) and (46), which is not conducive in practical application. Thus, the velocity window vd and course window χd of the USV are discretized in this paper, that is, vd is dispersed into m velocities, and χd is dispersed into n courses. In this way, a velocity vector vij usv is composed whose magnitude is vi(1 ≤i ≤m) and direction is χj(1 ≤j ≤n). The m × n velocities constitute a set of reachable velocities (RV) that can be reached by the USV within a duration 1t, as shown in Figure 9 (a). vd = {v\|v ∈[vc −˙v1t, vc + ˙v1t]} . (45) (45) The reachable course of the USV in a given time window 1t, which is called the course window, is χd = {χ\|χ ∈[χc −˙χ1t, χc + ˙χ1t]} . (46) (46) In (45), vc is the current velocity of the USV, and ˙v ≥0 is the velocity acceleration of the USV. In (46), χc is the current course of the USV, and ˙χ ≥0 is the course angular velocity of the USV. According to (45) and (46), the velocity window vd and the course window χd of the USV within a given time window 1t are determined. According to the encounter situation between the USV and the moving obstacle, the course selection of the USV is subject to the corresponding rules of COLREGs. In this paper, 207851 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment FIGURE 9. Schematic of RV , VREG, and RAV . Assuming that the latitude and longitude coordinates of the target vehicle in the WGS84 coordinate system are (λ, ϕ), the position in the Cartesian coordinate system is (x, y), and the origin of the coordinate system is (λ0, ϕ0). The Cartesian coordinate system of the ground plane takes the east as the X-axis and the north as the Y-axis. 4) OBTAINING USV COLLISION AVOIDANCE VELOCITY The conversion based on the Gauss–Kruger model is as follows:    x = a cos ϕ p 1 −e2 sin ϕ 1λ d y = S(ϕ) −S(ϕ0) (51) (51) where where where S(ϕ) = a(1 −e2)(A d ϕ −B 2 sin 2ϕ + C 4 sin 4ϕ −D 6 sin 6ϕ) (52) 1λ = λ −λ0 (53) (52) (53) (52) (53) 1λ = λ −λ0 (53) In (52) and (53), d = 180 π is the conversion index, a is the major axis of the earth, e2 is the ﬂatness of the earth, and A, B, C, and D are the indexes. In (52) and (53), d = 180 π is the conversion index, a is the major axis of the earth, e2 is the ﬂatness of the earth, and A, B, C, and D are the indexes. Two different moving vehicles are selected as the predic- tion objects of Case 1 – 2, which contain 2017 and 4441 tra- jectory points, respectively. Assuming that the moving obsta- cles do not change their motion states within the duration of 1t = 1 s, the time sequences of the velocity components of the moving obstacle are obtained according to Deﬁnition 1. FIGURE 9. Schematic of RV , VREG, and RAV . the set of velocity vectors prohibited by the USV based on COLREGs is deﬁned as VREG, which is the red part shown in Figure 9 (b). 2) SIMULATIONS OF TRAJECTORY PREDICTION The USV needs to adjust its velocity vusv out of the area of VOuo to avoid collision with the moving obstacle. Therefore, a set of reachable avoidance velocities (RAV) for the USV that can both avoid collision and meet the rules of COLREGs is obtained, which is shown in Figure 9 (c): The accuracy of the proposed algorithm in terms of prediction error is evaluated by the root mean square error (RMSE) metric deﬁned as RMSEx = v u u t 1 N N X i=1 (ˆxmo(i) −xmo(i))2) (54) RMSEy = v u u t 1 N N X i=1 (ˆymo(i) −ymo(i))2 (55) (54) RAV = {vusv\|vusv ∈RV, vusv /∈VO, vusv /∈VREG} . (50) The elements in RAV are calculated by the cost function in (47), and the element vusv(i) that minimizes the cost func- tion value is selected. When vusv(i) is selected as the control target for navigation, the USV can avoid the moving obstacle while minimizing the cost function and subjecting it to the kinematic constraints and the rules of COLREGs. (55) where N is the number of trajectories in the prediction dataset, ˆxmo(i) and ˆymo(i) are the components of the predicted position. xmo(i) and ymo(i) are the components of the actual position. RMSE is calculated in every prediction step. 1) DATA PREPARATION To verify the effectiveness of the proposed algorithm based on GMM-GPR, a real vehicle trajectory dataset for predicting the moving obstacle’s trajectory is performed. The position information of the target vehicle sent by the GPS sensor is based on the latitude and longitude data of the WGS84 geo- graphic coordinate system. To compare the predicted results accurately, it is essential to convert the latitude and longitude in the WGS84 coordinate system to the Cartesian coordinate system of the ground plane. As shown in Table 1, when the number of clusters is large, the calculation time is long. In contrast, when the number of clusters is small, the prediction error is large. Hence, to bal- ance the prediction effect and the calculation time, the cluster number is 15 for trajectory prediction in Case 1, and the cluster number is 20 for trajectory prediction in Case 2. 207852 207852 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment TABLE 1. Results of different cluster numbers. FIGURE 10. Schematic of the velocity prediction of the moving obstacle in Case 1. FIGURE 11. Comparison between the actual and predicted trajectories of the moving obstacle in Case 1. TABLE 1. Results of different cluster numbers. FIGURE 11. Comparison between the actual and predicted trajectories of the moving obstacle in Case 1. FIGURE 12. Schematic of the velocity prediction of the moving obstacle in Case 2. FIGURE 11. Comparison between the actual and predicted trajectories o the moving obstacle in Case 1. FIGURE 10. Schematic of the velocity prediction of the moving obstacle in Case 1. FIGURE 11. Comparison between the actual and predicted trajectories of the moving obstacle in Case 1. FIGURE 12. Schematic of the velocity prediction of the moving obstacle in Case 2. The prediction results of the moving obstacles’ veloc- ity based on GMM-GPR in Case 1 and Case 2 are shown in Figure 10 and Figure 12, respectively, where the blue solid line represents the mean values of the velocity, and the blue shaded portion represents the conﬁdence interval of the velocity based on the 3σ rule of thumb. Based on the predicted velocity information, the trajectories of the moving obstacles are calculated. The comparisons between the actual and predicted trajectories of the moving obstacles in Case 1 and Case 2 are shown in Figure 11 and Figure 13, respectively. B. SIMULATIONS OF USV COLLISION AVOIDANCE TRAJECTORY PLANNING To verify the effectiveness and feasibility of the USV col- lision avoidance trajectory planning algorithm in uncertain environments, two sets of simulations are conducted: an encounter situation between the USV and a single moving obstacle and an encounter situation between the USV and multiple moving obstacles. FIGURE 12. Schematic of the velocity prediction of the moving obstacle in Case 2. rules, we set up four sets of encounter cases. In Case 3 – 6, the USV will encounter a single moving obstacle. The ini- tial motion information of the USV and the obstacles in Case 3 – 6 are listed in Table 2. 1) SIMULATIONS OF ENCOUNTER SITUATIONS BETWEEN THE USV AND A SINGLE MOVING OBSTACLE After calculation, the collision time tc between the USV and the moving obstacle in Case 3 – 6 are 31 s. At colli- sion time tc, the radius of the circle D(P, rsum) of Obstacle 3 – 6 are 25 m with considering the position noise in the kinematic model of the USV and the motion uncertainty of In an uncertain environment, the starting point of the USV is (0, 0), and the target point is (500, 500). Meanwhile, to verify the effectiveness of the proposed algorithm on COLREGs 207853 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment TABLE 2. Initial motion information of the USV and obstacle. FIGURE 13. Comparison between the actual and predicted trajectories of the moving obstacle in Case 2. FIGURE 14. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 3 TABLE 2. Initial motion information of the USV and obstacle. FIGURE 13. Comparison between the actual and predicted trajectories of the moving obstacle in Case 2. the obstacle. For the USV, the velocity window vd is dispersed into 8 velocities, and the course window χd is dispersed into 16 courses The weights (w1 w2) are (100 1) in (47) The FIGURE 14. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 3. TABLE 2. Initial motion information of the USV and obstacle. TABLE 2. Initial motion information of the USV and obstacle. FIGURE 14. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 3. FIGURE 15. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 4. FIGURE 13. Comparison between the actual and predicted trajectories of he moving obstacle in Case 2. FIGURE 14. The trajectories for USV collision avoidance considering the ti t i t f th b t l i C 3 FIGURE 13. Comparison between the actual and predicted trajectories of the moving obstacle in Case 2. FIGURE 13. Comparison between the actual and predicted trajectories of the moving obstacle in Case 2. FIGURE 14. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 3. FIGURE 15. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 4. the obstacle. 1) SIMULATIONS OF ENCOUNTER SITUATIONS BETWEEN THE USV AND A SINGLE MOVING OBSTACLE The trajectory for the USV to avoid collision with five moving obstacles based on NLFVO. can obtain a collision-free trajectory that meets the rules of COLREG h d FIGURE 16. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 5. FIGURE 18. The trajectory for the USV to avoid collision with five moving obstacles based on NLVO. FIGURE 18. The trajectory for the USV to avoid collision with five moving FIGURE 18. The trajectory for the USV to avoid collision with five moving obstacles based on NLVO. FIGURE 16. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 5. FIGURE 19. The trajectory for the USV to avoid collision with five moving obstacles based on NLFVO. FIGURE 17. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 6. FIGURE 19. The trajectory for the USV to avoid collision with five moving obstacles based on NLFVO. FIGURE 17. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 6. can obtain a collision-free trajectory that meets the rules of COLREGs on head-on. USV and the moving obstacle is right crossing. According to the rules in COLREGs, the USV should adjust the course to its starboard side and sail along the tail of the moving obstacle. As shown in Figure 15, the USV can obtain a collision-free trajectory that meets the rules of COLREGs on right crossing. The encounter situation of Case 5 between the USV and the moving obstacle is left crossing. According to the rules in COLREGs, the USV should adjust the course to its port side and sail along the tail of the moving obstacle. As shown in Figure 16, the USV can obtain a collision-free trajectory that meets the rules of COLREGs on left crossing. The encounter situation of Case 6 between the USV and the moving obstacle is head-on. According to the rules in COLREGs, the USV should sail along the starboard side of the moving obstacle. As shown in Figure 17, the USV 1) SIMULATIONS OF ENCOUNTER SITUATIONS BETWEEN THE USV AND A SINGLE MOVING OBSTACLE For the USV, the velocity window vd is dispersed into 8 velocities, and the course window χd is dispersed into 16 courses. The weights (w1, w2) are (100, 1) in (47). The parameters in (49) are set as CD = 1 and A = 300. The observation dataset in trajectory prediction should select the data at equal time intervals to ensure the regular- ity of the prediction. Hence, the ﬁrst 70% of the historical velocity information of the moving obstacle is used as the observation dataset, and the remaining 30% is used as the prediction dataset. Figure 14–17 show the planned trajectories for USV col- lision avoidance considering the motion uncertainty of the obstacle in Case 3 – 6, respectively. In Figure 14–17, the loca- tion of the USV is represented by a blue pentagon every 4 seconds, and the trajectory of the obstacle is represented by a red line. The prohibited zone D(P, rsum) is represented as a yellow disk at tc, and the global path connecting the initial location of the USV and the location of the goal is represented by a dotted green line. FIGURE 15. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 4. As shown in Table 2, the encounter situation of Case 3 between the USV and the moving obstacle is over- taking. According to the rules in COLREGs described in Section IV, the USV should pass along the port side of the moving obstacle. As shown in Figure 14, the USV can obtain a collision-free trajectory that meets the rules of COLREGs on overtaking. The encounter situation of Case 4 between the moving obstacle. As shown in Figure 14, the USV can obtain a collision-free trajectory that meets the rules of COLREGs on overtaking. The encounter situation of Case 4 between the 207854 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment FIGURE 16. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 5. FIGURE 17. The trajectories for USV collision avoidance considering the motion uncertainty of the obstacle in Case 6. USV and the moving obstacle is right crossing. According FIGURE 18. The trajectory for the USV to avoid collision with five moving obstacles based on NLVO. FIGURE 19. VI. CONCLUSION To enable USVs to avoid collisions with moving obstacles in uncertain environments, a trajectory prediction algorithm based on GMM-GPR and a collision avoidance trajectory planning algorithm based on the NLFVO method are pro- posed in this paper. [8] D. Fox, W. Burgard, and S. Thrun, ‘‘The dynamic window approach to collision avoidance,’’ IEEE Robot. Autom. Mag., vol. 4, no. 1, pp. 23–33, Mar. 1997. [9] Y. Y. Zhang, ‘‘Dynamic obstacle avoidance for USV based on velocity obstacle and dynamic window method,’’ J. Shanghai Univ., Natural Sci. Ed., vol. 23, no. 1, pp. 1–16, 2017. First, to solve the uncertainty of the obstacle’s motion, the historical trajectories of moving obstacles are used to predict their trajectory. The motion patterns of the moving obstacle are modeled by GPR, and the GMM is used to select the future motion pattern with the maximum probability. Then, the predicted trajectory of the obstacle is calculated. The Gaussian distributions of the obstacle’s velocity repre- senting the uncertainty of the obstacle’s future motion are obtained. [10] G. Aoude, ‘‘Mobile agent trajectory prediction using Bayesian nonpara- metric reachability trees,’’ Infotech @ Aerosp., vol. 6, no. 1512, pp. 29–31, Mar. 2011. [11] S. Lefèvre, D. Vasquez, and C. Laugier, ‘‘A survey on motion prediction and risk assessment for intelligent vehicles,’’ Robomech J., vol. 1, no. 1, pp. 1–14, Dec. 2014. [12] C. K. Williams and C. E. Rasmussen, Gaussian Processes for Machine Learning. vol. 2, no. 3. Cambridge, MA, USA: MIT Press, 2006. [13] A. Y. Sun, D. Wang, and X. Xu, ‘‘Monthly streamﬂow forecasting using Gaussian process regression,’’ J. Hydrol., vol. 511, pp. 72–81, Apr. 2014. [14] D. Kong, Y. Chen, and N. Li, ‘‘Gaussian process regression for tool wear prediction,’’ Mech. Syst. Signal Process., vol. 104, pp. 556–574, May 2018. Second, the NLFVO method is proposed for USVs to effectively avoid obstacles with uncertain motion. Due to its own kinematic constraints, the velocity window and course window of the USV are determined by the DWA. COLREGs are also considered in the NLFVO method to meet the actual navigation requirements of USVs. [15] J. Yu, K. Chen, J. Mori, and M. M. Rashid, ‘‘A Gaussian mixture copula model based localized Gaussian process regression approach for long-term wind speed prediction,’’ Energy, vol. 61, pp. 673–686, Nov. 2013. [16] S. A. Goli, B. H. Far, and A. O. REFERENCES [1] H. Kim, S.-H. Kim, M. Jeon, J. Kim, S. Song, and K.-J. Paik, ‘‘A study on path optimization method of an unmanned surface vehicle under environ- mental loads using genetic algorithm,’’ Ocean Eng., vol. 142, pp. 616–624, Sep. 2017. g Figures 18 and 19 show the planned trajectories for the USV to avoid collision with ﬁve moving obstacles based on the NLVO and NLFVO methods, respectively. In Figure 18, the USV will collide with Obstacle 2 and Obstacle 3. In Figure 19, the USV will not collide with any obstacle because collision avoidance trajectory planning based on the NLFVO method will have more candidate velocities when encoun- tering multiple obstacles. With more candidate velocities, the USV sailing along the planned trajectory obtained by the NLFVO method can avoid collision with Obstacle 1 – 5 in an uncertain environment while constrained by its own kinemat- ics and COLREGs. [2] P. Fiorini and Z. Shiller, ‘‘Motion planning in dynamic environments using velocity obstacles,’’ Int. J. Robot. Res., vol. 17, no. 7, pp. 760–772, Jul. 1998. [3] Y. Huang, P. H. A. J. M. van Gelder, and Y. Wen, ‘‘Velocity obstacle algo- rithms for collision prevention at sea,’’ Ocean Eng., vol. 151, pp. 308–321, Mar. 2018. [4] Y. Kuwata, M. T. Wolf, D. Zarzhitsky, and T. L. Huntsberger, ‘‘Safe mar- itime autonomous navigation with COLREGS, using velocity obstacles,’’ IEEE J. Ocean. Eng., vol. 39, no. 1, pp. 110–119, Jan. 2014. [5] J. Van Den Berg, ‘‘Reciprocal n-body collision avoidance,’’ in Robotics Research. Berlin, Germany: Springer, 2011. 3-19. [6] M. Kim and J.-H. Oh, ‘‘Study on optimal velocity selection using velocity obstacle (OVVO) in dynamic and crowded environment,’’ Auto. Robots, vol. 40, no. 8, pp. 1459–1470, Dec. 2016. [7] S. Samavati, M. Zarei, and M. T. Masouleh, ‘‘An optimal motion planning and obstacle avoidance algorithm based on the ﬁnite time velocity obstacle approach,’’ in Proc. Artif. Intell. Signal Process. Conf. (AISP), Oct. 2017, pp. 200–255. 2) SIMULATIONS OF ENCOUNTER SITUATIONS BETWEEN THE USV AND MULTIPLE MOVING OBSTACLES The USV may encounter multiple moving obstacles that will collide with it. Therefore, a situation where the USV encounters multiple obstacles is designed in the simulation, and the proposed trajectory planning algorithm is used to enable the USV to avoid collisions with the obstacles. In the simulation, the USV will encounter moving obstacles whose initial motion information is listed in Table 3. The initial position of the USV is (0, 0), the position of the target point is (800, 800), the initial velocity of the USV is 12 m/s, and the initial course of the USV is 45◦. 207855 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment TABLE 3. Initial motion information for multiple moving obstacles. TABLE 3. Initial motion information for multiple moving obstacles. TABLE 3. Initial motion information for multiple moving obstacles. velocity vector. The position measurement noise of the USV needs to be processed by the corresponding ﬁltering algo- rithm in the subsequent study. After calculating, the USV will collide with Obstacle 1 – 5 at 32 s, 40 s, 42 s, 56 s, and 60 s, respectively. Hence, the USV needs to avoid collisions with the ﬁve moving obstacles simultaneously based on the NLVO and NLFVO methods, respectively. The motion uncertainties of the mov- ing obstacles are also considered in the simulations. VI. CONCLUSION Fapojuwo, ‘‘Vehicle trajectory prediction with Gaussian process regression in connected vehicle environment⋆,’’ in Proc. IEEE Intell. Vehicles Symp. (IV), Jun. 2018, pp. 550–555. [17] J. Joseph, F. Doshi-Velez, A. S. Huang, and N. Roy, ‘‘A Bayesian non- parametric approach to modeling motion patterns,’’ Auton. Robot., vol. 31, no. 4, p. 383, Nov. 2011. The simulation results show that the proposed algorithms can obtain the predicted trajectories of the moving obstacles and the USV collision avoidance trajectory in an uncertain environment. [18] Y. Chen, C. Liu, B. Shi, and M. Liu, ‘‘CoMoGCN: Coherent motion aware trajectory prediction with graph representation,’’ 2020, arXiv:2005.00754. [Online]. Available: http://arxiv.org/abs/2005.00754 In the future, an optimization algorithm that is both fast and accurate should be designed to obtain an optimized USV [19] D. A. Reynolds, ‘‘Gaussian mixture models,’’ Encyclopedia Biometrics, vol. 741, pp. 1–5, Jul. 2009. 207856 VOLUME 8, 2020 G. Xia et al.: USV Collision Avoidance Trajectory Planning in an Uncertain Environment [20] F. Large, S. Sckhavat, Z. Shiller, and C. Laugier, ‘‘Using non-linear veloc- ity obstacles to plan motions in a dynamic environment,’’ in Proc. 7th Int. Conf. Control, Autom., Robot. Vis., ICARCV, 2002, pp. 734–739. ZHIWEI HAN received the bachelor’s degree in electrical engineering and automation from North- east Agricultural University (NEAU), in 2015. He is currently pursuing the Ph.D. degree in con- trol science and engineering with Harbin Engi- neering University. His research interests include intelligent optimization algorithm, path planning and collision avoidance of unmanned surface vehi- cle, and thrust allocation of DP vessels. [21] COLREGs, Convention on the International Regulations for Preventing Collisions at Sea. International Maritime Organization, London, U.K., 1972. [22] G. P. Smeaton and F. P. Coenen, ‘‘Developing an intelligent marine naviga- tion system,’’ Comput. Control Eng. J., vol. 1, no. 2, pp. 95–103, Mar. 1990. [23] Y. Wang, X. Yu, X. Liang, and B. Li, ‘‘A COLREGs-based obstacle avoidance approach for unmanned surface vehicles,’’ Ocean Eng., vol. 169, pp. 110–124, Dec. 2018. [24] O. Brock and O. Khatib, ‘‘High-speed navigation using the global dynamic window approach,’’ in Proc. IEEE Int. Conf. Robot. Autom., May 1999, pp. 341–346. BO ZHAO BO ZHAO received the bachelor’s degree in automation and the master’s degree in naviga- tion, guidance and control from the Beijing Uni- versity of Aeronautics and Astronautics (BUAA), in 2006 and 2009, respectively, and the Ph.D. degree in marine cybernetics from the Norwegian University of Science and Technology, in 2015. From 2013 to 2018, he was with Global Maritime, as a Senior Marine System Advisor, and devel- oped hardware-in-the-loop testing for dynamic positioning systems. He is currently working as an Associate Professor with Harbin Engineering University. His research interests include applying advanced control and artiﬁcial intelligence in the control of vessel, underwa- ter robotics, and other marine systems. [25] H. Niu, Y. Lu, A. Savvaris, and A. Tsourdos, ‘‘An energy-efﬁcient path planning algorithm for unmanned surface vehicles,’’ Ocean Eng., vol. 161, pp. 308–321, Aug. 2018. [26] G. Xia, Z. Han, B. Zhao, and X. Wang, ‘‘Local path planning for unmanned surface vehicle collision avoidance based on modiﬁed quantum particle swarm optimization,’’ Complexity, vol. 2020, pp. 1–15, Apr. 2020. GUOQING XIA received the Ph.D. degree in con- trol theory and control engineering from Harbin Engineering University (HEU), in 2001. He is currently working as a Professor with HEU. His research interests include ship dynamic position- ing control technique, intelligent control theory, and system simulation technique. GUOQING XIA received the Ph.D. degree in con- trol theory and control engineering from Harbin Engineering University (HEU), in 2001. He is currently working as a Professor with HEU. His research interests include ship dynamic position- ing control technique, intelligent control theory, and system simulation technique. XINWEI WANG received the bachelor’s degree in electrical engineering and automation from Harbin Engineering University (HEU), in 2015, where he is currently pursuing the Ph.D. degree in control science and engineering. His research interests include ship motion control, nonlinear control the- ory, and intelligent control theory. 207857 VOLUME 8, 2020
https://openalex.org/W2608686001	https://europepmc.org/articles/pmc5416713?pdf=render	English	null	15q11.2 CNV affects cognitive, structural and functional correlates of dyslexia and dyscalculia	Translational psychiatry	2,017	cc-by	9,177	INTRODUCTION in these areas can lead to reading and/or math difﬁculties; the left fusiform gyrus has been shown to have less gray matter density and activation in individuals diagnosed with DLX.7 The angular gyrus8 has been shown to associate with high-level language and mathematical tasks, such as arithmetic fact retrieval.9 Speciﬁc learning disorders, such as dyslexia (DLX) and dyscalculia (DC), are challenging phenotypes to disentangle. DLX and DC refer to neurodevelopmental disorders manifested in learning difﬁcul- ties with impairment in acquiring skills in reading and arithmetic, respectively, not due to intellectual disabilities or other develop- mental or neurological disorders.1 Despite being highly heritable, h2 = 0.52 for DLX and 0.61 for DC,2 genome-wide association studies have failed to uncover sequence variants conferring risk of these speciﬁc learning disorders.3,4 Hence, larger genome-wide scans are needed to unravel how a conﬂuence of rare and common sequence variants confer risk and which biological pathways are affected. , Some rare copy number variants (CNVs) are associated with neuropsychiatric disorders. Although little is known about how these high-impact variants confer risk of disease, they provide a biologically deﬁned entry point for investigations into the mechanisms of brain function. These CNVs impact cognitive functions and learning and are probably the strongest identiﬁable factors contributing to the disease in affected carriers.10–12 An example is the 15q11.2(BP1–BP2) deletion that confers risk of neuropsychiatric disorders including speciﬁc learning difﬁculties. We have previously described the impact of the 15q11.2(BP1–BP2) deletion on cognitive abilities assessed by neuropsychological tests.11 Deletion carriers show modest impairments in most cognitive domains and the deletion confers high risk for DLX and DC. Here we investigate the effect of the 15q11.2(BP1–BP2) deletion on cognition, brain structures and functions of deletion carriers in a larger sample. Through neuropsychological testing, we establish that the cognitive proﬁle of the deletion carriers is similar to the cognitive proﬁle of the combined phenotype of dyslexia and dyscalculia. By using magnetic resonance imaging (MRI) and functional MRI (fMRI), we show that the deletion affects structural and functional correlates of DLX and DC. 1deCODE Genetics/Amgen, Reykjavik, Iceland; 2Faculty of Electrical and Computer Engineering, University of Iceland, Reykjavik, Iceland; 3Cardiff University Brain Imaging Research Center, Cardiff University, Cardiff, UK; 4Institute of Psychiatry, King's College, London, UK; 5Faculty of Physical Sciences, University of Iceland, Reykjavik, Iceland; 6Department of Psychiatry, Landspitali National University Hospital, Reykjavik, Iceland; 7Faculty of Medicine, University of Iceland, Reykjavik, Iceland; 8The State Diagnosis and Counselling Center, Kopavogur, Iceland; 9Röntgen Domus, Reykjavik, Iceland; 10School of Business, University of Reykjavik, Reykavik, Iceland; 11Department of Child and Adolescent Psychiatry and Psychotherapy, Psychiatric Hospital, University of Zurich, Zurich, Switzerland and 12Central Institute of Mental Health, University of Heidelberg Medical Faculty Mannheim, Mannheim, Germany. Correspondence: Dr K Stefansson or Dr MO Ulfarsson, deCODE Genetics/Amgen, Sturlugata 8, 101 Reykjavik, Iceland. E-mail: kstefans@decode.is or mou@hi.is Received 11 November 2016; revised 22 February 2017; accepted 23 February 2017 OPEN OPEN Citation: Transl Psychiatry (2017) 7, e1109; doi:10.1038/tp.2017.77 www.nature.com/tp ORIGINAL ARTICLE 15q11.2 CNV affects cognitive, structural and functional correlates of dyslexia and dyscalculia MO Ulfarsson1,2, GB Walters1, O Gustafsson1, S Steinberg1, A Silva3, OM Doyle4, M Brammer4, DF Gudbjartsson1,5, S Arnarsdottir1,6, GA Jonsdottir1, RS Gisladottir1, G Bjornsdottir1, H Helgason1,2, LM Ellingsen2, JG Halldorsson7, E Saemundsen7,8, B Stefansdottir1, L Jonsson1, VK Eiriksdottir1, GR Eiriksdottir1, GH Johannesdottir1, U Unnsteinsdottir1, B Jonsdottir9, BB Magnusdottir6,10, P Sulem1, U Thorsteinsdottir1,7, E Sigurdsson6,7, D Brandeis11,12, A Meyer-Lindenberg12, H Stefansson1 and K Stefansson1,7 Several copy number variants have been associated with neuropsychiatric disorders and these variants have been shown to also inﬂuence cognitive abilities in carriers unaffected by psychiatric disorders. Previously, we associated the 15q11.2(BP1–BP2) deletion with speciﬁc learning disabilities and a larger corpus callosum. Here we investigate, in a much larger sample, the effect of the 15q11.2(BP1–BP2) deletion on cognitive, structural and functional correlates of dyslexia and dyscalculia. We report that the deletion confers greatest risk of the combined phenotype of dyslexia and dyscalculia. We also show that the deletion associates with a smaller left fusiform gyrus. Moreover, tailored functional magnetic resonance imaging experiments using phonological lexical decision and multiplication veriﬁcation tasks demonstrate altered activation in the left fusiform and the left angular gyri in carriers. Thus, by using convergent evidence from neuropsychological testing, and structural and functional neuroimaging, we show that the 15q11.2(BP1–BP2) deletion affects cognitive, structural and functional correlates of both dyslexia and dyscalculia. Translational Psychiatry (2017) 7, e1109; doi:10.1038/tp.2017.77; published online 25 April 2017 INTRODUCTION p y Prevalence of DLX and DC range from 4 to 7% depending on the criteria used.5 These learning disorders co-occur much more frequently than expected by chance; the comorbidity rate has been estimated as high as 40%.6 Although DLX and DC occur more often separate from each other, and largely distinct brain systems handle reading and mathematics, certain brain regions are important for both. Regions that have been associated with both disorders include the fusiform gyrus (BA37), which lies below the lingual and parahippocampal gyri and above the inferior temporal gyrus, as well as the angular gyrus (BA39), which is located in the posterior part of the inferior parietal lobe. The fusiform gyrus is thought to be an important structure for discriminating between and within categories of objects and includes the left hemisphere ‘visual word form area’. Dysfunction Phenotypic heterogeneity, caused by many different biochem- ical perturbations, complicates the search for sequence variants Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al Figure 1. Association of the 15q11.2(BP1–BP2) deletion group and subgroups of NoCNV with cognitive traits, GAF, ARHQ, AMHQ and functional MRI test scores. (a) Average standardized scores for 15q11.2(BP1–BP2) deletion, DLX&DC and NoCNV without (w/o) learning difﬁculties. (b) Average standardized scores for DLXonly, DConly and NoCNV w/o learning difﬁculties. (c) Mean accuracy for fMRI phonological lexical decision tasks (words) and multiplication veriﬁcation tasks (mult) for 15q11.2(BP1–BP2) deletion, DLXonly, DConly, DLX&DC and NoCNV. The tests are verbal IQ (V-IQ); performance IQ (P-IQ); logical memory I and II (LM I and II); letter ﬂuency (LF); category ﬂuency (CF); Stroop (the difference between the time it takes to name the color of the ink of a word that is actually the name of another color and to name the color of colorpads); trail making test (TMT), TMT trail B–TMT trail A; perseverative errors in the Wisconsin card sorting test (Pers. Err); spatial working memory (SWM); rapid visual information processing (RVIP); TMT trail A (TMT-A); Str-bl (Stroop: time it takes to read the names of colors written in black ink); global assessment of function (GAF); adult reading history questionnaire (ARHQ); adult mathematical history questionnaire (AMHQ). Word experiment: orthographic familiar forms of Icelandic nouns (W); phonologically correct but orthographically unfamiliar forms of the same word (PH); phonologically and orthographically unfamiliar forms (PW); false fonts (FF). Multiplication experiment: correct equation (C); incorrect equation (NC); false equation (F). MATERIALS AND METHODS A total of 71 subjects carrying the 15q11.2(BP1–BP2) deletion in the absence of a schizophrenia, bipolar disorder, autism or intellectual disability diagnosis were recruited along with 643 controls not carrying CNVs associated with psychiatric disorders (NoCNV). Participants were assessed with a battery of neuropsychological tests measuring cognitive traits, the global assessment of functioning scale and self-reported questionnaires on reading (adult reading history questionnaire, ARHQ) and mathematics (adult mathematical history questionnaire, AMHQ). Psychologists and others phenotyping the study subjects were blind to the genotype. Large lists of CNV-carriers and non-carriers were sent to a clinic overseeing the phenotyping. Identiﬁers were encrypted and sent back to researchers working on the genetic data. A detailed deﬁnition of the tests and questionnaires is given in a previous study (see also Figure 1).11 Supplementary Table 2 shows the sample sizes for the tests and questionnaires. To investigate the deletion group with respect to INTRODUCTION See previous study11 for more information about tests in a and b, and the functional MRI section below for more information about tests in c. Error bars represent standard error. Impairment is in s.d. units. The sample size given in the ﬁgure legend for a and b refer to the number of subjects with available scores. Some individual scores are missing. The sample size for each test is given in Supplementary Table 2. CNV, copy number variation; DC, dyscalculic; DConly, dyscalculic but not dyslexic; DLX, dyslexic; DLX&DC, dyslexic and dyscalculic; DLXonly, dyslexic but not dyscalculic; IQ, intelligence quotient; MRI, magnetic resonance imaging. 2 Figure 1. Association of the 15q11.2(BP1–BP2) deletion group and subgroups of NoCNV with cognitive traits, GAF, ARHQ, AMHQ and functional MRI test scores. (a) Average standardized scores for 15q11.2(BP1–BP2) deletion, DLX&DC and NoCNV without (w/o) learning difﬁculties. (b) Average standardized scores for DLXonly, DConly and NoCNV w/o learning difﬁculties. (c) Mean accuracy for fMRI phonological lexical decision tasks (words) and multiplication veriﬁcation tasks (mult) for 15q11.2(BP1–BP2) deletion, DLXonly, DConly, DLX&DC and NoCNV. The tests are verbal IQ (V-IQ); performance IQ (P-IQ); logical memory I and II (LM I and II); letter ﬂuency (LF); category ﬂuency (CF); Stroop (the difference between the time it takes to name the color of the ink of a word that is actually the name of another color and to name the color of colorpads); trail making test (TMT), TMT trail B–TMT trail A; perseverative errors in the Wisconsin card sorting test (Pers. Err); spatial working memory (SWM); rapid visual information processing (RVIP); TMT trail A (TMT-A); Str-bl (Stroop: time it takes to read the names of colors written in black ink); global assessment of function (GAF); adult reading history questionnaire (ARHQ); adult mathematical history questionnaire (AMHQ). Word experiment: orthographic familiar forms of Icelandic nouns (W); phonologically correct but orthographically unfamiliar forms of the same word (PH); phonologically and orthographically unfamiliar forms (PW); false fonts (FF). Multiplication experiment: correct equation (C); incorrect equation (NC); false equation (F). See previous study11 for more information about tests in a and b, and the functional MRI section below for more information about tests in c. Error bars represent standard error. Impairment is in s.d. units. The sample size given in the ﬁgure legend for a and b refer to the number of subjects with available scores. Some individual scores are missing. INTRODUCTION The sample size for each test is given in Supplementary Table 2. CNV, copy number variation; DC, dyscalculic; DConly, dyscalculic but not dyslexic; DLX, dyslexic; DLX&DC, dyslexic and dyscalculic; DLXonly, dyslexic but not dyscalculic; IQ, intelligence quotient; MRI, magnetic resonance imaging. conferring risk of DLX and DC. Here we focus on the impact conferred by the 15q11.2(BP1–BP2) deletion and show that the sequence variant confers risks of both DLX and DC and the carriers have cognitive, structural and functional aberrations that are considered to be correlates of both conditions. used a subset of the NoCNV group of subjects without any large CNVs (PopCtrl). Supplementary Table 1 shows the population characteristics for subjects participating in the neuroimaging experiments. All the partici- pants signed informed consent approved by the National Bioethics Committee of Iceland. Translational Psychiatry (2017), 1 – 8 Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al 3 effects on gray and white matter volume were reported as signiﬁcant when a whole-brain family-wise error-corrected P-value was less than 0.05. Owing to the intrinsic spatial smoothness of the data, the Bonferroni correction procedure is overly conservative. Therefore, we use the random ﬁeld theory19,20 correction method as implemented in the SPM8 toolbox to correct for multiple comparisons. reading and math, the NoCNV group was further separated into six subgroups deﬁned by using a score greater than 0.43 on the ARHQ,14 and a score greater than 12 on the AMHQ11 as a surrogate for dyslexia and dyscalculia, respectively. These subgroups contain (i) 80 dyslexic but not dyscalculic (DLXonly), (ii) 69 dyscalculic but not dyslexic (DConly), (iii) 42 dyslexic and dyscalculic (DLX&DC), (iv) 452 NoCNV without speciﬁc learning difﬁculties, (v) 123 dyslexic (without regard to the dyscalculic status) (DLX), (vi) 111 dyscalculic (without regard to the dyslexic status). Supplementary Table 3 presents the carrier status and the number of individuals in each subgroup. reading and math, the NoCNV group was further separated into six subgroups deﬁned by using a score greater than 0.43 on the ARHQ,14 and a score greater than 12 on the AMHQ11 as a surrogate for dyslexia and dyscalculia, respectively. These subgroups contain (i) 80 dyslexic but not dyscalculic (DLXonly), (ii) 69 dyscalculic but not dyslexic (DConly), (iii) 42 dyslexic and dyscalculic (DLX&DC), (iv) 452 NoCNV without speciﬁc learning difﬁculties, (v) 123 dyslexic (without regard to the dyscalculic status) (DLX), (vi) 111 dyscalculic (without regard to the dyslexic status). Supplementary Table 3 presents the carrier status and the number of individuals in each subgroup. Functional MRI The subjects listed in Supplementary Table 1 participated in two fMRI experiments, the ﬁrst involving a phonological lexical decision task (word experiment), and the second involving a multiplication veriﬁcation task (multiplication experiment). fMRI multiplication paradigm During fMRI scanning, the participants were asked to verify whether a visually presented multiplication equation was correct or not. There were 132 stimuli consisting of three types: 44 correct (C) multiplication equations, for example, 5 × 9 = 45; 44 incorrect (NC) multiplication equations, for example, 6 × 6 = 21; 44 false (F) equations, for example, 9 q 4 = lv. The correct equations were selected from the 10 × 10 multi- plication table. Subjects were excluded from the analysis according to the same exclusion criteria as in the word experiment. A total of 117 participants were scanned with 110 subjects passing quality checks. Statistical analysis of cognitive traits The scores from each cognitive test or questionnaire were inverse normally transformed and then adjusted for gender, age at testing and where indicated, intelligence quotient (IQ) based on data from controls only. The scores were shifted and scaled so that controls had a mean of 0 and a standard deviation of 1, and also arranged so that higher scores indicated greater impairment. Fisher’s exact test and the DLX and DC subgroup information in Supplementary Table 3 was used to estimate the deletions’ risk of DLX, DC and DLX&DC. A result was judged as signiﬁcant when the P- value was less than 0.05 Bonferroni corrected for the number of cognitive tests or questionnaires. Functional MRI acquisition The data were collected using a 1.5 T Philips Achieva MRI scanner. Two hundred and sixty-ﬁve volumes (185 volumes in the short version; see below) and 215 volumes were acquired for the words and multiplication experiments, respectively. In both experiments, 28 axial brain slices were acquired using an echo planar imaging pulse sequence (TR = 3 s; TE = 55 ms; image matrix = 64 × 64; voxel size = 3.75 × 3.75 × 5 mm3; ﬂip angle = 90°; slice order = ascending, sequential; coverage = whole brain). Structural MRI data acquisition Statistical analysis of the fMRI data The data were analyzed using SPM12. The statistical analysis described here is only for the word experiment, the analysis was similarly performed for the multiplication test. The data were ﬁrst realigned to the mean, using a rigid model, followed by a slice timing correction. After that, the mean image of each echo planar imaging time series was spatially normalized to the SPM's MNI152 template. The images were then spatially resampled to 2 × 2 × 2 mm3. Finally, the images were spatially smoothed using a 9 mm full-width at half-maximum Gaussian kernel. fMRI word experiment The subjects listed in Supplementary Table 1 were scanned using an MRI scanner (1.5 T Philips Achieva, Philips Medical Systems, Eindhoven, Netherlands). The scans were performed with a three-dimensional fast T1-weighted gradient echo sequence (TR = 8.6 ms, TE = 4 ms, ﬂip angle = 8 degrees, slice thickness 1.2 mm, matrix = 192 × 192, ﬁeld of view = 240 × 240 mm). This MRI protocol was selected as it yields good contrast between white matter (WM), gray matter (GM) and cerebrospinal ﬂuid. Quality control consisted of visual inspection as well as a test of homogeneity of the image covariance, which is a part of the voxel-based morphometry (VBM) protocol described below. A total of 716 participants were scanned with 707 subjects passing quality checks. During the fMRI scanning, the participants were asked to decide whether a visually presented letter string sounded like a real word or not. The experimental design follows a previous fMRI study21 closely with the main difference being that it was modiﬁed for Icelandic native speakers. There were two versions of the experiment: the long version and the short version. In the long version, there were 176 stimuli, lasting one second each, consisting of four types: 44 orthographi- cally familiar forms of Icelandic nouns (W), 44 pseudohomophones that were phonologically correct but an orthographically unfamiliar form of the same word (PH), phonologically and orthographically unfamiliar forms (PW) and 44 false fonts (FF). In addition, there were 59 null events where only the ﬁxation cross was presented. In the short version, there were 120 stimuli, 30 for each stimuli type (W, PH, PW and FF) and 43 null events. Subjects were excluded from the analysis if they (i) exceeded a priori maximum movement criterion (±3 mm translation or ± 3° rotation), (ii) performed poorly in the phonological lexical decision task (o60% accuracy in one or more stimuli type) or (iii) had poor quality MR data. A total of 337 participants were scanned with 284 subjects passing quality checks. Voxel-based morphometry Voxel-based morphometry By using VBM,15,16 we analyzed the allele dose-dependent effect of CNV on white and gray matter tissue across subjects, while controlling for age and gender. VBM is a technique that allows investigation of regional differences in the brain anatomy. In this study, the T1-weighted structural brain MRIs were analyzed using VBM as implemented by the VBM8 software (http://dbm.neuro.uni-jena.de (version r351)), which is integrated into the SPM8 software (Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK, (http://www.ﬁl.ion.ucl.ac.uk/spm)) implemented in MATLAB R2013b (Mathworks, Sherborn, MA, USA). Brieﬂy, each T1-weighted structural brain MRI is tissue-segmented into WM, GM and cerebrospinal ﬂuid images, which are then registered to the MNI (Montreal Neurological Institute)17 space using an afﬁne transformation. After the tissue segmentation step, a spatial normalization step was performed where each tissue segment is brought into a common stereotactic space. This step uses the DARTEL algorithm18 and a brain template derived from 550 healthy control subjects in the IXI-database (http://www.brain-development.org). Finally, the maps from the normal- ization step were modulated, that is, intensity-corrected for local volume changes during the spatial normalization. To accommodate for noise and registration errors, the modulated maps were smoothed with a 12 mm full- width half-maximum Gaussian ﬁlter. fMRI multiplication paradigm Participants Subjects carrying the 15q11.2(BP1–BP2) deletion, and controls not carrying CNVs associated with psychiatric disorders (NoCNV), were recruited from a large genotyped sample of approximately 160 000 subjects representing half of the Icelandic population. Only subjects aged between 18 and 65 were included in this study. Subjects were excluded: if they had ICD-10 or DSM-IV diagnoses for schizophrenia, schizoaffective or bipolar disorder; if they were diagnosed with autism, intellectual disability or developmental delay at the State Diagnostic and Counselling Centre of Iceland serving children and adolescents with a disability; if they met psychoses criteria on the MINI13 interview; if they were diagnosed with schizophrenia, schizoaffective, bipolar disorder, autism, intellectual disability or develop- mental delay according to self-reports (or reports from parents); if they were using antipsychotic medication. In the neuroimaging experiment, we Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al Statistical analysis of the structural brain MRI data Multiple regression analysis was used to test carrier status effects on brain volume on a voxel-wise basis using SPM8. We examined regions throughout the entirety of the brain for volume differences using the model: Volume ¼ ðbaselineÞβ0+ðcarrierstatus)β1+ðageÞβ2+ðgenderÞβ3+noise: A two-stage model was used for statistical analysis assuming a mixed- effect design. In the ﬁrst stage, event types representing the correct responses for each stimuli type (W, PH, PW and FF) were modeled using the standard SPM hemodynamic function with its temporal derivative. The incorrect stimuli responses were modeled the same way and were also included in the model. The model also included six movement regressors from the realignment step. The carrier status was modeled by using a regressor coding deletion as 0, PopCTRL as 1 and duplication as 2. Age and gender were added in the statistical model as covariates of no interest. Furthermore, we inspected the effects of adding intracranial volume as a covariate of no interest but it had minor effect on the results and did not change the conclusions. The carrier status effects were tested using one-sided t-tests and the voxel-wise Translational Psychiatry (2017), 1 – 8 Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al 4 Table 1. Statistical analysis of the structural brain MRI data In the second stage, the following contrasts W, PH, PW, FF, W vs FF, FF vs W, PH vs W, W vs PH, W vs PW and PW vs W, were tested using a one-sided t-test using the following multiple regression model: worse on neuropsychological tests and are more likely to suffer from speciﬁc learning disorders than population controls.11 Hence, when learning difﬁculties are considered, the deletion is likely to be fully penetrant although the expressivity may vary substan- tially. The deletion confers high risk of DLX (odds ratio = 3.0, P = 2.2 × 10 −4) and DC (odds ratio = 3.4, P = 4.9 × 10 −5) when using a score greater than 0.43 on the ARHQ14 and a score greater than 12 on the AMHQ11 as a surrogate for dyslexia and dyscalculia, respectively. The deletion confers a greater risk when considering the comorbid phenotype DLX&DC (odds ratio = 4.4, P = 1.3 × 10 −4). Contrast ¼ ðbaselineÞβ0+ðcarrier status)β1+ðageÞβ2+ðgenderÞβ3+ðscan lengthÞβ4+noise; where the carrier status was modeled using a regressor coding deletion as 0, PopCTRL as 1 and duplication as 2, and the scan length was modeled by coding the long-word version with 1 and the short-word version with 2; scan length was not included as a covariate in the regression model for the multiplication test. Given our a priori hypothesis and the brain-wide signiﬁcant results from the structural analysis, we constructed a mask using the Automatic Anatomic Labeling atlas22 consisting of the left fusiform gyrus, left parahippocampal gyrus, inferior parietial lobule, angular gyrus, and supramarginal gyrus. We reported voxel-wise carrier status effects on the contrasts signiﬁcant when the family-wise error-corrected19,20 (within this mask) P-value was less than 0.05. In the case of the multiplication test, the contrasts C, F, F vs C, C vs F, NC, NC vs C, C vs NC, F vs NC and NC vs F were analysed. p yp Considering all the tests in Figure 1a except for the ARHQ and the AMHQ tests, it can be seen that the deletion group and the DLX&DC group have a similar proﬁle (Spearman’s correlation between mean proﬁle scores = 0.58, P = 0.042). Statistical analysis of the structural brain MRI data Carrier status-dependent functional, gray and white matter volume changes Hemisphere MNI coordinates (x, y, z) Effect (%) 95% CI P-value (corrected) Brodmann area sMRI: gray matter Fusiform gyrus Left (−35, −36, −15) +3.0 (2.9, 3.1) 0.045 BA37 Superior occipital Left (−22, −78, 24) −4.8 (−5.0, −4.6 ) 0.016 BA19 Superior frontal Right (20, 30, 52) −5.0 (−5.2, 4.8 ) 0.016 BA8 sMRI: white matter Cerebellum cruz 1 Right (28, −72, −32) +7.7 (7.6, 7.9) 6.84 × 10 −5 Paracentral lobule Right (10, −30, 54) +4.6 (4.5, 4.7) 6.93 × 10 −4 Superior temporal Left (−52, −12, 13) +5.0 (4.5, 5.1) 1.94 × 10 −3 Anterior corpus callosum NA (4, 0, 22) −4.6 (−4.7, −4.5) 6.84 × 10 −4 Amygdala Right (26, 2, −17) −4.7 (−4.8, −4.6) 5.57 × 10 −3 fMRI word paradigm: PW vs W Fusiform gyrus Left (−28, −36, −14) +68.2 (63.7, 72.8) 0.007a BA37 fMRI multiplication paradigm: C vs F Angular gyrus Left (−50, −66, 24) +87.2 (80.8, 93.8) 2.08 × 10 −4a BA39 Abbreviations: C, correct equation; CI, conﬁdence interval; F, false equation; fMRI, functional MRI; MNI, Montreal Neurological Institute; MRI, magnetic resonance imaging; NA, not available; PW, phonologically and orthographically unfamiliar forms; sMRI, structural brain MRI; W, orthographic familiar forms of Icelandic nouns. aP-values marked with a are corrected with respect to a region of interest (the left occipito-temporal lobe and the left parietal lobe). All the brain regions highlighted in Figures 1–3 are listed here. The sample sizes are n = 707 for sMRI, n = 284 for the fMRI word paradigm and n = 110 for the fMRI multiplication paradigm. The effects are calculated as (carrier status effect −mean)/\|mean\|. Table 1. Carrier status-dependent functional, gray and white matter volume changes Abbreviations: C, correct equation; CI, conﬁdence interval; F, false equation; fMRI, functional MRI; MNI, Montreal Neurological Institute; MRI, magnetic resonance imaging; NA, not available; PW, phonologically and orthographically unfamiliar forms; sMRI, structural brain MRI; W, orthographic familiar forms of Icelandic nouns. aP-values marked with a are corrected with respect to a region of interest (the left occipito-temporal lobe and the left parietal lobe). All the brain regions highlighted in Figures 1–3 are listed here. The sample sizes are n = 707 for sMRI, n = 284 for the fMRI word paradigm and n = 110 for the fMRI multiplication paradigm. The effects are calculated as (carrier status effect −mean)/\|mean\|. Correlations between the brain imaging and the cognitive phenotypes The Pearson’s correlation measure between each of the cognitive tests/ questionnaire score in Figure 1 and the brain imaging phenotype in Table 1 for both the NoCNV group and the 15q11.2(BP1–BP2) deletion group was computed. All the scores were corrected for gender and age before the correlation was computed. The brain phenotypes were the raw volume scores (for the VBM data) and contrast scores (for the fMRI data) at the locations indicated in Table 1. A result was judged as signiﬁcant when the P-value was less than 0.05 Bonferroni corrected for the number of correlations computed. The deletion group was compared with the combined group of DLXonly, DConly, DLX&DC and NoCNV without learning difﬁcul- ties. The largest impairment is observed on the ARHQ (0.58 s.d., P = 1.5 × 10 −4) and the AMHQ scores (0.75 s.d., P = 7.4 × 10 −7). However, the deletion group also shows impairment on other scores. When the scores are corrected for performance IQ and verbal IQ, the impairments on ARHQ (0.44 s.d., P = 0.0044), and AMHQ (0.64 s.d., P = 3.5 × 10 −5) remain, whereas the impairments measured by the other scores are not signiﬁcant compared with a Bonferroni threshold of P = 0.05/30 = 0.0017 accounting for the 15 tests with and without the IQ adjustment. Translational Psychiatry (2017), 1 – 8 Statistical analysis of the structural brain MRI data The impairments (again excluding the ARHQ and the AMHQ tests) present in the DLXonly, and the DConly phenotypes combine additively to produce the impairments in the DLX&DC phenotype (Spearman’s correlation between the sum of the DLXonly and DConly means and the DLX&DC means = 0.63, P = 0.023). The DLXonly and DConly groups (Figure 1b) clearly have different proﬁles, especially with regards to the IQ scores, perseverative errors in the Wisconsin card sorting test, spatial working memory and trail making test A. Structural MRI phenotypes Structural MRI phenotypes We obtained structural brain MRI of 51 carriers of the 15q11.2 (BP1–BP2) deletion not diagnosed with deﬁned neuropsychiatric disorders, 104 carriers of the reciprocal duplication and 552 controls not carrying CNVs associated with psychiatric disorders and without large CNVs (PopCtrl). A total of twenty nine 15q11.2(BP1–BP2) deletion carriers, 191 PopCtrl subjects and 60 duplication carriers took part in the word experiment. The participants were asked to decide whether a visually presented letter string sounded like a real word or not. There were four types of letter strings: (i) orthographically familiar forms of Icelandic nouns (W), (ii) pseudohomophones that were phonologically correct but orthographically unfamiliar forms of the same words (PH), (iii) pseudowords that were phonologically and orthographically unfamiliar forms (PW) and (iv) false fonts (FF). The experimental design follows that of van der Mark et al.,21 with the main difference being the translation to Icelandic. g p We examined regions throughout the entirety of the brain and analysed the result using whole-brain family-wise error multiple comparison correction. For both GM and WM, the carriers of a deletion vs duplication showed mirrored effects, that is, the deletion carriers show opposite changes to the duplication carriers (Supplementary Figures 1 and 2). The 15q11.2(BP1–BP2) deletion carriers have less GM volume in the left fusiform gyrus extending into the parahippocampal gyrus, and greater GM volume in the superior occipital gyrus (which is a part of the visual association area) and the superior frontal regions (Figure 2 and Table 1). The area in the superior frontal gyri has been implicated in visual attention.23 The deletion carriers have less WM volume in the right cerebellum, the right paracentral lobule and the left superior temporal lobe. On the other hand, the deletion carriers have greater WM volume in the anterior corpus callosum and the right amygdala (Figure 3 and Table 1). We observed a signiﬁcant interaction between carrier status of the CNV and gender in the right caudate nucleus (Supplementary Figure 3), that is, the female deletion carriers have greater GM volume than female duplication carriers, and on the other hand, male deletion carriers have less GM volume than the male duplication carriers (Supplementary Figure 4). As with the structural MRI results, the effect of deletion vs duplication carrier status on brain volumes was mirrored (Supplementary Figure 5). Neuropsychology The same neuropsychiatric CNV often confers risk of a range of neurodevelopmental phenotypes including schizophrenia, autism, intellectual disability, attention deﬁcit hyperactivity disorder and epilepsy. The 15q11.2(BP1–BP2) deletion has been associated with schizophrenia as well as speciﬁc learning disorders.11 The deletion group shows impairments in reading and mathematics, based on the phonological lexical decision task (PW) and the multiplication task (NC; see Figure 1c for a deﬁnition) used to assess how well carriers recognize words and understand mathematical equations. The deletion carriers perform worse on both tests than controls (Figure 1c). Our previous results show that 15q11.2(BP1–BP2) deletion carriers unaffected by neuropsychiatric disorders do perform Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al 5 Figure 2. The effect of the CNV carrier status (deletion, PopCtrl, duplication) on gray matter volume difference. The T-scores for the CNV carrier status are displayed where ﬁndings are highlighted in red or blue if Po0.001 with red indicating less gray matter volume for the 15q11.2 (BP1–BP2) deletion carriers, and blue indicating greater volume. The ﬁrst three ﬁgures are sagittal slices while the last ﬁgure shows a coronal slice where the location of the sagittal slices are denoted by vertical lines. CNV, copy number variation. Figure 2. The effect of the CNV carrier status (deletion, PopCtrl, duplication) on gray matter volume difference. The T-scores for the CNV carrier status are displayed where ﬁndings are highlighted in red or blue if Po0.001 with red indicating less gray matter volume for the 15q11.2 (BP1–BP2) deletion carriers, and blue indicating greater volume. The ﬁrst three ﬁgures are sagittal slices while the last ﬁgure shows a coronal slice where the location of the sagittal slices are denoted by vertical lines. CNV, copy number variation. Figure 3. The effect of the CNV carrier status (deletion, PopCtrl, duplication) on white matter volume change. The T-scores for the CNV carrier status are displayed where ﬁndings are highlighted in red or blue if Po0.001 with red indicating less white matter volume for the 15q11.2 (BP1–BP2) deletion carriers, and blue indicating greater volume. The ﬁrst ﬁve images are axial slices (inferior to superior). The rightmost image shows the locations of the axial slices on a sagittal view. CNV, copy number variation. Figure 3. The effect of the CNV carrier status (deletion, PopCtrl, duplication) on white matter volume change. Neuropsychology The T-scores for the CNV carrier status are displayed where ﬁndings are highlighted in red or blue if Po0.001 with red indicating less white matter volume for the 15q11.2 (BP1–BP2) deletion carriers, and blue indicating greater volume. The ﬁrst ﬁve images are axial slices (inferior to superior). The rightmost image shows the locations of the axial slices on a sagittal view. CNV, copy number variation. Subjects carrying 15q11.2(BP1–BP2) duplications were also investigated with respect to the aforementioned tests. No signiﬁcant impairments in the 15 tests presented in Figures 1a and b were detected in the 15q11.2(BP1–BP2) duplication carriers as compared with the rest of the NoCNV group. Functional MRI phenotypes Based on our a priori hypothesis, we performed two fMRI experiments: a phonological lexical decision task (word experi- ment) and a multiplication veriﬁcation task (multiplication experiment). Given the results of the structural analysis, we restricted our fMRI analysis to the left occipito-temporal cortex and the left parietal lobe. Based on our a priori hypothesis, we performed two fMRI experiments: a phonological lexical decision task (word experi- ment) and a multiplication veriﬁcation task (multiplication experiment). Given the results of the structural analysis, we restricted our fMRI analysis to the left occipito-temporal cortex and the left parietal lobe. Structural MRI phenotypes The results show that the deletion carriers have less PW vs W contrast in the left fusiform gyrus (Figure 4, Table 1). A previous report demonstrated the presence of phonological and orthographic familiarity effects in non- dyslexic children. Non-dyslexic children showed higher activation for unfamiliar (PH and PW) rather than familiar (W) word-forms, whereas this effect was absent in children with dyslexia.21 The results presented here are in line with this, showing that the phonological/orthographic familiarity effect is decreased in the left fusiform gyrus of deletion carriers. The multiplication experiment was performed on 18 deletion carriers, 40 PopCTRL and 52 duplication carriers. They were asked to determine whether a visually presented multiplication equation Translational Psychiatry (2017), 1 – 8 Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al 6 Figure 4. Carrier status-dependent functional difference. (a) The word experiment. A sagittal slice (left), x = −28, showing the location, MNI = (−28, −36, −14) of signiﬁcant carrier status effect on the contrast PW vs W. (b) The multiplication experiment. A sagittal slice (left), x = −50, showing the location, MNI = (−50, −66, 24), of signiﬁcant carrier status effect on the contrast C vs F. The right images on both a and b show the location of the sagittal slice on a coronal view. C, correct equation; F, false equation; MNI, Montreal Neurological Institute; PW, phonologically and orthographically unfamiliar forms; W, orthographic familiar forms of Icelandic nouns. Figure 4. Carrier status-dependent functional difference. (a) The word experiment. A sagittal slice (left), x = −28, showing the location, MNI = (−28, −36, −14) of signiﬁcant carrier status effect on the contrast PW vs W. (b) The multiplication experiment. A sagittal slice (left), x = −50, showing the location, MNI = (−50, −66, 24), of signiﬁcant carrier status effect on the contrast C vs F. The right images on both a and b show the location of the sagittal slice on a coronal view. C, correct equation; F, false equation; MNI, Montreal Neurological Institute; PW, phonologically and orthographically unfamiliar forms; W, orthographic familiar forms of Icelandic nouns. was correct or not. The equations were either correct (C), incorrect (NC) or false (F). structures in the brain and is associated with speciﬁc changes in function relative to controls. Structural MRI phenotypes In particular, the deletion affects the left fusiform gyrus and the left angular gyrus, brain structures that have been associated with both DLX and DC. The 15q11.2(BP1–-BP2) deletion carriers have less C vs F contrast in the left angular gyrus (Figure 4, Table 1). It has been proposed that the left angular gyrus supports the retrieval of mathematical facts such as the multiplication table24 and also the usage of previously learned facts.25 The lower GM volume in the left fusiform gyrus of the deletion carriers is of particular interest as it has been reported that this region has a major role in reading and mathematical processing.26 The fusiform gyrus is a part of the ventral temporal cortex,27 and is generally thought to be a key structure for high-level visual processing including face perception,28 reading29 and object recognition.30 Alterations in this region have also been associated with DLX in both structural,31–33 and functional studies,21,34,35 as well as with DC36 in morphometry and tractography studies. A recent meta-analysis of brain dysfunction in both DLX children and adults noted that the fusiform gyrus was the only brain region affected in both the groups.24 Correlations between the brain imaging and the cognitive phenotypes The Pearson’s correlation measure between each of the cognitive tests/questionnaire scores in Figure 1 and the brain imaging phenotypes in Table 1 for both the NoCNV group and the 15q11.2 (BP1–BP2) deletion group was computed (Supplementary Tables 6 and 8). There are signiﬁcant correlations within the cognitive tests/ questionnaire scores and also within the brain imaging pheno- types. But no signiﬁcant correlation between the brain imaging phenotypes and the cognitive tests/questionnaire scores. The failure to detect signiﬁcant association between the cognitive tests/questionnaire scores and the brain imaging phenotypes could, at least in part, be explained by the loss of statistical power due to lower sample size for the intersection of the cognitive tests/questionnaire scores and brain phenotypes than for them separately (Supplementary Tables 7 and 9). The decreased WM volume observed in the temporal lobe and cerebellum, and the greater WM volume in the corpus callosum replicate previous ﬁndings.11,37 Overall, we note that the WM ﬁndings are stronger than the GM ﬁndings. As the left fusiform gyrus is thought to support skilled and ﬂuid reading, and the left angular gyrus to support retrieval of mathematical facts such as the multiplication table, the structural and functional alterations in those areas may be the cause of the speciﬁc learning disorders found in the deletion carriers as some abnormalities predate literacy38 and numeracy, but may also reﬂect a lack of reorganization due to emerging literacy39,40 and/ or numeracy. Although caudate GM volume alterations in DLX have been described previously,41 the interaction with gender is a novel ﬁnding and may reﬂect differences in articulatory compensation.42 The ﬁnding of decreased WM in the cerebellum could lend support to the cerebellar deﬁcit theory,43 which states that dyslexia is characterized by a general cerebellar abnormality resulting in impaired ability to perform tasks automatically thereby negatively affecting language and reading. Overall, the data support that the 15q11.2(BP1–BP2) CNV maps to a multifocal neurobiological proﬁle and the implicated structures ﬁt well with those identiﬁed in studies on reading and/or math problems. REFERENCES 1 American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders (DSM5), vol. 5. American Psychiatric Publishing: Arlington, VA, USA, 2013. 2 Plomin R, Kovas Y. Generalist genes and learning disabilities. Psychol Bull 2005; 131: 592–617. 3 Bishop DV. The interface between genetics and psychology: lessons from developmental dyslexia. Proc Biol Sci 2015; 282: 20143139. This study adds to the emerging understanding of the impact conferred by the 15q11.2(BP1–BP2) deletion on brain structure and function. The deletion confers high risk of the DLX&DC phenotype (odds ratio = 4.4, P = 1.4 × 10 −4), and the results demonstrate signiﬁcant volume changes in WM and GM brain structures in addition to a decrease in brain activation in regions important for reading and arithmetic. Overall, our ﬁndings shed light on the role of this CNV in typical and atypical brain development. The deletion allele impacts cognitive function and learning and is probably the strongest factor contributing to the DLX&DC phenotype in the affected carriers. 4 Pettigrew KA, Fajutrao Valles SF, Moll K, Northstone K, Ring S, Pennell C et al. Lack of replication for the myosin-18B association with mathematical ability in independent cohorts. Genes Brain Behav 2015; 14: 369–376. independent cohorts. Genes Brain Behav 2015; 14: 369–376. 5 Landerl K, Fussenegger B, Moll K, Willburger E. Dyslexia and dyscalculia: two learning disorders with different cognitive proﬁles. J Exp Child Psychol 2009; 103: 309–324. 6 Wilson AJ, Andrewes SG, Struthers H, Rowe VM, Bogdanovic R, Waldie KE. Dyscalculia and dyslexia in adults: cognitive bases of comorbidity. Learn Individ Differ 2015; 37: 118–132. 7 Clark D. The Brain and Behavior. Cambridge University Press: Cambridge, 2005. 8 Seghier ML. The angular gyrus: multiple functions and multiple subdivisions. Neuroscientist 2013; 19: 43–61. Brain structure is largely shaped by sequence variants that exert lasting inﬂuences on its function and several common variants have been associated with subcortical structures.49,50 The conﬂuence of common variants predicting subcortical structures does, however, not predispose to brain diseases like schizophrenia.51 Thus, although brain structure volumes show high heritability, there may not be a direct correlation with diseases. Hence, although subcortical volumes may differentiate patients from controls, the explanation may not necessarily be rooted in their genomes. Many brain disorders are heterogeneous groups of disorders at the level of genetic etiology and clinical presentation. REFERENCES Through high-impact variants, the relationship between genotype and phenotype may be disentangled, which, in turn, may help determine which brain phenotypes, associated with a disease, are a cause or consequence of the disease. For instance, larger putamen and pallidum volumes associate with duration of illness in schizophrenia,51 a consequence of the disease that can be combatted with antipsychotic drugs. 9 Grabner RH, Ischebeck A, Reishofer G, Koschutnig K, Delazer M, Ebner F et al. Fact learning in complex arithmetic and ﬁgural-spatial tasks: the role of the angular gyrus and its relation to mathematical competence. Hum Brain Mapp 2009; 30: 2936–2952. 10 Rosenfeld JA, Coe BP, Eichler EE, Cuckle H, Shaffer LG. Estimates of penetrance for recurrent pathogenic copy-number variations. Genet Med 2013; 15: 478–481. 11 Stefansson H, Meyer-Lindenberg A, Steinberg S, Magnusdottir B, Morgen K, Arnarsdottir S et al. CNVs conferring risk of autism or schizophrenia affect cognition in controls. Nature 2014; 505: 361–366. 12 Singh T, Walters JTR, Johnstone M, Curtis D, Suvisaari J, Torniainen M et al. Rare schizophrenia risk variants are enriched in genes shared with neuro- developmental disorders. bioRxiv 2016. 13 Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E et al. The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry 1998; 20 (59 Suppl): 22–33. 14 Bjornsdottir G, Halldorsson JG, Steinberg S, Hansdottir I, Kristjansson K, Stefansson H et al. The Adult Reading History Questionnaire (ARHQ) in Icelandic: psycho- metric properties and factor structure. J Learn Disabil 2014; 47: 532–542. 15 Wright IC, McGuire PK, Poline JB, Travere JM, Murray RM, Frith CD et al. A voxel- based method for the statistical analysis of gray and white matter density applied to schizophrenia. Neuroimage 1995; 2: 244–252. Here we have demonstrated by using convergent evidence from neuropsychological testing and structural and functional neuroimaging that a high-impact sequence variant provides insight into the causes of variability in human brain structure and function. Although the 15q11.2 CNV alleles confer mirror effects on both brain structure and function, only the deletion affects cognition with large effect. Sequence variants inﬂuencing brain structures may reveal new biological mechanisms under- lying cognition and neuropsychiatric illness. 16 Ashburner J, Friston KJ. Voxel-based morphometry—the methods. Neuroimage 2000; 11(6 Pt 1): 805–821. 17 Evans AC, Collins DL, Mills SR, Brown ED, Kelly RL, Peters TM. CONFLICT OF INTEREST 20 Cao J, Worsley KJ. Applications of random ﬁelds in human brain mapping. In: Spatial Statistics: Methodological Aspects and Applications, vol. 159. Springer-Verlag: New York, 2001, pp 170–182. MOU, GBW, OG, SS, DFG, SA, GAJ, RSG, GB, HH, BS, LJ, VKE, GRE, GHJ, UU, PS, UT, HS and KS are employees of deCode genetics/Amgen. BJ is an employee of Röntgen domus. The remaining authors declare no conﬂict of interest. 21 van der Mark S, Bucher K, Maurer U, Schulz E, Brem S, Buckelmuller J et al. Children with dyslexia lack multiple specializations along the visual word-form (VWF) system. Neuroimage 2009; 47: 1940–1949. REFERENCES 3D statistical neuro- anatomical models from 305 MRI volumes. Proceedings of the Nuclear Science Symposium and Medical Imaging Conference, 1993. 1993 IEEE Conference Record, 31 October–6 November 1993. 18 Ashburner J. A fast diffeomorphic image registration algorithm. Neuroimage 2007; 38: 95–113. 19 Nichols T, Hayasaka S. Controlling the familywise error rate in functional neuro- imaging: a comparative review. Stat Methods Med Res 2003; 12: 419–446. Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al Impact of 15q11.2 CNV on dyslexia and dyscalculia MO Ulfarsson et al 7 widely in the central nervous system, while TUBGCP5 is highly expressed in the subthalamic nucleus.46 Yoon et al.46 took a multifaceted approach to investigate why the 15q11.2(BP1–BP2) deletion confers risk of neuropsychiatric disorders. They used human iPSC-derived neural progenitors carrying the deletion and noticed deﬁcits in adherens junctions and apical polarity. They claim that these results from haploinsufﬁciency of CYFIP1 encoding a subunit of the WAVE complex. They also demonstrated that in the developing mouse cortex, deﬁciency in CYFIP1 and WAVE signaling similarly affects radial glial cells, leading to their ectopic localization outside of the ventricular zone.46 Bozdagi et al.47 furthermore reported that haploinsufﬁency of CYFIP1 produces fragile X-like phenotypes in mice. Thus, haploinsufﬁency of CYFIP1 may contribute to the neuro- developmental origins of the disorders associated with the 15q11.2(BP1–BP2) deletion.10,46,48 at the University of Iceland) for his assistance in selecting Icelandic words for the lexical decision task. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreements’ no. 115008 (NEWMEDS) and no. 115300 (EUAIMS) of which resources are composed of EFPIA in-kind contribution and ﬁnancial contribution from the European Union’s Seventh Framework Programme (EU-FP7/2007-2013), EU-FP7 funded grant no. 602450 (IMAGEMEND) and EU funded FP7-People-2011-IAPP grant agreement no. 286213 (PsychDPC). Approval for this study was obtained from the National Bioethics Committee of Iceland and the Icelandic Data Protection Authority. at the University of Iceland) for his assistance in selecting Icelandic words for the lexical decision task. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreements’ no. 115008 (NEWMEDS) and no. 115300 (EUAIMS) of which resources are composed of EFPIA in-kind contribution and ﬁnancial contribution from the European Union’s Seventh Framework Programme (EU-FP7/2007-2013), EU-FP7 funded grant no. 602450 (IMAGEMEND) and EU funded FP7-People-2011-IAPP grant agreement no. 286213 (PsychDPC). Approval for this study was obtained from the National Bioethics Committee of Iceland and the Icelandic Data Protection Authority. DISCUSSION It has been argued that a complex set of impairments in brain function account for comorbidity of DLX and DC.6 Here we demonstrate that the same variant confers risk of both DLX and DC. It is, however, important to keep in mind that this variant encompasses several genes. Haploinsufﬁciency of the genes affected by the 15q11.2(BP1–BP2) deletion impacts both cognitive traits and brain structure in a pattern consistent with the cognitive, structural and functional correlates of DLX and DC. The results show that the cognitive proﬁle of the subjects carrying the 15q11.2(BP1–BP2) deletion resembles the cognitive proﬁle of the DLX&DC subjects and, even after correcting for IQ, associates with the ARHQ and AMHQ scores. As the neuropsycho- logical proﬁles of the DLXonly and DConly groups are clearly different and only when combined produce the impairments in the DLX&DC phenotype, it can be inferred that impairments in DLX and DC are additive, suggesting that the cognitive processes involved in DLX and DC are largely independent. However, the DLX and the DC phenotypes are clearly not independent since that would mean (assuming 7% prevalence rate of DLX and DC) that the prevalence of the comorbid phenotype would be 0.49%, which is not the case. This indicates that some unknown factor affects both the DLX and the DC cognitive processes in the DLX&DC phenotype. The neuroimaging data show that the 15q11.2(BP1–BP2) deletion affects both gray and white matter The 15q11.2(BP1–BP2) CNV shows an allele dose-dependent (mirrored) effect on both the structure and function of the human brain, that is, duplication carriers show reciprocal changes in exactly the same brain regions as the deletion. However, this was not observed for the cognitive traits. Although the deletion negatively impacts performance on cognitive tests, the duplica- tion carriers performed on par with controls. A similar asymmetry between neuroimaging and cognitive phenotypes have been reported44,45 for the 16p11.2 CNV where the cognitive perfor- mance is negatively impacted by both the deletion and the reciprocal duplication. The BP1–BP2 region, spanning approximately 500 kb, contains four highly conserved, non-imprinted genes: TUBGCP5, NIPA1, NIPA2 and CYFIP1. NIPA1, NIPA2 and CYFIP1 are highly expressed Translational Psychiatry (2017), 1 – 8 ACKNOWLEDGMENTS 22 Tzourio-Mazoyer N, Landeau B, Papathanassiou D, Crivello F, Etard O, Delcroix N et al. Automated anatomical labeling of activations in SPM using a macroscopic anatomical parcellation of the MNI MRI single-subject brain. Neuroimage 2002; 15: 273–289. We are grateful to the participants and we thank the nurses and staff at the Research Recruitment Center and technicians and staff at Röntgen Domus (in particular Gunnhildur Sigurdardottir). We also thank the staff at deCODE genetics core facilities and all our colleagues for their important contribution to this work. We also acknowledge Eirikur Rognvaldsson (Professor of Icelandic Language and Linguistics 23 Volz KG, Schubotz RI, von Cramon DY. Variants of uncertainty in decision-making and their neural correlates. Brain Res Bull 2005; 67: 403–412. 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https://openalex.org/W2771446934	https://content.sciendo.com/downloadpdf/journals/adhi/2/1/article-p88.pdf	German	null	Akteure im Dazwischen	Administory. Zeitschrift für Verwaltungsgeschichte	2,017	cc-by	20,126	Akteure im Dazwischen Griechisch-orthodoxe Stifterinnen und Stifter zwischen Wien und dem Osmanischen Reich (1750–1918) NATHALIE PATRICIA SOURSOS, ANNA RANSMAYR A D M I N I S T O R Y Z E I T S C H R I F T F Ü R V E R W A L T U N G S G E S C H I C H T E B A N D 2 , 2 0 1 7 S E I T E 8 8 – 1 1 1 D O I : 1 0 . 2 4 7 8 / A D H I - 2 0 1 8 - 0 0 1 7 B A N D 2 , 2 0 1 7 S E I T E 8 8 – 1 1 1 D O I : 1 0 . 2 4 7 8 / A D H I - 2 0 1 8 - 0 0 1 7 © 2017 Nathalie Patricia Soursos, Anna Ransmayr. Published by Sciendo. This work is licensed under the Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0 © 2017 Nathalie Patricia Soursos, Anna Ransmayr. © 2017 Nathalie Patricia Soursos, Anna Ransmayr. Published by Sciendo This work is licensed under the Creative Commons Attribution 4 0 license (https://creativecommons org/licenses/by/4 0/) © 2017 Nathalie Patricia Soursos, Anna Ransmayr. Published by Sciendo. This work is licensed under the Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/). Bewegen zwischen zwei Räumen. Osmanische Untertanen im habsburgischen Wien Im habsburgischen Wien existierten seit dem 18. Jahr- hundert zwei ›griechische Gemeinden‹.10 Es handelte sich um Kirchengemeinden der griechisch nicht unier- ten Konfession (in der Monarchie später als griechisch- orientalisch11 oder orthodox bezeichnet), die sich nach dem Kriterium der Staatsangehörigkeit ihrer Mitglieder entweder zum Osmanischen Reich oder zur Habsburger- monarchie unterschieden. Erstere waren der Gemeinde zum Heiligen Georg am Hafnersteig, Letztere der Ge- meinde zur Heiligen Dreifaltigkeit am Alten Fleischmarkt zugeordnet. Obwohl die erstmals 1776 von Maria There- sia für die Gemeinde zum Heiligen Georg und 1787 von Joseph II. für die Gemeinde zur Heiligen Dreifaltigkeit verliehenen Privilegien die kirchliche Definition der Ge- meinden festschrieben, bewegte sich deren Charakter als Körperschaften zwischen einem Zusammenschluss von Personen ökonomisch-professioneller, religiöser und ethnischer Natur.12 Sie wurden je nach zeitlichem und sachlichem Kontext als Vertretungsorganisationen der Balkanhändler, als Pfarrgemeinden der nicht unierten Griechen und als Organisationen von ethnischen Grie- chen wahrgenommen. Im Rahmen dieses Beitrages sollen die vielseitigen stiftungskulturellen Aktivitäten der Wiener Griechen als ein Agieren innerhalb mehrerer Räume interpretiert werden. Zunächst werden dazu in den ersten zwei Ab- schnitten die beiden Wiener griechischen Gemeinden als rechtliche und religiöse Teilräume innerhalb Wiens untersucht, insbesondere in ihrer Aufgabe als Vermitt- lungsinstanzen zwischen den griechisch-orthodoxen Händlern und der staatlichen Verwaltung. Der rechtliche Rahmen, in welchem sich die Gemeinden zum Heiligen Georg und zur Heiligen Dreifaltigkeit bewegten, wird anhand der ihnen verliehenen Privilegien, der Geschäfts- ordnungen und der religiöse Minderheiten betreffenden Gesetze analysiert. Außerdem wird die Verbindung zwi- schen den Privilegien und Geschäftsordnungen und der Entwicklung der Stiftungen, insbesondere der Verwal- tung der Stiftungskapitalien, dargelegt. Der dritte Teil widmet sich der Verwaltungspraxis der Stiftungen inner- halb der Gemeinden sowie den Bemühungen seitens des Staates, Stiftungen zu beaufsichtigen und zu katalogisie- ren. Insbesondere interessieren der Umgang staatlicher Stellen mit der räumlichen Freizügigkeit von Menschen und deren Versuche, Stiftungen möglichst eindeutig zu identifizieren und zu beaufsichtigen. Ziel des Beitrages ist, die Einordnung von Stiftungen als »totales soziales Phänomen«9 und ihre damit verbundene Durchdringung Die gegenseitige Handelsfreiheit, die im Friedensver- trag von Karlowitz (1699)13 in Artikel 14 festgeschrieben worden war, wurde durch den Friedensvertrag von Passarowitz (1718) sowie einen weiteren Handelsvertrag (1718), der die wechselseitige Handelsfreiheit zu einem privilegierten Zoll von 3 Prozent garantierte, erweitert.14 Bei den osmanischen Händlern handelte es sich zum größten Teil um orthodoxe Christen. A D I N I S T O R Y 2 / 2 0 1 7 A D I N I S T O R Y 2 / 2 0 1 7 von Ökonomischem, Sozialem, Politischem und Rechtli- chem durch das transnationale Element zu erweitern. gründeten die Griechisch-Orthodoxen Wiens Stiftungen zugunsten ihrer Heimatgemeinden im Osmanischen Reich. Zweitens bewegten sie sich im abgegrenzten Raum der beiden griechisch-orthodoxen Gemeinden in Wien, indem sie arme und kranke Glaubensgenossen, die beiden griechisch-orthodoxen Kirchen und die grie- chische Schule in Wien unterstützten. Drittens handelte es sich bei den zumeist wohlhabenden Wohltätern um Akteure und Funktionsträger der habsburgischen Politik, Verwaltung, Wissenschaft und Kunst. Sie agierten daher durch Stiftungen für Wiener Institutionen und außer- konfessionelle Wohltätigkeitszwecke auch als Angehörige des Wiener Bürgertums, für welches das karitative und gemeinnützige Stiften, Schenken und Fördern ein »Inst- rument bürgerlichen Handelns«7 war. Ein gemeinsames Charakteristikum des Großteils der griechisch-orthodo- xen Stiftungen war ihre Verwaltung durch die Vorsteher der Gemeinden zum Heiligen Georg und zur Heiligen Dreifaltigkeit, die sich nach dem Kriterium der Staats- angehörigkeit ihrer Mitglieder unterschieden.8 Einleitung In seinem Testament vom 24. August 1799 sowie einem Kodizill vom 2. Dezember 18001 bedachte der griechisch- orthodoxe Händler Christoph von Nako Institutionen in seinem Heimatort Dojran im osmanischen Makedonien, im ungarischen Nagyszentmiklós (heute Sânnicolau Mare in Rumänien) und Pest (Budapest) sowie in Wien. Nako war gemeinsam mit seinem Bruder Kyrill von Dojran zunächst ins ungarische Banat gegangen, wo sie landwirt- schaftliche Flächen pachteten und mit den dort erzeug- ten Rohprodukten handelten.2 1784 erwarben sie die Grundherrschaften Nagyszentmiklós und Terémi (das heutige Teremia Mică in Rumänien) und wurden als Grafen von Nagyszentmiklós in den ungarischen Adels- stand erhoben.3 Von dort zog Christoph von Nako über Wien bis nach Chemnitz und starb schließlich in Un- garn. Doch nicht nur als Händler war er ein Akteur zwischen mehreren staatlichen Räumen, auch seine Stiftungen spiegeln seine transnationale Biografie. In seinen letztwilligen Verfügungen stiftete Christoph von Nako für die Schule in Dojran, die Einrichtung einer Landesindustrieschule in Nagyszentmiklós, eine zu er- richtende griechische Nationalschule in Wien, außerdem für das Bürgerspital und das griechisch-wallachische National-Spital in Pest. Er überschritt auch konfessio- nelle Grenzen, indem er »einer jeden katholischen, griechisch nicht unirten und protestantischen Kirche«4 auf seinen Gütern im Banat 100 Gulden vermachte. Seine Verbindung zu Chemnitz5 fand Ausdruck in dem Plan, auf den ungarischen Gütern »zur Auflebung der Landesindustrie« eine Kolonie »mit lauter gebornen Sachsen«6 anzusiedeln. Christoph von Nako ist sowohl durch seine Handels tätigkeit als auch durch sein wohltätiges Tun paradigma- tisch für andere griechisch-orthodoxe Stifterinnen und Stifter, deren transnationale Biografien und familiäre sowie wirtschaftliche Netzwerke sich ab dem 18. Jahr- hundert vermehrt über den Balkan bis nach Mitteleuropa streuten. Charakteristisch an der vielseitigen Stiftungs- kultur der griechisch-orthodoxen Wohltäterinnen und Wohltäter war, dass sie in drei sozialen, ökonomischen und kulturellen Bezugsräumen gleichzeitig agierten, die in diesem Beitrag in ihrem Verhältnis zueinander be- schrieben werden. Erstens wirkten sie im transnationa- len Raum. Durch die Erfahrungen der Migration geprägt, 017 Nathalie Patricia Soursos, Anna Ransmayr. blished by Sciendo. This work is licensed under the Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/). A D I N I S T O R Y 2 / 2 0 1 7 Plöchls Definition dieser Körperschaft als religiöse In- stitution nach orientalischem Kirchenrecht zu kurz.24 Bei der Bruderschaft zum Heiligen Georg handelte es sich nicht nur um eine religiöse Institution, sondern auch um einen kommerziellen Zusammenschluss der in Wien anwesenden ›griechischen‹ Händler aus dem Osmanischen Reich. Daher war die Bruderschaft auch nicht von der Abschaffung der religiösen Bruderschaf- ten 1783 durch Joseph II. betroffen.25 Die Mitgliedschaft war nach Firmen organisiert; bei der Wahl der Vorste- her wurde darauf geachtet, dass jeweils ein Firmenver- treter von jeder Region, aus der die Händler stammten, in der Dodekas vertreten war.26 Die Mitglieder waren verpflichtet, 1 Promille des jährlichen Umsatzes als Bei- trag für die Bruderschaft zu leisten.27 Dabei handelte es sich bei der Bruderschaft zum Heiligen Georg nicht um eine Handelskompanie, die selbst Handel betrieb, wie dies bei den ersten Organisationen von griechischen Händlern in Siebenbürgen und Ungarn der Fall war,28 sondern sie übernahm die Vertretung der Händler ge- genüber den österreichischen Behörden in rechtlichen Angelegenheiten.29 Die Bruderschaft (αδελφότης) oder auch Gemeinde (κοινότης) zum Heiligen Georg – eine strikte Trennung der beiden Begriffe, die teilweise syn- onym verwendet wurden, ist nicht möglich30 – grenzte sich also über die Kriterien der griechisch nicht unierten Konfession, der osmanischen Staatsangehörigkeit und des Berufsstandes des Händlers nach außen hin ab. In Bezug auf das Kriterium der Konfession entspricht dies der Organisation der Bevölkerung nach Religionsgrup- pen, dem sogenannten ›millet‹-System, im Osmanischen Reich, das den rechtlichen Status nicht muslimischer Religionsgemeinschaften regelte und ihnen Schutz durch den Sultan garantierte.31 Auf Firmenebene wurde jedoch durchaus mit Orienthändlern anderer Konfessionen zu- sammengearbeitet, mit denen die ›griechischen‹ Händler die Herkunftsregionen im Osmanischen Reich teilten.32 Die durch die Konfession, in der Folge auch durch die Staatsangehörigkeit konstruierten abgegrenzten Räume wurden also im Zuge der Handelstätigkeit dieser Akteure vielfach überschritten. Die Gemeinde zum Heiligen Georg befand sich in ihrer Eigenschaft als Gemeinde von osmanischen Unter des Griechischen einherging.17 Sie handelten vorwiegend mit Rohbaumwolle, deren Nachfrage durch die neu eta blierte österreichische Textilindustrie um die Mitte des 18. Jahrhunderts anstieg, aber auch mit Produkten wie Leder oder Rotgarn.18 Bis ins 19. Jahrhundert hinein blieb die Gruppe durch eine hohe professionelle Homogenität gekennzeichnet. Der von ihr besetzte Raum definierte sich zunächst nicht über ethnische Kriterien, sondern über die gemeinsame Herkunft aus dem Osmanischen Reich, den Beruf des Händlers sowie die Zugehörigkeit zur griechisch nicht unierten Konfession. Bewegen zwischen zwei Räumen. Osmanische Untertanen im habsburgischen Wien Diese sogenannten ›griechischen Handelsmänner‹,15 welche meist aus den Balkangebieten des Osmanischen Reiches (insbesondere aus den Regionen Makedonien, Thessalien und Epirus) kamen, hatten nicht zwingend das Griechische als Mut- tersprache – ein erheblicher Anteil war aromunischer16 Herkunft –, doch sie verwendeten es als Verkehrssprache des Handels und folgten dem griechischen Bildungspara- digma, bei dem der soziale Aufstieg mit der Verwendung 89 A D I N I S T O R Y 2 / 2 0 1 7 Untertanen behandelt wurde. Im Verlauf des 19. Jahr- hunderts erfolgte eine immer stärkere Angleichung der beiden Gemeinden, sodass schließlich die Notwendigkeit der Existenz zweier getrennter Gemeinden nach dem Kriterium der Staatsangehörigkeit infrage gestellt wur- de.38 Dennoch blieb die strikte Abgrenzung der beiden Gemeinden zueinander und somit die Konstruktion von zwei gesonderten Teilräumen bis 1918 aufrecht. Die Er- weiterung der Rechte der Bruderschaft zum Heiligen Ge- org fiel mit dem Unabhängigkeitskrieg in Griechenland zusammen, der mit der Ausrufung des neuen Staates im Rahmen des Londoner Protokolls von 1830 beendet wurde. Zudem wurden innenpolitisch die Rechte für akatholische Religionsgemeinschaften erweitert, als es durch die Märzverfassung vom 4. März 1849 jeder ge- setzlich anerkannten Kirche möglich wurde, die Verwal- tung ihrer Angelegenheiten selbstständig zu regeln.39 auch darin, dass sie ihren Privilegien gemäß nominell dem auf dem Herrschaftsgebiet der Habsburgermonar- chie residierenden serbischen Metropoliten von Kar- lowitz (heute Sremski Karlovci) unterstellt war, da die habsburgischen Behörden eine Unterordnung unter ein ausländisches kirchliches Oberhaupt, wie es der Patri- arch von Konstantinopel gewesen wäre, nicht duldeten. Die Metropolie von Karlowitz war bis 1864 die einzige orthodoxe Metropolie innerhalb der Habsburgermon- archie.33 Die Unterordnung unter den Metropoliten war jedoch rein formaler Natur, und die Privilegien erlaub- ten ihm keinerlei Einmischung in die Gemeindeverwal- tung und die Gemeindefinanzen, sodass sie kirchenhier archisch de facto autonom war. Durch die im Laufe der Zeit vorgenommenen Veränderungen in Hinblick auf die Behandlung ausländischer Untertanen wurden auch die Rechte der Gemeinde immer wieder angepasst oder neu ausgehandelt. Die administrative Autonomie der Gemeinde war für die Verwaltung der Stiftungen, somit für den finan- ziellen Erhalt der Gemeinde und für die Fortführung der sozialen Wohltätigkeit, von zentraler Bedeutung. Angesichts der Wichtigkeit solcher Stiftungen für das Selbstverständnis und den finanziellen Erhalt der Ge- meinde erscheint es erstaunlich, dass Stiftungen in den Privilegien nicht erwähnt wurden. Auch die finan- zielle Verwaltung des Gemeindekapitals wurde darin nur am Rande geregelt.40 Wichtig war einzig, dass die Bruderschaft keine Schulden anhäufen und für die Un- kosten der Kapelle sowie für das Personal durch frei- willige Almosen oder durch außerordentliche Beiträ- ge der Mitglieder aufkommen sollte. Tatsächlich hatte der Vorstand der Gemeinde zum Heiligen Georg 1771, fünf Jahre vor dem ersten Privilegium, in einem Schrei- ben an den kaiserlichen Hof betont, dass diese weder Grundstücke, Stiftungen noch sonstiges Eigentum in Österreich besäße. A D I N I S T O R Y 2 / 2 0 1 7 Die ältere der beiden Gemeinden ist jene zum Heili- gen Georg der osmanischen Untertanen, die in Form einer sogenannten ›Bruderschaft‹ entstand.19 Das älteste er- haltene Dokument, das die Existenz einer Gottesdienst- stätte der nicht unierten Griechen in Wien belegt, ist ein Hofkriegsratsbescheid aus dem Jahr 1726,20 der im Zuge eines Konflikts zwischen dem orthodoxen serbischen Klerus der Habsburgermonarchie und den aus dem Os- manischen Reich stammenden griechischen Händlern entstand.21 Darin wird die ungestörte Fortsetzung des ›exercitium religionis‹ der »Türckhischen unterthanen und forestiers der Griechischen Religion alhier« bestä- tigt. 1776 verlieh Maria Theresia den »gesamten in Unser K. K. Residenz Stadt Wienn sich aufhaltenden Griechi- schen Handelsleuten, und türkischen Unterthanen« ein Privilegium, das dazu dienen sollte, den Gottesdienst in der zu diesem Zeitpunkt im Steyrerhof befindlichen Ka- pelle zum Heiligen Georg zu ordnen und zu regulieren.22 Die Gemeinde legitimierte sich also aufgrund eines vom österreichischen Kaiserhaus erlassenen Privilegiums und stellte insofern, obwohl sie von ausländischen Unterta- nen gebildet wurde, eine österreichische Institution dar. Als Gremium, das für die Verwaltung der Kapelle zu- ständig war, wurde im Privilegium die »aus denen der Ottomanischen Pforte unterworfenen allhier Handeln- den Griechen der nicht unirten orientalischen Kirche allein bestehende Bruderschaft« genannt. Im Jahr 1782 bestätigte Joseph II. das Privilegium von Maria Theresia für die Bruderschaft zum Heiligen Georg, wobei die Be- stimmungen in Hinblick auf die Verwaltung der Kapelle durch die Nennung eines von der Bruderschaft zu wäh- lenden Ausschusses, der Dodekas (›Zwölferrat‹), präzi- siert wurden.23 Ob hl i h di P i il i f di ki hli h Die Gemeinde zum Heiligen Georg befand sich in ihrer Eigenschaft als Gemeinde von osmanischen Unter- tanen, die aber in Wien angesiedelt war, lange Zeit in einer gesetzlichen Ausnahmestellung und agierte aus einer Art Zwischenposition heraus. Dies äußerte sich 90 Na Obwohl sich die Privilegien nur auf die kirchlichen Aufgaben der Bruderschaft bezogen, greift Willibald 90 A D I N I S T O R Y 2 / 2 0 1 7 für wohltätige Zwecke in den Heimatgemeinden der Stifterinnen und Stifter im Osmanischen Reich errichtet. Stiftungsinteressen gingen jährlich oder halbjährlich nach Dojran, Metsovo, Melnik, Kastoria, Siatista, Tsari tsani, Përmet und Ioannina.43 Welche Schwierigkeiten sich aus diesen grenzüberschreitenden Stiftungen er- geben konnten, illustrieren die Formulierungen in den Testamenten, welche die Auszahlung der Zinsen aus den in Wien angelegten Kapitalien auch im Falle eines möglichen Krieges mit der Hohen Pforte garantieren wollten.44 Wien galt hinsichtlich der Anlage und Ver- waltung der Gelder als sicher. Deshalb stiftete diese erste Generation zwar für ihre Heimatgemeinden, das Kapital wurde jedoch in Wien deponiert. Die Interessen wurden jährlich oder halbjährlich transferiert, meist gemeinsam mit weiteren Zuwendungen für dieselbe Ge- meinde. Die habsburgischen Behörden mussten sich mit der Tatsache auseinandersetzen, dass »das Kapital zu ewigen Zeiten unaufkündlich in hiesigen Kreditfonds, so wie die Obligationen bei der Stiftungshauptkassa liegenbleiben, die Interessen aber in die türkischen Lande verschickt werden dürfen«.45 Diese Stiftungen für Empfänger im Osmanischen Reich mussten als eine Kontrollmaßnahme des Staates zusätzlich eine Geneh- migung der Hofkanzlei erbitten. Demeter Theocharides bestimmte für seine Stiftung so- gar, dass die Mietzinseinnahmen des gestifteten Hauses in der Biberbastei jährlich zur Hälfte für die Armen der Heiligen Dreifaltigkeit und zur Hälfte für die Armen des Heiligen Georg verwendet werden sollten.50 Damit wurde die klare Trennung der Teilräume, die in der Verwaltung der Gemeinden bestand, von den Stiftern überschritten. Eine weitere Komplikation hinsichtlich des Status der ›Griechen‹ in Wien und ihrer Stiftungen ergab sich mit der Gründung des neuen Staates Griechenland, der von der Habsburgermonarchie im Oktober 1832 anerkannt wurde.51 1835 wurde ein Handels- und Schifffahrts-Trak- tat geschlossen, dessen Additional-Artikel vom 22. August 1856 besagte, dass die Gerichte jenes Staates, welchem der Verstorbene als Untertan angehört hatte, für die Entscheidung strittiger Erbansprüche zuständig seien.52 Außerdem war es möglich, das Vermögen aus Österreich nach Griechenland zu übertragen, »ohne dieser Übertra- gung wegen einer was immer für außergewöhnlichen Steuer oder anderen Auflage unterworfen zu seyn«.53 Diese Steuerbefreiung zeigte jedoch nur geringe Auswir- kungen für die Gemeinde zum Heiligen Georg. Die meis- ten Mitglieder waren weiterhin osmanische Untertanen, da deren Herkunftsregionen erst wesentlich später an den griechischen Staat angegliedert wurden: Thessalien 1881, Makedonien und Epirus 1912. Ebenso lagen die Empfängerorte der Stiftungen außerhalb des griechischen Staatsgebietes. Diese Veränderungen an der politischen Landkarte Südosteuropas, aber auch der starke Rück- gang der Zahl der Gemeindemitglieder prägten die Stif- tungslandschaft der Gemeinde zum Heiligen Georg am Ende des 19. A D I N I S T O R Y 2 / 2 0 1 7 Das Kapital sei durch Almosen und Sammlungen unter den Mitgliedern zusammengetragen worden.41 Der Erhalt von Stiftungen war also zunächst nicht selbstverständlich. Eine große Einschränkung für die Gemeinde zum Heiligen Georg stellte das Verbot des Immobilienbesit- zes dar. Aus diesem Grund wurde ihr nicht erlaubt, das Haus, in dem 1803 die Kirche zum Heiligen Georg in Form eines Toleranzbethauses34 errichtet wurde, zu er- werben.35 Osmanischen Untertanen war aufgrund des Prinzips der Reziprozität der Besitz von Immobilien in der Habsburgermonarchie nicht gestattet, da dies auch Ausländern im Osmanischen Reich bis 1856 verboten war.36 Die Gemeinde behalf sich mit einer von den habs- burgischen Behörden ausdrücklich tolerierten Umge- hungskonstruktion: Der griechisch-orthodoxe k. k. Un- tertan Georg Johann von Karajan kaufte das Haus am Hafnersteig und wurde anstelle der Bruderschaft ins Grundbuch eingetragen. Nach Karajans Tod am 2. Juni 1813 waren dessen Erben jedoch unzufrieden mit der Pro-forma-Eintragung im Grundbuch. Die Vorsteher der Gemeinde sprachen daher am 9. Juli 1829 beim Kaiser vor und erbaten eine Erweiterung ihrer Rechte.37 Am 24. Januar 1834 wurde der Bruderschaft zum Heiligen Georg von Franz I. eine Erweiterung ihrer Privilegien gewährt, welche ihr den eigentümlichen Besitz ihres Kirchenhauses gestattete. Diese Privilegienerweiterung markiert eine wichtige Zäsur, indem die der Gemeinde aufgrund ihrer Eigenschaft als Organisation von osma- nischen Untertanen auferlegten Einschränkungen aufge- weicht wurden und sie nun ähnlich wie ihre Schwester- gemeinde zur Heiligen Dreifaltigkeit der habsburgischen Die erste zu verwaltende Stiftung erhielt die Ge- meinde 1785 vom griechischen Händler und osmani- schen Untertanen Demeter Sava. Dieser hatte in seinem Testament vom 17. August 1769 insgesamt 20.000 Gul- den für Arme und Schulen in Ioannina sowie zur jähr- lichen Erlösung eines Sklaven bestimmt.42 Dem Vorbild von Demeter Sava folgend, wurden weitere Stiftungen 91 A D I N I S T O R Y 2 / 2 0 1 7 müsste. Daraufhin wurde der Statthalterei versichert, bei der »Pfarre St. Georg« handle es sich zwar um osma- nische Untertanen, man würde aufgrund der Verringe- rung der Mitgliederzahl aber auch andere Untertanen aufnehmen wollen.66 Eine solche Aufnahme wurde je- doch aufgrund des Protests der Schwestergemeinde zur Heiligen Dreifaltigkeit, die seit 1893 auch für die Staats- bürger anderer Staaten zuständig war,67 bis 1918 nicht verwirklicht.68 Die Stiftung Betly blieb einstweilen lie- gen, bis nach dem Ersten Weltkrieg und dem Ende der Habsburgermonarchie seitens des Ministeriums für Kul- tus und Unterricht beschlossen wurde, die Stiftungsgelder, deren Bestimmungsort Kastoria seit 1912 zum griechi- schen Staat gehörte, nach Griechenland zu transferieren. Grund dafür war demnach einerseits die Grenzverschie- bung und damit die Sicherheit, dass die Gelder nicht der osmanischen Verwaltung übertragen würden. Anderer- seits verwies man auf den Friedensvertrag von Saint- Germain-en-Laye vom 10. September 1919. Dieser traf in § 169 eine Bestimmung hinsichtlich der Übergabe von Stiftungen an die alliierten und assoziierten Mächte: dem Staatsgrundgesetz vom 21. Dezember 1867 wurde in der Habsburgermonarchie die Unabhängigkeit in der Verwaltung von Stiftungen in Artikel 15 erneut festge- legt.58 Gemeindeintern wurde mit der Geschäftsordnung von 1907 die selbstständige Verwaltung bereits in den einleitenden Bestimmungen erneut betont:59 § 1) Die griechisch-orientalische Gemeinde zum heiligen Georg in Wien ordnet und verwaltet ihre Kirchen- und Wohltätigkeitsangelegenheiten und die hiezu bestimmten Stiftungen und Fonds auf Grund der am 3. August 1782 und am 8. Oktober 1796 mit k. k. Hofdekret gewährten Privilegien selbstständig.60 § 1) Die griechisch-orientalische Gemeinde zum heiligen Georg in Wien ordnet und verwaltet ihre Kirchen- und Wohltätigkeitsangelegenheiten und die hiezu bestimmten Stiftungen und Fonds auf Grund der am 3. August 1782 und am 8. Oktober 1796 mit k. k. Hofdekret gewährten Privilegien selbstständig.60 Außerdem wurde in § 17 dem Ausschuss die »Wahrung der Wohlfahrt der Gemeinde und die Sorge für die Er- haltung der Kirche«61 übertragen. Der Großteil der Stiftungen und Legate war in der Verwaltung unproblematisch, die Administration über die Gemeinde zum Heiligen Georg routiniert, und die Gelder kamen meist pünktlich bei den Empfängern an, wie mithilfe der Bücher und Quittungen aus dem Archiv der Gemeinde nachgewiesen werden kann. A D I N I S T O R Y 2 / 2 0 1 7 Jahrhunderts. Hatte sich gegen Ende des 18. Jahrhunderts die Zahl der in der Bruderschaft organi- sierten Händler auf ungefähr 100 bis 200 Personen be- laufen,54 so zählte die Gemeinde im Jahr 1900 nur noch 12 Mitglieder.55 Als Folge dieser Entwicklungen wurden immer weniger Legate für wohltätige Zwecke in den Hei- matorten gewidmet.56 Ein Großteil der von der Gemeinde zum Heiligen Georg verwalteten Stiftungen und Legate war für wohltätige Zwecke in Wien gedacht.57 92 Nathalie Patricia Soursos, Anna Ransmayr — Akteure im Dazwis Problematisch war jedoch weniger der Umgang mit diesen ›Stiftungen für auswärtige Zwecke‹, sondern die Grenzüberschreitung zwischen den griechisch-orthodo- xen Teilräumen innerhalb Wiens, als nämlich die Ge- meinde zum Heiligen Georg Stiftungen und Legate (klei- nere Zuwendungen durch letztwillige Verfügungen) von griechisch-orthodoxen habsburgischen Untertanen erhal- ten sollte. Der Handeltreibende Georg Johann von Karajan legierte 1811 500 Gulden für die Gemeinde,46 während der Hausinhaber Markus Darvar zwölf Jahre später 200 Gulden Conventionsmünze47 für die Kapelle zum Heiligen Georg legierte. Beides wurde von der niederösterreichi- schen Landesregierung mit der Begründung abgelehnt, dass die Kapitalien aus Stiftungen und Legaten dann in osmanisches Eigentum übergehen würden, was gesetz- lich nicht zulässig sei.48 Erst mit der Erweiterung der Privilegien durch Franz I. 1834 wurde die Annahme von Geschenken und Nachlässen von österreichischen Unter- tanen gestattet.49 In den folgenden Jahren gab es viele, die sowohl für die Gemeinde zum Heiligen Georg als auch für die Gemeinde zur Heiligen Dreifaltigkeit stifteten. Rechtlich wurde wiederholt die Autonomie der Ge- meinde zum Heiligen Georg festgelegt. Die Märzverfas- sung von 1849 sowie das Silvesterpatent 1851 bestätigten das Selbstverwaltungsrecht religiöser Gemeinden und damit auch die selbstständige Verwaltung der Stiftun- gen und Fonds der Gemeinde zum Heiligen Georg. Mit 92 A D I N I S T O R Y 2 / 2 0 1 7 hatten, und des Entstehens neuer politischer Räume in Form der Nationalstaaten am Balkan sowie der Republik Österreich. Familie je nach Staatsangehörigkeit Mitglieder der ei- nen oder der anderen Gemeinde. So bildeten die abge- grenzten Teilräume der beiden Gemeinden wiederum einen übergreifenden gemeinsamen Raum. Die griechische Gemeinde zur Heiligen Dreifaltig- keit hatte sich wenige Jahre nach dem von Joseph II. für die Protestanten und nicht unierten Griechen erlasse- nen Toleranzpatent von 1781 formiert. Sie errichtete 1787 eine Kirche am Alten Fleischmarkt, im Herzen des Viertels, in dem die griechischen Händler lebten und arbeiteten, durch die Griechengasse nur wenige Meter entfernt von der Kirche zum Heiligen Georg und den Gemeinderäumlichkeiten am Hafnersteig.74 Diese neue Gemeinde erhielt von Joseph II. ein Privilegium, das dem der Bruderschaft zum Heiligen Georg nachempfunden war.75 In den Texten der Privilegien für die beiden grie- chischen Gemeinden wurden aus ökonomischen Über- legungen die eng definierten Rechte überschritten, die akatholischen Konfessionellen sonst zugedacht waren. Nicht nur erhielten die Gemeinden eine weitgehende Autonomie in Bezug auf ihre Verwaltung und somit Un- abhängigkeit von kirchlichen Obrigkeiten, sie durften darüber hinaus auch Priester aus dem Osmanischen Reich berufen, also ausländische Staatsangehörige. Die bereits erwähnte Unterordnung unter den Metropoliten von Karlowitz war rein formaler Natur und gestattete ihm keinerlei Einmischung in die Gemeindeverwaltung. Besonders deutlich äußerte sich die Privilegierung der Gemeinde zur Heiligen Dreifaltigkeit in der entgegen den Bestimmungen des Toleranzpatents erteilten Er- laubnis zum Bau eines Glockenturms, der somit auch im physischen Wiener Stadtraum ein sichtbares Zeichen darstellte.76 Außerdem war der Gemeinde zur Heiligen Dreifaltigkeit sowohl der Besitz von Immobilien als auch der Erhalt von Stiftungen und Legaten gestattet. Ein Grund für die Privilegierung war, dass es sich bei den Mitgliedern um wohlhabende Händler handelte. Für die ökonomische Bedeutung der Griechen für den Habsbur- gerstaat spricht auch das finanzielle Volumen der Stif- tungen dieser Gemeinde. Aus einer Note des Hofagenten Joseph Hartl vom 12. März 1793 geht hervor, dass sie bereits kurz nach ihrer Gründung Stiftungen mit einem Gesamtkapital von über 150.000 Gulden besessen hat- te.77 Hartl wurde im Zuge der strittigen Frage der neu- erlichen Bestätigung der kaiserlichen Privilegien durch Franz II. um seine Meinung gebeten, wobei wohl auch A D I N I S T O R Y 2 / 2 0 1 7 Einzelne Stiftungen bargen jedoch durch wohl unbeabsichtigte Formulierungen in den Testamenten der Stifterinnen und Stifter ein Konfliktpotenzial, wie exemplarisch anhand der 1838 errichteten Stiftung des osmanischen Unterta- nen Demeter Betly für die Erhaltung dreier Kirchen in Kastoria sowie für die dortige Schule gezeigt werden kann.62 Betly hatte als Verwalter nicht die Gemeinde zum Heiligen Georg, sondern das k. k. Landrecht – den Gerichtshof für die Provinz Niederösterreich – bestimmt. Dieses wollte das Depositum jedoch nicht behalten und beabsichtigte das Kapital dem türkischen Konsulat aus- zuliefern.63 Das Kapital blieb nach Protesten der Ge- meinde zum Heiligen Georg und der Legatarin Juliana Betly, die inzwischen österreichische Untertanin gewor- den war, in Wien,64 wurde jedoch nicht der Gemeinde übertragen. 1912 wurde das Thema der Verwaltung der Stiftung erneut aufgegriffen. Man verwies seitens der Behörde auf Rudolf Herrmann von Herrnritts Erklä- rung, nach der »eine im Inland errichtete Stiftung, wel- che ausschließlich für ausländische Percipienten be- stimmt wäre, also die staatliche Verwaltungstätigkeit gewissermaßen in den Dienst ausländischer Interessen stellen würde, sogar als nicht annehmbar erscheinen«65 Österreich verpflichtet sich, alle Gegenstände, Werte oder Urkunden, die Staatsangehörigen der alliierten oder assoziierten Mächte gehört haben und etwa von den österreichischen Behörden zurückbehalten sind, unverzüglich nach Inkrafttreten des gegenwärtigen Vertrages zurückzustellen.69 Am 9. Januar 1921 wurde der Beschluss vom Bezirks gericht Innere Stadt Wien durchgeführt, das Vermögen der Demeter-Betly-Stiftung wurde der griechischen Re- gierung übergeben.70 Die Betly-Stiftung zeigt eine viel- schichtige Problematik der Grenzüberschreitung: Die habsburgischen Behörden wollten nicht für Stiftungen außerhalb des Staatsgebiets zuständig sein, während die Gemeinde zum Heiligen Georg die Übertragung in osmanische Verwaltung ablehnte. Dennoch entschloss sich die Statthalterei nicht zur Übertragung der Stiftungs- verwaltung an die Gemeinde. Erst durch die Inklusion des Empfängerortes in den griechischen Staat schien der Erhalt garantiert. Dieses Ende einer grenzüberschrei- tenden Stiftung zeigt beispielhaft die Auswirkungen des Untergangs der beiden Imperien – des Osmanischen Reichs und der Habsburgermonarchie –, welche die Le- benswelten der Wiener Griechen räumlich strukturiert 93 A D I N I S T O R Y 2 / 2 0 1 7 den erwünschten Umgang mit den Stiftungen. Aufgabe des Ausschusses war es demnach, die Zinsen aus dem angelegten Stiftungskapital zu kassieren und nach dem Willen der Stifterin oder des Stifters auszubezahlen.83 Ausdrücklich unterschieden wurden die Legate, Ver- mächtnisse und Stiftungsgelder von den Spenden und Beiträgen, den Sammlungen und Hauszinsen.84 Eine genauere Regelung wurde durch die zweite, 1861 verab- schiedete Geschäftsordnung der Gemeinde zur Heiligen Dreifaltigkeit geschaffen.85 In mehreren Paragrafen, je- doch in eher unstrukturierter Form, regelte sie die Ver- waltung von Stiftungen durch die Gemeinde.86 seine Funktion als Beisitzer der Wohltätigkeits-Hofkom- mission eine Rolle spielte.78 Zu Punkt 5 des Privilegiums, der die Anstellung von Seelsorgern aus dem Osmani- schen Reich betraf, nannte Hartl das Argument, dass ein solcher Seelsorger Anreiz für »die Uibersiedlung der vermöglichsten Familien«79 biete, welche sich dann auch als Stifter hervortäten: Nicht minder hat die gute Verfassung der hiesigen griechischen Kirche schon mehrere ihrer vermög- lichen Religionsgenossen bewogen, fromme Ver- mächtnisse für die Kirche und zum Besten der Armen zu machen, welche Vermächtnisse alle in hiesigen k. k. Banko für ewige Zeiten unaufkündlich gegeben wurden, und seit der kurzen Zeit des allhier geschehe- nen Kirchenbaues sich schon über 150000 f belaufen, und nächstens, wie es bereits bekannt ist, mit einer ansehnlichen Summe werden vermehret werden.80 Wie bereits dargelegt, finden sich schon wenige Jah- re nach der Verleihung von Privilegien an die beiden griechisch-orthodoxen Gemeinden in Wien Stiftungen und Legate. Die Stiftungskapitalien wurden angelegt und die Zinsen jährlich oder halbjährlich an Bedürftige im In- und Ausland ausbezahlt, was durch entsprechen- de Belege dokumentiert wurde. Arme Kranke wurden an das Allgemeine Krankenhaus, das Krankenhaus der Barmherzigen Brüder oder das Blindeninstitut ver- mittelt, wo Stiftungsbetten und Betreuungsplätze für griechisch-orthodoxe Arme vorhanden waren.87 Neben diesen von den beiden Gemeinden verwalteten waren manche Stiftungen von Griechisch-Orthodoxen auch unabhängig. Darunter fallen das Gros jener von Ni- colaus Dumba,88 die von Eugen Dusy von Laczkowa,89 Athanas Szekeres, Georg Poppovic sowie von Alexander Graf von Nako und Maria Karkaleky.90 Sollte aber der Privilegientext der Gemeinde zur Heiligen Dreifaltigkeit umgeändert werden, wie es der serbische Metropolit von Karlowitz, der seinen Einfluss auf die Ge- meinde zu vergrößern wünschte, forderte, dann würden diese Geldmittel dem Staat entgehen, so Hartl. Die Note verdeutlicht, dass das erhebliche Volumen der von der Gemeinde zur Heiligen Dreifaltigkeit verwalteten und in der Habsburgermonarchie angelegten Stiftungskapitali- en als politisches Argument genutzt werden konnte, um Gemeindeinteressen durchzusetzen. Ankommen im Raum. Griechisch- orthodoxe k. k. Untertanen Den Hintergrund für die durchaus ungewöhnliche Tat- sache, dass in Wien zwei separate Gemeinden der grie- chischen Balkanhändler gegründet wurden, bildete der vermehrte Wechsel der Staatsangehörigkeit durch grie- chische Händler ab dem letzten Viertel des 18. Jahrhun- derts.71 Diese Entwicklung wurde durch die josephini- sche Politik forciert, welche sich darum bemühte, dem habsburgischen Staat neue ›nützliche‹ Untertanen zu- zuführen, wie es die griechischen Händler waren. Das in der Politik der Bevölkerungsvermehrung begründete österreichische Staatsbürgerschaftsrecht72 war inklusiv und von einem territorialen Verständnis gekennzeichnet; osmanische Untertanen waren aufgrund des Prinzips der Reziprozität eindeutig schlechter gestellt. So gab es für griechische Händler aus dem Osmanischen Reich eine Reihe von gesetzlichen Vorteilen, wenn sie sich entschie- den, habsburgische Untertanen zu werden. Dazu zählte das Recht, Großhandlungs-, Fabriks- und andere Befug- nisse zu erhalten.73 Besonders hervorzuheben ist das Recht auf Immobilienerwerb, das nicht unierten Grie- chen durch das josephinische Toleranzpatent von 1781 gewährt wurde. Voraussetzung für die Verleihung der Staatsangehörigkeit war, dass der osmanische Handel- treibende seine Frau und seine Kinder nach Österreich brachte. Die Entscheidung für die osmanische oder die habsburgische Staatsangehörigkeit wurde zu einem Un- terscheidungsmerkmal, das die Identitäten der Wiener griechischen Händler signifikant prägte und durch das Bestehen von zwei getrennten Kirchengemeinden zur Formierung zweier Teilräume innerhalb der Griechisch- Orthodoxen in Wien führte. Während die Trennung dieser Teilräume jedoch auf der Ebene der Gemeinden streng befolgt wurde, wurde sie auf der Ebene der Han- delsfirmen und der familiären Netzwerke überschritten, indem häufig gemeinsame Unternehmen von osmani- schen und habsburgischen Untertanen gegründet wur- den, um von den Vorteilen beider Staatsangehörigkei- ten zu profitieren. Auch waren häufig Angehörige einer A D I N I S T O R Y 2 / 2 0 1 7 die Vererbung oder das Stiften eines solchen Hauses an die Gemeinde gewinnt durch den Zusammenhang von Haus und Translokalität zusätzlich an Bedeutung. Paradigmatisch für eine Verbindung zwischen dem Stifter und dem Stiftungshaus ist die Stiftung von Con- stantin Panadi, der in seinem Testament verfügte, dass ein Porträt von ihm und seiner Frau mit dem gestifte- ten Haus in der Beschäftigungsanstalt für erwachsene Blinde, dem Bestimmungsort seiner Stiftung, zu hängen habe.97 Weitere Möglichkeiten für ein solches Verorten im Raum waren die öffentliche Erinnerung an die Per- son des Stifters oder der Stifterin durch Gedenktafeln, wie sie im Eingangsbereich der beiden Kirchen bis heu- te angebracht sind, die Pflege der Grabstätten der Stifter am Friedhof zu St. Marx oder das religiöse Gedenken in jährlichen Seelenmessen. neben den Einkünften aus den Stiftungen und Legaten das ›Armenhaus‹ auf der Biberbastei Nr. 1175 (später Biberbastei 2, dann Dominikanerbastei 17). Dieses wur- de von Demeter Theocharides in seinem Testament von 1836 zu gleichen Teilen den Armen der Gemeinden zur Heiligen Dreifaltigkeit und zum Heiligen Georg gestiftet und bis zu seinem Verkauf an Katharina Kermpotich im März 1886 von der Gemeinde zur Heiligen Dreifaltigkeit verwaltet.93 Über den Kirchenfonds wurden auch die in Stiftungen vorgesehenen Seelenmessen administriert. Die dafür bestimmten Tage wurden in den letztwilligen Verfügungen oft so gewählt, dass sie mit dem Sterbe- tag der Stifterinnen und Stifter oder ihrer Verwandten zusammenfielen. Der Schulfonds bestritt die Ausgaben der Griechischen Nationalschule. Im Laufe des 19. Jahrhunderts rückte die Verwal- tung der Stiftungen vermehrt in den Mittelpunkt des Gemeindelebens, wofür verschiedene Gründe zu nen- nen sind. Einerseits war die Gefahr des Zugriffs auf die Besitzungen der Kirchengemeinde durch die 1873 gegründete Metropolie von Czernowitz, der sie infolge des Österreichisch-Ungarischen Ausgleichs seit 1883 an- stelle der Metropolie von Karlowitz formal unterstellt war,94 mit der Angst vor dem Verlust ihrer (finanziellen) Autonomie verknüpft. Andererseits nahm die Zahl der Stiftungen und Legate und damit der Verwaltungsauf- wand zu, insbesondere aufgrund von gestifteten Häu- sern. Die Tatsache, dass die Gemeinde zur Heiligen Drei- faltigkeit mehrere Stiftungshäuser zu verwalten hatte, stellt einen wesentlichen Unterschied zur Gemeinde der osmanischen Untertanen dar und illustriert das endgül- tige Ankommen derjenigen Griechen, die die habsburgi- sche Staatsangehörigkeit angenommen hatten, in Wien. A D I N I S T O R Y 2 / 2 0 1 7 So wurde das Privi- legium schließlich 1796 durch Franz II. bestätigt.81 Die von den Gemeinden verwalteten Kapitalien für wohltätige Zwecke wurden nach Zwecken gebündelt, ohne dass dabei die Kapitalanlagen zusammengelegt wurden. Auch blieben die Namen der Stifterinnen und Stifter in den Verwaltungsdokumenten erhalten. Die Ge- meinde zur Heiligen Dreifaltigkeit bündelte die Stiftun- gen und Legate in Fonds (›Armenfonds‹, ›Kirchenfonds‹, ›Schulfonds‹ und ›Fromme Stiftungen an Auswärtige‹), während die Gemeinde zum Heiligen Georg in solche für die Kirche und für Arme trennte und die weiteren nach Empfangsorten organisierte. Größere Stiftungen, wie die Heiratsausstattungs- und Stipendienstiftung von Ju- liana Betly, wurden separat gehandhabt.91 Ab wann die Einteilung in Fonds vorgenommen wurde, konnte nicht eindeutig festgestellt werden. Vermutlich aber wurde zunächst der Armenfonds mit der Stiftung von Brutus Edler von Zettiry gegründet.92 Dieser Fonds verwaltete 95 Nathalie Patricia Soursos, Anna Ransmayr — Ak Trotz der genannten Bedeutung von Stiftungen ent- hält der Text jedoch keinen Hinweis auf deren finanzielle Verwaltung. Stattdessen wird, wie bereits im Privilegium von Joseph II. (1787), betont, dass keine Schulden ge- macht werden sollten und die Almosen von den Epitro- pen (Kirchenvorstehern) zu verwalten seien. Die Kosten der Kirchenverwaltung und die Besoldung der Pfarrer sollten über freiwillige Almosen und, wenn nötig, durch Beiträge der Gemeindemitglieder bestritten werden. Die Lücken in Bezug auf die Regelung der Administration der Stiftungen der Gemeinde zur Heiligen Dreifaltigkeit wur- den im Laufe des 19. Jahrhunderts durch Statuten und Geschäftsordnungen geschlossen. Die am 1. April 1805 beschlossenen »Statuten der Griechisch-nicht unirten Kir- che sämmtlicher in Wien ansässigen Griechen, und Wa- lachen und k. k. Unterthanen«82 erklären unter Punkt 13 95 A D I N I S T O R Y 2 / 2 0 1 7 Die Anlage von Stiftungskapitalien in Immobilien zeigt, dass ethnische und religiöse Grenzen im Stiftungsver- halten keine Rolle spielten und die Stifterinnen und Stif- ter in ihrer Rolle als Wiener Bürgerinnen und Bürger handelten.95 Wichtig ist zu bedenken, dass sie durch die Verortung der Liegenschaften in Wien das Gedächtnis an ihre Person mit dem Raum verbanden. Nach Simone Derix »ist das gebaute Haus der Inbegriff lokaler Ver- ortung und Sesshaftigkeit und bildet vordergründig ei- nen Kontrapunkt zu Migration und mobilen Lebensfor- men«.96 Der Ankauf von unbeweglichem Vermögen, der per se ein Privileg war, das nur den habsburgischen Un- tertanen unter den Wiener Griechen gestattet war, und Weitgehend autonom agierte die von der Gemeinde zur Heiligen Dreifaltigkeit erhaltene Griechische Natio- nalschule, die 1804 durch ein Hofdekret legitimiert wur- de und bereits um die Zeit ihrer Gründung zahlreiche Stiftungen und Legate erhielt. Gestiftet wurden nicht nur Geldbeträge, sondern auch Bücher.98 Die Finanzierung der Schule basierte zunächst auf einer Stiftung des ein- gangs genannten Christoph von Nako und denen von Georg Johann von Karajan, doch erst mit dem Empfang des Hauses von Kyriak Polyzou konnte die Erhaltung der Schule nachhaltig gesichert werden. Neben der finanzi- ellen Verwaltung des mobilen Vermögens waren die Verwalter des Schulfonds auch für die Schulstiftungs- häuser zuständig. Es handelte sich dabei um Zinshäuser, die für die Griechische Nationalschule gestiftet worden waren und deren Erträge dem Schulfonds zugutekamen, nämlich die Häuser von Kyriak Polyzou in der Unteren Bäckerstraße Nr. 748 (später Sonnenfelsgasse 17/Schön- laterngasse 2) und von Anna Alexander in der Leopold stadt Nr. 512 (später Praterstraße 29/Komödiengasse 10). Das geerbte Haus in der Unteren Bäckerstraße be- reitete der Gemeinde und Nationalschule jedoch einige Schwierigkeiten. Als der griechische Handelsunterneh- mer Kyriak Polyzou am 17. Dezember 1811 in Wien ver- starb,99 ernannte er in Punkt 13 seines Testaments vom 29. September 1811 die Griechische Nationalschule zu seinem Universalerben und vermachte ihr damit auch sein Haus in der Unteren Bäckerstraße. Als Bedingung dafür legte er die »sichere Verwaltung und Anlegung die- ses Vermögens« durch die Kirchenvorsteher und seine 96 A D I N I S T O R Y 2 / 2 0 1 7 Abbildung 1: Titelbild des Privilegiums von Franz II./I. mit dem Schulfondhaus, 1822 (Quelle: Von Seiner Majestät Kaiser Franz des Zweyten, huldreichst verliehene Privile- gien, Wien 1822). A D I N I S T O R Y 2 / 2 0 1 7 »Verwendung zum Besten dieser Schule« fest.100 Der von den Behörden sanktionierte Unterricht konnte im Jahr 1811 aufgenommen werden,101 allerdings befand sich die Schule in den Folgejahren hinsichtlich der Finanzen und des pädagogischen Niveaus in einem schlechten Zustand.102 Erst als 1822 mit Abschluss der Verlassen- schaftsabhandlung von Kyriak Polyzou das Haus in der Bäckerstraße in den Besitz der Schule überging, verbes- serte sich zumindest die finanzielle Situation der Schule wesentlich.103 Das Haus wurde im Jahr 1822 auf einen Wert von 75.000 Gulden geschätzt.104 Die Mietzinsein- nahmen seiner neun, später elf Wohnungen kamen dem Schulfonds zugute (Abb. 1).105 Dieser Fall zeigt Unklarheiten in Bezug auf die for- male Definition von Stiftungen. Tatsächlich handelte es sich bei der Vererbung des Hauses an die Schule nicht um eine von Polyzou in seinem Testament errichtete Stiftung, sondern um ein Erbe mit einer Bedingung. Daher existiert in diesem Fall auch kein Stiftbrief. Den- noch wurde es in späteren Jahren auch als ›Schulstift- haus‹ bezeichnet. Diese unklare rechtliche Einordnung verkomplizierte um 1900 den Verkauf des Hauses, das über Jahre hinweg keine bedeutenden Mietzinserträge mehr eingebracht hatte,106 sodass das Anwesen 1908 für 200.000 Kronen an die Gemeinde Wien abgegeben wurde.107 Da es sich nicht um eine auf die Ewigkeit an- gelegte Stiftung handelte, war die Möglichkeit gegeben, die Immobilie zu veräußern. Trotzdem kam es im Zuge des Verkaufs zu rechtlichen Schwierigkeiten. Es wurde nicht nur bezweifelt, ob die Gemeinde wirklich berech- tigt sei, das Gebäude ohne behördliche Genehmigung zu veräußern, sondern es erwies sich auch als Problem, dass als Besitzerin nicht die Gemeinde, sondern die Grie- chische Nationalschule im Grundbuch eingetragen war. Dies warf wiederum die Frage auf, inwieweit es sich bei der Schule um eine eigenständige Körperschaft han- delte und ob die Gemeinde das in deren Besitz befind- liche Haus verkaufen durfte. Als 1920 die Griechische Nationalschule »infolge des Mangels an Schülern grie- chischer Nationalität« geschlossen wurde,108 bekämpfte ihr Direktor Eugen Zomarides die Schließung, die wohl auch mit der Verarmung der Gemeinde infolge des Ers- ten Weltkriegs zusammenhängende finanzielle Gründe hatte.109 In diesem Konnex warf er der Gemeinde vor, dass sie »ohne zwingenden Grund« das Schulfondshaus, »welches jetzt einen ungeheuren Wert hätte«, veräußert Abbildung 1: Titelbild des Privilegiums von Franz II./I. mit dem Schulfondhaus, 1822 (Quelle: Von Seiner Majestät Kaiser Franz des Zweyten, huldreichst verliehene Privile- gien, Wien 1822). A D I N I S T O R Y 2 / 2 0 1 7 Stiftungsempfänger zu leiden. So wurden Zinsen aus dem Stiftungskapital während der Balkankriege und während des Ersten Weltkriegs nur unregelmäßig abge- hoben. 1913 wurde aus der griechischen Schule in Doj ran, an die jährlich die Interessen zweier Stiftungen überwiesen wurden, ein Zollhaus. Der Bericht der Gene- ralversammlung der Gemeinde zur Heiligen Dreifaltig- keit bemerkte hierzu: zwar die Rechte der freien Religionsausübung, jedoch nicht der Verwaltungsautonomie bestimmt waren. Diese wurde im ersten Paragrafen der neuen Statuten festgelegt: §. 1. Die griechisch-orientalische Gemeinde »Zur heili- gen Dreifaltigkeit« in Wien ordnet und verwaltet ihre Kirchen-, Unterrichts- und Wohltätigkeits-Angelegen- heiten und die hiezu bestimmten Stiftungen und Fonds auf Grund der ihr mit Allerhöchstem Handschreiben vom 29. Jänner 1787 und vom 8. Oktober 1796, bezie- hungsweise mit k. k. Hofdekrete vom 19. Mai 1804 gewährten Privilegien selbständig.113 Zum ersten Male blieben die Zinsen einer Stiftung für die griechischen Schulen in Dogriani [Dojran] im Jahresbetrage von K 500,- unbehoben. Die Ursa- che mag in der von Serbien, an welchen Staat das in Mazedonien gelegene Dogriani gefallen ist, geübten Entnationalisierung liegen. Aus Zeitungen war zu ent- nehmen, daß aus der griechischen Schule in Dogriani ein serbisches Zollamt »gemacht« wurde. Für dieses serbische Zollamt wird selbstredend die griechische Schulstiftung nicht zu dienen haben und es wird uns obliegen, unter Mitwirkung der Behörden die Stiftungszinsen ihrem ursprünglichen oder tunlichst ähnlichen Zwecke zuzuführen.118 Dabei bezog man sich nicht auf das Staatsgrundgesetz von 1867 und die Autonomie der Kirchen, vielmehr auf die kaiserlichen Privilegien. In § 9 wurde außerdem ge- klärt, dass man außerordentliche Beiträge der Gemein- demitglieder heranziehen werde, sollten die Einnahmen aus den Almosen und Stiftungen nicht genügen.114 Wie lange diese Einnahmen ausreichten, um die Kosten der Gemeinde zu decken, zeigt sich darin, dass Kultusbei- träge erst in der Generalversammlung vom 9. April 1922 eingeführt wurden.115 98 Nathalie Patricia Soursos, Anna Ransmayr — Akteure im Dazwischen Die Gemeinde zur Heiligen Dreifaltigkeit fungierte nicht nur als Vermittlungsinstanz für in Wien verortete Stiftungen, sondern verwaltete ebenso wie ihre Schwes- tergemeinde zum Heiligen Georg auch Stiftungen für auswärtige Zwecke und war mit vergleichbaren Proble- men bei der grenzüberschreitenden Verschickung der Gelder konfrontiert. Die sogenannten ›Frommen Stiftun- gen an Auswärtige‹ wurden nahezu zeitgleich mit den ebenfalls für Zwecke im Osmanischen Reich gedachten Stiftungen der Gemeinde zum Heiligen Georg gegründet. A D I N I S T O R Y 2 / 2 0 1 7 Die Stiftungsinteressen gingen nach Ioannina, Argyro- kastro (Gjirokastra), Kronstadt (Brașov), Serres, Metsovo und Kastoria.116 Später wurden unter diesem Sammel- begriff auch Stiftungen der Familien Dumba, Curti und Vranyi, welche erst Ende des 19. Jahrhunderts errichtet wurden, verwaltet.117 Wie bei der Gemeinde zum Heiligen Georg ergaben sich auch in der Verwaltung der Stiftun- gen der Gemeinde zur Heiligen Dreifaltigkeit Schwierig- keiten durch die Verwirrung über die rechtliche Position um den neuen griechischen Staat sowie allgemein des- halb, weil viele der Empfängerorte nun in unterschied- lichen Staatsterritorien zu finden waren, die sich zudem durch Kriege weiter veränderten. Darunter hatten die Die Entscheidung über die Weiterführung der Schul- stiftung in Dojran wurde im Jahr darauf aufgrund der Kriegswirren verschoben, da die Zinsen für auswärtige Stiftungen nicht ausbezahlt werden konnten. Während bei der Gemeinde zum Heiligen Georg, wie anhand der Demeter-Betly-Stiftung gezeigt wurde, das Überschrei- ten der Grenzen in der Verwaltung der Stiftung prob- lematisch war, zeigt das Beispiel der Schulstiftung für Dojran die Auswirkungen auf den Fortbestand der Stif- tungen durch die Veränderung der Räume, in welchen sie wirkten. In den Kriegsjahren entstanden Komplikati- onen erstens beim Transfer der Gelder von auswärtigen Stiftungen, während durch die Grenzverschiebungen auf dem Balkan zweitens der Stiftungszweck, die grie- chische Schule in Dojran, nicht mehr erhalten blieb. A D I N I S T O R Y 2 / 2 0 1 7 habe.110 Tatsächlich hätte eine Renovierung, wozu die Gemeinde aber die Mittel nicht aufbringen konnte, an- stelle des Verkaufs den Bestand der Griechischen Natio- nalschule möglicherweise besser sichern können. Zu Anfang des 20. Jahrhunderts musste die Gemeinde zur Heiligen Dreifaltigkeit aufgrund von Streitigkeiten um die von ihr verwalteten Stiftungshäuser sowie wegen einzelner strittiger Stiftungen vor Gericht ihren Rechts- status neuerlich begründen. Die kaiserlichen Privilegien, auf welche sie sich dabei berief, trafen jedoch in Bezug auf die Stiftungen keine Aussage. So legte erst der Ent- wurf einer neuen Satzung im Jahr 1909 schließlich die Autonomie der Gemeinde in der Verwaltung der Stiftun- gen fest.111 Man ließ zudem vom Advokaten Nathaniel Thumim ein Gutachten erstellen, das als Grundlage für die neue Geschäftsordnung von 1909 diente.112 In der umfangreichen Stellungnahme werden zentrale Fragen der Stiftungsverwaltung angeführt. Die Schwierigkeiten der Gemeinde sah der Gutachter darin, dass in den Pri- vilegien, auf die sich die Gemeinde noch immer berief, A D I N I S T O R Y 2 / 2 0 1 7 Stiftungswesen im Zuge der Reform der Armenpflege, schuf 1785 die Stiftungsoberdirektion und übertrug das Stiftungswesen an die Landesstellen.125 Ansprechpartner für Wiener Stiftungen waren folglich auf Landesebene die niederösterreichische Statthalterei und auf Bezirks ebene der Wiener Magistrat sowie die Bezirksgerichte.126 In der zweiten Hälfte des 19. Jahrhunderts waren die griechisch-orthodoxen Stiftungen dem Ministerium für Kultus und Unterricht unterstellt.127 Gerichtlich vertreten wurden sie nach 1853 von der Finanzprokuratur. Auf- grund der behördlichen Kontrolle und durch manche Verzögerung bei der Verlassenschaftsabhandlung konn- ten zwischen der Willenserklärung der Stifterinnen oder Stifter und dem Beginn der Stiftung manchmal mehrere Jahre liegen. Problematisch ist hierbei, dass die Erforschung des Stif- tungswesens in Österreich einerseits aufgrund der kom- plizierten Rechtslage und andererseits aufgrund der fehlenden historischen Studien zur Stiftungsgeschichte erschwert wird.119 Die Rechtshistoriker sind sich einig, dass das österreichische Stiftungsrecht bis nach dem Zweiten Weltkrieg höchst lückenhaft und uneinheitlich war. Das Allgemeine Bürgerliche Gesetzbuch (ABGB) von 1811 unterschied in § 646 zwar Stiftungen von Substituti- onen und Fideikommissen, bot aber keine Klarheit über deren rechtliche Stellung oder Verwaltung. Stiftungen beziehen nach diesem Passus ihre »Einkünfte von Capi- talien, Grundstücken oder Rechten zu gemeinnützigen Anstalten« und widmen diese für »geistliche Pfründen, Schulen, Kranken- oder Armenhäuser; oder, zum Unter- halte gewisser Personen auf alle folgende Zeiten«.120 Neben dem ABGB sind Vorschriften über Stiftungen in den politischen Verordnungen zu finden, also in »unzäh- ligen Hofkanzleidekreten, Allerhöchsten Entschließun- gen, Ministerial- und Statthaltereierlässen, Reichs- und Landesgesetzen sowie anderen monarchischen Rechts- quellen«.121 Diese bestimmten, dass Stiftungskapitalien unveräußerlich sind und von einer Stifterin oder einem Stifter zu Lebzeiten oder durch eine letztwillige Verfü- gung vermacht werden. Die Erträgnisse sind zur Erfül- lung eines wohltätigen, gemeinnützigen oder ideellen Zwecks einzusetzen.122 Zentral sind außerdem die Selbst- ständigkeit des Vermögens sowie die Stellung der Stif- tung als juristische Person.123 Das Vertretungsorgan der Stiftung hatte die Aufgabe des Vollzugs, das heißt der Zu- schreibung der Stiftungserträge an die Empfänger, und der Kommunikation mit der staatlichen Aufsicht. Verorten und katalogisieren. Das Stiftungswesen in Österreich Die Verwaltung der Stiftungen Griechisch-Orthodoxer in Wien muss als ein Handeln innerhalb des Rechts- raumes der Habsburgermonarchie untersucht werden. 98 A D I N I S T O R Y 2 / 2 0 1 7 Gemeinde zur Heiligen Dreifaltigkeit angefertigt. Als Be- gründung wurde angegeben: beiden griechisch-orthodoxen Gemeinden und der nie- derösterreichischen Statthalterei herangezogen werden. Von staatlicher Seite wurden für den »Kataster der in Wien und Niederösterreich verwalteten Stiftungen« Informationen zu den Stiftungen gesammelt, auch bei den griechisch-orthodoxen Gemeinden. Die Grundlage dafür bildete die Kanzleireform des niederösterreichischen Statthalters Erich Graf Kielmansegg, die mit dem Erlass der niederösterreichischen Statthalterei vom 6. August 1893 veranlasst wurde.132 Im Zuge der Reform wurden zunächst Fragebögen an die Stiftungsverwalter geschickt. In einem zweiten Schritt wurde die im Statthaltereiarchiv aufbewahrte Original-Stiftbriefsammlung ergänzt, feh- lende Stiftbriefe wurden nachträglich erstellt.133 Erstes Ergebnis war eine statistische Übersicht, verfasst von Ferdinand Schmid und publiziert in der Zeitschrift der Statistischen Central-Commission.134 Zugleich wurden die baufälligen oder wenig ertragreichen Stiftungshäuser – davon gab es etwa 30 in Wien und Niederösterreich – zum Teil verkauft oder neu erbaut, wie etwa das Blindenins- titut, dessen Gebäude von dem Griechen Constantin Pa- nadi vermacht worden war.135 Schließlich wurden die gesammelten Informationen im »Kataster der in Wien und Niederösterreich verwalteten Stiftungen« aufgelis- tet. Dieser nennt 32 griechisch-orthodoxe Stifterinnen und Stifter. Die dort genannten Informationen beruhen auf zwei Listen: Die Gemeinde zur Heiligen Dreifaltig- keit übersandte einen ausgefüllten Fragebogen mit 25 Stiftungen, wovon zehn in den Kataster übernommen wurden, die Gemeinde zum Heiligen Georg wies in ei- nem Schreiben vom 12. April 1894 vierzehn Stiftungen aus, wovon acht eingetragen wurden. Mehrere von den beiden Gemeinden genannte Stiftungen wurden nicht im Kataster angeführt. Diese Diskrepanz hat verschie- dene Gründe: Erstens wurden im Kataster keine Seelen- messstiftungen angeführt,136 zweitens fehlen Stiftungen für auswärtige Zwecke, drittens wurden Fonds – nach der Definition des ABGB § 646 –, die als Stiftungen be- handelt worden waren, ausgeschieden. So zeigen die nachträglichen Durchstreichungen und die Vermerke »keine Stiftung« in den von der Gemeinde zur Heiligen Dreifaltigkeit eingesandten Fragebögen, dass die ange- führten »Diversi ohne Benennung« für den Armen-, Kirchen- und Schulfonds von der Statthalterei nicht als Stiftungen gewertet wurden.137 Am 26. Mai 1906 wurde ein Stiftbrief des Armenfonds von den Vorstehern der Es habe der Ausschuss der griechisch-orientalischen Kirchengemeinde zur heil. Dreifaltigkeit in Wien in seiner Sitzung am 22. A D I N I S T O R Y 2 / 2 0 1 7 Wiener Stiftungen wurden in Sammlungen aufgelis- tet, wie etwa der »Chronologisch-geschichtlichen Samm- lung« von Johann Nepomuk von Savageri (1832), dem »Handbuch der Wiener Stiftungen« von Carl Ferdinand Mautner Ritter von Markhof (1895) und dem »Kataster der in Wien und Niederösterreich verwalteten Stiftun- gen« der Statistischen Zentralkommission (1893).128 Au- ßerdem werden die Stiftbriefe der griechisch-orthodoxen Stifterinnen und Stifter im Niederösterreichischen Lan- desarchiv sowie in den Archiven der beiden griechisch- orthodoxen Gemeinden aufbewahrt.129 Was jedoch nach einer Recherche in den genannten Quellen auffällt, ist die Diskrepanz zwischen der Anzahl an Stiftungen, wel- che von den Gemeinden verwaltet wurden, und den in den Stiftungs-Sammlungen von Wien und Niederöster- reich angeführten griechisch-orthodoxen Stiftungen. Ein Grund hierfür ist die unklare Trennung zwischen Stiftun- gen und Legaten oder Spenden. So sind die diesbezügli- chen Listen, wie sie von den beiden Gemeinden geführt wurden, deutlich länger als jene der in den Sammlungen angeführten Stiftungen. In den Archiven der beiden Ge- meinden befinden sich für die Zeit zwischen 1785 und 1918 Quellen zu über 140 Wohltäterinnen und Wohl- tätern. Neben Stiftbriefen findet man ab der Mitte des 19. Jahrhunderts nahezu jährlich Ausweise130 und Über- sichten,131 außerdem Aufstellungen von Seelenmessen sowie Rechnungsbücher und Bilanzen zur finanziellen Verwaltung der Stiftungen. Es ist jedoch oft nicht einfach festzustellen, wann es sich um eine Stiftung handelt und wann wir es mit einem Legat oder einer Spende zu tun haben. Dazu muss die Kommunikation zwischen den 99 Nathalie Patricia Soursos, Anna Ransmay Eine solche staatliche Kontrolle beinhaltete neben der Genehmigung der Stiftung ein Aufsichtsrecht so- wie eine Schutzpflicht. »Der Staat«, so die Rechtshistorike- rin Gabriele Schneider, »soll Stiftungen vor allem Schutz bieten, da Stiftungen als verselbständigte Vermögens- massen, die der Verfolgung eines bestimmten Zweckes dienen, aufgrund ihrer Eigentümerlosigkeit sowie ihrer hohen sozialen Bedeutung naturgemäß besonderen Ge- fahren ausgesetzt sind«.124 Zur Sicherstellung des Schut- zes verstärkte sich die staatliche Überwachung seit dem 18. Jahrhundert, insbesondere mit der Gründung der Stiftungs-Hofkommission 1750, der Stiftungsbuchhaltung und Stiftungshauptkasse für Wien und Niederösterreich 1752 unter Maria Theresia. Joseph II. reformierte das 99 A D I N I S T O R Y 2 / 2 0 1 7 eine Gemeinde der osmanischen und eine Gemeinde der habsburgischen Untertanen zwei Teilräume, die jedoch miteinander kommunizierten und so einen gemeinsa- men übergreifenden Raum bildeten. Dies manifestierte sich in der Tatsache, dass häufig für die jeweils andere Gemeinde gestiftet wurde und Stiftungen für beide Ge- meinden errichtet wurden. Besonders deutlich zeigte sich der Aspekt der Grenzüberschreitung bei der Gemeinde zum Heiligen Georg, die sich aus osmanischen Unterta- nen zusammensetzte, im Hinblick auf ihren rechtlichen Status aber eigentlich eine Institution der Habsburger- monarchie darstellte. Insgesamt kann anhand dieser Stiftungs-Sammlungen konstatiert werden, dass die Trennung zwischen den bei- den griechisch-orthodoxen Gemeinden zwar die Verwal- tung der Stiftungen betraf, doch stifteten viele der ange- führten Wohltäterinnen und Wohltäter – gleichgültig ob habsburgische oder osmanische Untertanen – für beide Gemeinden.140 Ähnlich waren bereits bei den Listen von Spendern für die Kirchenneubauten 1787 zur Heiligen Dreifaltigkeit und 1803 zum Heiligen Georg Mitglieder der jeweils anderen Gemeinde angeführt worden.141 Die Stifter Demeter Theocharides, Constantin Bellio, Kyro Nicolitz, Juliana Betly, Cosmas Liveropulos und Theodor Dumba wurden 1893 in den Stiftungslisten beider Ge- meinden angeführt. Dies zeigt, dass sich die Griechen in Wien trotz der Spaltung in zwei durch die Staatsan- gehörigkeit getrennte Teilräume in einem gemeinsamen Raum bewegten. Dieser durch die Zugehörigkeit zur grie- chisch-orthodoxen Konfession definierte Raum war aber auch durchlässig zur ihn umgebenden Wiener bürgerli- chen Gesellschaft. So bedachten Bellio, Liveropulos und Dumba außerdem auch Institutionen und Einrichtungen außerhalb der beiden griechischen Gemeinden.142 Die Gemeinden in ihrer Rolle als Stiftungsverwalter fungierten als Vermittlungsinstanzen zwischen dem Willen der Stifterinnen und Stifter, der internen Ver- waltung, den habsburgischen Behörden und den Stif- tungsempfängern. Sie übernahmen die Funktion eines Zwischenraumes, der durch diese Position die jahrelange Verwaltung der Stiftungen überhaupt erst ermöglichte. Das vielseitige stiftungskulturelle Verhaltensmuster der Wiener Griechen verband die griechisch-orthodoxen Stifterinnen und Stifter durch ihr wohltätiges Handeln sowohl mit der Stadt Wien als auch mit den Stationen ihrer Handels- und Familiennetzwerke in Südosteuropa sowie ihren Heimatgemeinden. Stifterinnen und Stifter als handelnde Personen überschritten vielfach Grenzen zwischen verschiedenen Handlungsräumen. Nicht nur überquerten sie die physischen Grenzen zwischen dem Osmanischen Reich und der Habsburgermonarchie, sie wechselten auch die Staatsangehörigkeit und damit die Mitgliedschaft in den beiden Wiener griechischen Ge- meinden. Als Händler überschritten sie wiederum kon- fessionelle Grenzen, indem mit Orienthändlern anderer Konfessionen zusammengearbeitet wurde. Auch dies äußerte sich in den Empfängerinstitutionen, für die ge- stiftet wurde, wie sich exemplarisch an der Biografie des Christoph von Nako zeigt. A D I N I S T O R Y 2 / 2 0 1 7 Februar 1906 einhellig be- schlossen, für den aus Schenkungen ohne nähere Wid- mung und aus frei seitens Mitgliedern und Förderern dieser Gemeinde vermachten Legaten herrührenden Vermögensteil des Armenfonds [...] eine Stiftung zu dem Zwecke zu errichten, damit in gleicher Weise wie seit dem jeweiligen Anfalle dieses Vermögensteiles die Beteilung dürftiger griechisch-orientalischer Glau- bensgenossen aus den Erträgnissen desselben auch für alle Zeiten sichergestellt werden.138 Die Armenfonds-Stiftung wurde einen Monat später von der Statthalterei genehmigt. Stiftbriefe für den Kirchen- und den Schulfonds wurden nicht erstellt. Das Problem der Gemeinde zum Heiligen Georg bei der Bekanntgabe der Informationen für den »Kataster der Stiftungen« war dagegen, dass sie bei kleineren ge- spendeten Geldbeträgen für Messen oder für die Unter- stützung von Armen keine Stiftbriefe vorweisen konnte: In solchen Fällen übersenden oder überbringen dann die Hinterbliebenen statt des legierten Betrages zumeist schon die dafür angeschafften Staatsrenten. Ein Stiftbrief oder sonstige Wid- mungsurkunde wird nicht errichtet, deshalb sind wir auch nicht in der Lage die verlangten Wid- mungsurkunden hierunter anzulegen. Auch sind wir nicht im Stande nähere Details über die in den Fragebogen ausgewiesenen 14 Stiftungen auszuge- ben, da diese Stiftungen ungefähr vor 30–50 Jahren in der erwähnten Weise errichtet wurden. Die Legate werden bei der Staatsschuldencassa hinterlegt, Abschriften der Quittungen angefertigt; außer den Abschriften der Quittungen gibt es keine die Stiftungen betreffenden Urkunden bei der Gemeinde zum Hl. Georg.139 Die Argumente der Gemeindevorsteher wurden jedoch von der Statthalterei für nicht ausreichend befunden, und die fehlenden Stiftbriefe mussten nachträglich ver- fasst werden. A D I N I S T O R Y 2 / 2 0 1 7 Was mit den Stiftungen nach der letzten Erhebung von 1893 geschah und wie viele von den Gemeinden nach dem Ende der Habsburgermonarchie und dem Friedens- vertrag von Saint-Germain verwaltet wurden, ist ein For- schungsdesiderat. Einen Großteil ereilte, da die Gelder in Staatsanleihen angelegt waren, ein ähnliches Schicksal wie die anderen österreichischen Stiftungen, die mit der Wirtschaftskrise einen rapiden Wertverlust erlitten. Die Gemeinde zur Heiligen Dreifaltigkeit verwaltete zwar weiterhin die Fonds, doch war deren Kapital nahezu wertlos geworden.143 Stiftungsrechtlich war es erst 1925 erlaubt, Stiftungen, deren Stammvermögen weniger als 1000 Schilling ausmachte, mit anderen Stiftungen des gleichen Zwecks zusammenzufassen.144 Die räumliche Verortung in der Habsburgermonar- chie und insbesondere in der Stadt Wien zeigt sich in zahlreichen griechisch-orthodoxen Stiftungen für in Wien angesiedelte Institutionen. Die Stifterinnen und Stifter agierten in dieser Hinsicht als Angehörige des Wiener Bürgertums. Dabei spielten Stiftungshäuser eine zentrale Rolle, wie anhand des Hauses von Kyriak Polyzou veranschaulicht wurde. Tatsächlich stellten der Besitz und die Verwaltung von Stiftungshäusern den Conclusio Die Stiftungsaktivitäten der Wiener Griechen zeigen, wie selbige als Akteure innerhalb mehrerer Räume und Teil- räume handelten, wobei es zu vielfältigen Grenzüber- schreitungen kam. Bereits die beiden Gemeinden selbst, die als Vermittlungsinstanzen die Stiftungen verwalte- ten, bildeten aufgrund ihrer strengen Unterscheidung in Sekundärliteratur A n a g n o s t o p o u l o s , Constantin: »Geschichte einer längst ver- schwundenen griechischen Kolonie zu Chemnitz«, in: Hellas-Jahrbuch. Organ der Deutsch-Griechischen Gesellschaft und der Griechisch-Deut- schen Vereinigungen in Athen und Thessaloniki 6 (1940), S. 11–19. B a r t o n , Peter F.: »›Das‹ Toleranzpatent von 1781. Edition der wich- tigsten Fassungen«, in: Peter F. Barton (Hg.): Im Zeichen der Toleranz. Aufsätze zur Toleranzgesetzgebung des 18. Jahrhunderts in den Reichen Joseph II., ihren Voraussetzungen und ihren Folgen. Eine Festschrift, Wien 1981, S. 152–202. B e i n h a u e r , Gunter: »Das österreichische Stiftungsrecht«, in: Österreichische Juristen-Zeitung 27 (1972), S. 378– 382. B o r g o l t e , Michael: »Interkulturelle Perspektiven«, in: Michael Borgolte et al. (Hg.): Enzyklopädie des Stiftungswesens in mittelalterli- chen Gesellschaften, Bd. 1: Grundlagen, Berlin 2014, S. 19–23. B u r g e r , Hannelore: »Paßwesen und Staatsbürgerschaft«, in: Waltraud Heindl et al. (Hg.): Grenze und Staat. Paßwesen, Staatsbürgerschaft, Heimatrecht und Fremdengesetzgebung in der österreichischen Monarchie 1750–1867, Wien 2000, S. 1–172. C i c a n c i , Olga: »Les statuts et les règlements de fonctionnement des compagnies grecques de Transylvanie (1636–1746). La compagnie de Sibiu«, in: Revue des Études Sud-Est Européennes 14 (1976), S. 477–496. • »Le statut juridique et le régime de fonctionnement de la compagnie de commerce de Braşov«, in: Revue des Études Sud-Est Européennes 17 (1979), S. 241–255. D e r i x , Simone: »Haus und Trans- lokalität: Orte der Macht – Orte der Sehnsucht«, in: Joachim Eibach et al. (Hg.): Das Haus in der Geschichte Europas. Ein Handbuch, Berlin 2015, S. 589–604. 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V e s q u e v o n P ü t t l i n g e n , Johann: Die gesetzliche Behandlung der Ausländer in Oesterreich nach den daselbst gültigen Civilrechts-, Straf-, Commerzial-, Militär- und Polizei-Normen, nebst einer einleitenden Abhandlung über die österreichische Staatsbürgerschaft, Wien 1842. W u r z b a c h , Constant von: Biographisches Lexikon des Kaiserthums Österreich, enthaltend die Lebensskizzen der denkwürdi- gen Personen, welche seit 1750 in den österreichischen Kronländern geboren wurden oder darin gelebt und gewirkt haben, Bd. 7, Wien 1861. Allgemeines bürgerliches Gesetzbuch für die gesammten deutschen Erbländer der Oesterreichischen Monarchie, Wien 1811. • Bundesge- setzblatt für die Republik Österreich (1925, 1993). • Die Bauthätigkeit der k. k. niederösterreichischen Statthalterei als Verwalterin von Stiftungen von 1889 bis 1903, Wien 1904. • »Die griechische Schule und Kirche zu Wien«, in: Oesterreichische Zeitschrift für Geschichts- und Staatskunde 3 (1837), S. 370–371. • Die von Seiner Majestät dem römischen Kaiser Joseph dem II. denen in der kaiserl. Residenzstadt Wien handelnden, der ottomanischen Pforte unterthänigen nicht unirten Griechen, in Betreff ihres Gottesdienstes in der Kapelle des heil. Georgius im Steyerhof aller- gnädigst ertheilte Freyheit, Wien 1783. • »Επιστολή λογίου των εν Βιέννη προς τον εαυτού φίλον κύριον Φ. – Π. εις την 30. Σεπτεμβριου 1816« [Schreiben eines Wiener Intellektuellen an seinen Freund, Herrn F. P., am 30. September 1816], in: Ερμής ο Λόγιος 20 (15. 10. 1816), S. 355– 362. • Griechisch-orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rechnungs- abschlüsse (1887, 1900, 1914, 1922, 1926). • Κανονισμός των εργασιών δια την έκκλητον της Ελληνοβλαχικής των Κ.Β. υπηκόων Κοινότητος της Αγ. Τριάδος συντεθειμένος εκ των από του έτους 1786 εν τοις αρχείοις της Κοινότητος εγκατατεθειμένων αποφάσεων. Επικεκυρωμένος εν τη γενική συνελεύσει την 8/20. Ιανουαρίου 1861. Geschäftsordnung für den Ausschuss der Gemeinde k. k. Unterthanen griechisch nicht unirten Glaubensbekenntnisses zur heiligen Dreifaltigkeit in Wien, zusammen- gestellt aus den in den Acten der Gemeinde seit dem Jahre 1786 nieder- gelegten Beschlüssen derselben. Genehmigt in der Generalversamm- lung der Gemeinde am 8/20. Jänner 1861, Wien 1861. • Kataster der in Niederösterreich verwalteten weltlichen Stiftungen nach dem Stande des Jahres 1893, Wien 1898. • Reichs-Gesetz-Blatt für das Kaiserthum Oesterreich (1856, 1864, 1867). Publikationen mit Quellencharakter • Sammlung der Gesetze für das Erzher- zogthum Oesterreich unter der Ens, Bd. 16: Jahr 1834, Wien 1837. • Sammlung der Kaiserlich-Königlichen Landesfürstlichen Gesetze und Verordnungen in Publico-Ecclesiasticis vom Jahre 1782 bis 1783, Bd. 2, Wien 1784. • »1699 I 26 Friede von Karlowitz. Kaiser, Osmanisches Reich«, in: Europäische Friedensverträge der Vormoderne online, online unter: http://www.ieg-friedensvertraege.de/treaty/1699%20I%2026%20 Friede%20von%20Karlowitz/t-428-1-de.html?h=1 (8. 9. 2017). • Staatsge- setzblatt für die Republik Österreich (1920). • Von Seiner Majestät Kaiser Franz des Zweyten, huldreichst verliehene Privilegien, denen in der k. k. Haupt- und Residenz-Stadt Wien ansässigen Griechen und Wallachen von der orientalischen Religion, k. k. Unterthanen, in Betreff ihres Gottes- dienstes in der Pfarrkirche zur heiligen Dreyfaltigkeit am alten Fleisch- markt, Wien 1822. H e r r m a n n v o n H e r r n r i t t , Rudolf: Das österreichische Stiftungsrecht. Mit Berücksichtigung der ausländischen Gesetzgebung und mit Benützung amtlicher Quellen dargestellt, Wien 1896. K i e l m a n s e g g , Erich von: Kaiserhaus, Staatsmänner und Politi- ker. Aufzeichnungen des k. k. Statthalters, hg. von Walter Goldinger, Wien 1966. M a u t n e r v o n M a r k h o f , Carl Ferdinand / G u g l i a , Eugen: Die Wiener Stiftungen. Ein Handbuch, Wien 1895. M a y r h o f e r , Ernst: Handbuch für den politischen Verwaltungsdienst in den im Reichsrathe vertretenen Königreichen und Ländern mit besonderer Berücksichtigung der diesen Ländern gemeinsamen Gesetze und Verordnungen, Bd. 3, Wien 41881. N o r a d o u n g h i a n , Gabriel: Recueil d’actes internatio- naux de l’Empire Ottoman. Traités, conventions, arrangements, déclara- tions, protocoles, procès-verbaux, firmans, bérats, lettres patentes et autres documents relatifs au droit public extérieur de la Turquie, Bd. 1, Paris 1897. S a v a g e r i , Johann Nepomuk von: Chronologisch-geschicht- liche Sammlung aller bestehenden Stiftungen, Institute, – öffentlichen Erziehungs- und Unterrichts-Anstalten der k. k. österreichischen Monar- chie mit Ausnahme von Italien, Bd. 1, Brno 1832. S c h m i d , Ferdinand: A D I N I S T O R Y 2 / 2 0 1 7 »Statistik der in Nieder-Oesterreich verwalteten Stiftungen nach dem Stande vom December 1893«, in: Statistische Monatsschrift 23 (1897), S. 205–301. V e s q u e v o n P ü t t l i n g e n , Johann: Die gesetzliche Behandlung der Ausländer in Oesterreich nach den daselbst gültigen Civilrechts-, Straf-, Commerzial-, Militär- und Polizei-Normen, nebst einer einleitenden Abhandlung über die österreichische Staatsbürgerschaft, Wien 1842. W u r z b a c h , Constant von: Biographisches Lexikon des Kaiserthums Österreich, enthaltend die Lebensskizzen der denkwürdi- gen Personen, welche seit 1750 in den österreichischen Kronländern geboren wurden oder darin gelebt und gewirkt haben, Bd. 7, Wien 1861. A D I N I S T O R Y 2 / 2 0 1 7 auch die Auszahlung von Stiftungsgeldern von Griechen, die wie Peter Darvar und Georg Johann von Karajan habsburgische Untertanen geworden waren, an die Ge- meinde zum Heiligen Georg verhindert, da das Kapital dadurch in osmanischen Besitz übergegangen wäre. Demeter Betly dagegen hatte das Landrecht mit der Verwaltung der Stiftung beauftragt, einer Aufgabe, der sich diese Gerichtsbehörde nicht gewachsen sah, wes- wegen eine Überweisung des Stiftungsvermögens an die osmanischen Behörden in Betracht gezogen wurde. Die Gemeinden spielten also bei den vielfältigen Grenzüber- schreitungen, die die Stiftungsverwaltung notwendig machte, eine entscheidende Vermittlerrolle. Die Insta- bilität der davon betroffenen geopolitischen Räume am Balkan wirkte sich jedoch weniger auf die Stiftungen aus, als man vermuten könnte, da die Heimatregionen der Wiener Griechen erst spät vom Osmanischen Reich abgetrennt wurden. So zeigten sich die Stiftungen, so- lange die beiden Imperien Osmanisches Reich und Habsburgermonarchie noch existierten, erstaunlich sta- bil. Erst mit dem Ende des Ersten Weltkriegs und dem Untergang der Habsburgermonarchie fand auch die Verwaltung der auswärtigen Stiftungen und damit die Grenzüberschreitung der Gelder ihr Ende. zentralen Unterschied zwischen den beiden Gemeinden dar, da der Erwerb von Immobilien nur habsburgischen Untertanen erlaubt war. Diese waren konkrete Mani- festationen des dauerhaften Ankommens der aus dem Osmanischen Reich stammenden Griechen in Wien. Das zeigt sich auch darin, dass die Probleme in der Verwal- tung der Stiftungshäuser und der Wertverfall dieser Besitztümer, die schließlich zum Verkauf führten, sich nicht von anderen Wiener Stiftungshäusern unterschie- den, darunter jene, die von der niederösterreichischen Statthalterei verwaltet wurden. 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Österreichische Wirtschaftsgeschichte vom Mittelalter bis zur Gegenwart, Wien 1995. S a r a c i n o , Stefano: »Wit- wen als Stifterinnen in den Wiener griechischen Gemeinden während des 19. Jahrhunderts«, in: Archiv für Kulturgeschichte 98 (2016), S. 315–357. A D I N I S T O R Y 2 / 2 0 1 7 5 Christoph von Nako trug gemeinsam mit mehreren anderen aus Ma- kedonien stammenden Griechen zur Entwicklung der Textilindustrie in Chemnitz bei; vgl. Constantin Anagnostopoulos: »Geschichte ei- ner längst verschwundenen griechischen Kolonie zu Chemnitz«, in: Hellas-Jahrbuch. Organ der Deutsch-Griechischen Gesellschaft und der Griechisch-Deutschen Vereinigungen in Athen und Thessaloniki 6 (1940), S. 11–19, hier S. 14. 17 Ioannis Zelepos: »Kulturtransfer und europäische Identität. Zur Bedeutung des Griechischen im vornationalen Südosteuropa«, in: Christian Voß / Wolfgang Dahmen (Hg.): Babel Balkan? Politische und soziokulturelle Kontexte von Sprache in Südosteuropa, München 2014, S. 19–28. 6 NÖLA, GSbS, Kt. 94, Griechische Schule; AHD, S 2, Fasz. 16, Testa- ment Christoph Nako. 6 NÖLA, GSbS, Kt. 94, Griechische Schule; AHD, S 2, Fasz. 16, Testa- ment Christoph Nako. 7 Dieter Hein: »Das Stiftungswesen als Instrument bürgerlichen Han- delns im 19. Jahrhundert«, in: Bernhard Kirchgässner / Hans-Peter Becht (Hg.): Stadt und Mäzenatentum. 33. Arbeitstagung 1994, Sig- maringen 1997, S. 75–92. 7 Dieter Hein: »Das Stiftungswesen als Instrument bürgerlichen Han- delns im 19. Jahrhundert«, in: Bernhard Kirchgässner / Hans-Peter Becht (Hg.): Stadt und Mäzenatentum. 33. Arbeitstagung 1994, Sig- maringen 1997, S. 75–92. 18 Roman Sandgruber: Ökonomie und Politik. Österreichische Wirt- schaftsgeschichte vom Mittelalter bis zur Gegenwart, Wien 1995, S. 179–184; Olga Katsiardi-Hering: Τεχνίτες και τεχνικές βαφής νημάτων: από τη Θεσσαλία στην Κεντρική Ευρώπη (18ος – αρχές 19ου αι.). Επίμετρο: Η Αμπελακιώτικη συντροφιά (1805) [Handwer- ker und Techniken der Garnfärbung: von Thessalien nach Mitteleu- ropa (18. – Anfang 19. Jh.). Anhang: Die ambelakiotische Kompanie (1805)], Athina 2003, S. 53–95; Andrea Komlosy: »Einleitung«, in: Leopoldine Hokr: Groß Siegharts – Schwechat – Waidhofen/Thaya. Das Netzwerk der frühen niederösterreichischen Baumwollindustrie, Frankfurt am Main 32007, S. 15–39. 8 Im Rahmen des FWF-Projekts AP2714021 »Soziales Engagement in den Wiener griechischen Gemeinden (18. – 20. Jh.)« unter der Lei- tung von Maria A. Stassinopoulou konnten 2014 bis 2017 in den Archiven der griechisch-orthodoxen Gemeinden zum Hl. Georg und zur Hl. Dreifaltigkeit in Wien sowie im Wiener Stadt- und Landesar- chiv, dem Niederösterreichischen Landesarchiv und dem Österrei- chischen Staatsarchiv über 260 Stiftbriefe und mehr als 220 Testa- mente in den Sprachen Deutsch, Griechisch, Latein und Italienisch katalogisiert und zu einem großen Teil transkribiert werden. 19 Olga Katsiardi-Hering: »Αδελφότητα, κομπανία, κοινότητα. Για μια τυπολογία των ελληνικών κοινοτήτων της κεντρικής Ευρώπης, με αφορμή το άγνωστο καταστατικό του Miskolc (1801)« [Bruder- schaft, Kompanie, Gemeinde. A D I N I S T O R Y 2 / 2 0 1 7 Zu einer Typologie der griechischen Gemeinden von Mitteleuropa, aus Anlass der unbekannten Statuten von Miskolc (1801)], in: Εώα και εσπέρια 7 (2007), S. 247–310. 9 Michael Borgolte baut in der Bezeichnung von Stiftungen als »to- tales soziales Phänomen« auf der Mauss’schen Beschreibung des Gabentausches auf; vgl. Michael Borgolte: »Interkulturelle Perspek- tiven«, in: Michael Borgolte et al. (Hg.): Enzyklopädie des Stiftungs- wesens in mittelalterlichen Gesellschaften, Bd. 1: Grundlagen, Berlin 2014, S. 19–23, hier S. 22. 20 Österreichisches Staatsarchiv (Wien) [ÖStA], Kriegsarchiv, Zentral- stellen, Wiener Hofkriegsrat, Hauptreihe Bücher, Bd. 581, fol. 844; ediert bei Ion I. Nistor: »Bisericile şi şcoala greco-română din Vi- ena«, in: Analele Academiei Române. Memoriile Secţiunii Istorice 3. Serie 3 (1932), S. 69–108, hier S. 98. Allerdings ist das Datum nicht wie bei Nistor angegeben der 9. 5., sondern der 9. 6. 1726. 10 Zur Geschichte der Wiener Griechen allgemein Vassiliki Seirinidou: Έλληνες στη Βιέννη (18ος – μέσα 19ου αιώνα) [Griechen in Wien (18. – Mitte 19. Jahrhundert)], Athina 2011. 11 Die Umbenennung erfolgte mittels Verordnung vom 29. 11. 1864: Reichs-Gesetz-Blatt für das Kaiserthum Oesterreich (1864), S. 307. 12 Anna Ransmayr: ›Untertanen des Sultans oder des Kaisers‹. Struktur und Organisationsformen der beiden Wiener griechischen Gemein- den von den Anfängen im 18. Jahrhundert bis 1918, unpublizierte Dissertation, Universität Wien 2016, S. 376. 21 Ransmayr: Untertanen, S. 29–46. 21 Ransmayr: Untertanen, S. 29–46. 22 Archiv der griechischen Gemeinde zum Hl. Georg (Wien) [AHG], G 1, Fasz. 1, Privilegium, 3. 3. 1776; Text bei Willibald M. Plöchl: Die Wiener Orthodoxen Griechen. Eine Studie zur Rechts- und Kulturgeschichte der Kirchengemeinden zum Hl. Georg und zur Hl. Dreifaltigkeit und zur Errichtung der Metropolis von Austria, Wien 1983, S. 133–136. 13 Text bei Gabriel Noradounghian: Recueil d’actes internationaux de l’Empire Ottoman. Traités, conventions, arrangements, déclarations, protocoles, procès-verbaux, firmans, bérats, lettres patentes et au- tres documents relatifs au droit public extérieur de la Turquie, Bd. 1, Paris 1897, S. 182–193. Digitalisate einer zeitnahen Abschrift: »1699 I 26 Friede von Karlowitz. Kaiser, Osmanisches Reich«, in: Europäi- sche Friedensverträge der Vormoderne online, online unter: http:// www.ieg-friedensvertraege.de/treaty/1699%20I%2026%20Frie- de%20von%20Karlowitz/t-428-1-de.html?h=1 (8. 9. 2017). 23 Eine dreisprachige Ausgabe des Privilegientextes wurde 1783 ge- druckt: Die von Seiner Majestät dem römischen Kaiser Joseph dem II. denen in der kaiserl. Residenzstadt Wien handelnden, der ottoma- nischen Pforte unterthänigen nicht unirten Griechen, in Betreff ihres Gottesdienstes in der Kapelle des heil. Georgius im Steyerhof aller- gnädigst ertheilte Freyheit, Wien 1783. A D I N I S T O R Y 2 / 2 0 1 7 Teil: Kopitars Briefwechsel mit Karl Georg Rumy, München 1942. Z e l e p o s , Ioannis: »Kulturtransfer und europäische Identität. Zur Bedeutung des Griechischen im vornationalen Südosteu- ropa«, in: Christian Voß / Wolfgang Dahmen (Hg.): Babel Balkan? Politische und soziokulturelle Kontexte von Sprache in Südosteuropa, München 2014, S. 19–28. A D I N I S T O R Y 2 / 2 0 1 7 1 Niederösterreichisches Landesarchiv [NÖLA], Landesfürstliche Ver- waltung Selekte, Geistliche Stiftbriefsammlung [GSbS], Kt. 94, Griechi- sche Schule; Archiv der griechischen Gemeinde zur Hl. Dreifaltigkeit (Wien) [AHD], S 2, Fasz. 16, Testament Christoph Nako, Wien, 24. 8. 1799; Kodizill Christoph Nako, Pest, 2. 12. 1800. 1 Niederösterreichisches Landesarchiv [NÖLA], Landesfürstliche Ver- waltung Selekte, Geistliche Stiftbriefsammlung [GSbS], Kt. 94, Griechi- sche Schule; Archiv der griechischen Gemeinde zur Hl. Dreifaltigkeit (Wien) [AHD], S 2, Fasz. 16, Testament Christoph Nako, Wien, 24. 8. 1799; Kodizill Christoph Nako, Pest, 2. 12. 1800. 1718, West Lafayette 2011, S. 53–62; Jovan Pešalj: »Making a Pros- perous Peace: Habsburg Diplomacy and Economic Policy at Passa- rowitz«, in: Charles Ingrao / Nikola Samardžić / Jovan Pešalj (Hg.): The Peace of Passarowitz, 1718, West Lafayette 2011, S. 141–157. 15 Vassiliki Seirinidou: »›Griechischer Handelsmann‹: Anatomizing a Collective Subject«, in: Herbert Kröll (Hg.): Austrian-Greek Encounters over the Centuries. History, Diplomacy, Politics, Arts, Economics, Innsbruck 2007, S. 129–137; Traian Stoianovich: »The Conquering Balkan Orthodox Merchant«, in: The Journal of Economic History 20 (1960), S. 234–313. 2 Maximilian Demeter Peyfuss: »Aromanian Landlords in the Banat around 1800«, in: Revista istorică 14 (2003), S. 59–82, hier S. 77–78. 2 Maximilian Demeter Peyfuss: »Aromanian Landlords in the Banat around 1800«, in: Revista istorică 14 (2003), S. 59–82, hier S. 77–78. 3 Hans Haas: Das Adelsgeschlecht der Nákó de Nagy-Szentmiklós. Auf- stieg und Niedergang einer Grafendynastie, Reşiţa 2011, S. 15–16. 3 Hans Haas: Das Adelsgeschlecht der Nákó de Nagy-Szentmiklós. Auf- stieg und Niedergang einer Grafendynastie, Reşiţa 2011, S. 15–16. g g g y ş 4 NÖLA, GSbS, Kt. 94, Griechische Schule; AHD, S 2, Fasz. 16, Testa- ment Christoph Nako. 16 Bei den Aromunen (in den zeitgenössischen Quellen als ›Wallachen‹ bezeichnet) handelt es sich um Sprecher einer nicht kodifizierten, mit dem Rumänischen verwandten romanischen Sprache auf dem Gebiet der südlichen Balkanhalbinsel; vgl. Maximilian Demeter Pey- fuss: Die Aromunische Frage. Ihre Entwicklung von den Ursprüngen bis zum Frieden von Bukarest (1913) und die Haltung Österreich- Ungarns, Wien 1974. 5 Christoph von Nako trug gemeinsam mit mehreren anderen aus Ma- kedonien stammenden Griechen zur Entwicklung der Textilindustrie in Chemnitz bei; vgl. Constantin Anagnostopoulos: »Geschichte ei- ner längst verschwundenen griechischen Kolonie zu Chemnitz«, in: Hellas-Jahrbuch. Organ der Deutsch-Griechischen Gesellschaft und der Griechisch-Deutschen Vereinigungen in Athen und Thessaloniki 6 (1940), S. 11–19, hier S. 14. A D I N I S T O R Y 2 / 2 0 1 7 39 Otto Stammer: Handbuch des österreichischen Stiftungs- und Fondswesens, Eisenstadt 1983; Ernst Mayrhofer: Handbuch für den politischen Verwaltungsdienst in den im Reichsrathe vertretenen Königreichen und Ländern mit besonderer Berücksichtigung der diesen Ländern gemeinsamen Gesetze und Verordnungen, Bd. 3, Wien 41881, S. 74. Verordnungen in Publico-Ecclesiasticis vom Jahre 1782 bis 1783, Bd. 2, Wien 1784, S. 100–104. 26 Sofronios Efstratiadis: Ο εν Βιέννη ναός του Aγίου Γεωργίου και η κοινότης των οθωμανών υπηκόων [Die Kirche zum Hl. Georg in Wien und die Gemeinde der osmanischen Untertanen], hg. von Cha- ralambos Chotzakoglou, Athina 21997, S. 70. 40 Der Text des josephinischen Privilegiums von 1782 weicht in den Punkten zur Verwaltung von Geldern kaum vom Privilegium Maria Theresias ab. In der Folge wurde das Privilegium von Leopold II. am 30. 11. 1791 und Franz II. am 10. 1. 1794 ohne Änderung des Textes bestätigt. 27 Efstratiadis: Ο εν Βιέννη ναός του Aγίου Γεωργίου, S. 175, 180, 197. 28 Katsiardi-Hering: »Αδελφότητα«, S. 267; Despoina-Eirini Tsourka-Pa- pastathi: Η ελληνική εμπορική κομπανία του Σιμπίου Τρανσυλβανίας 1636–1848. Οργάνωση και δίκαιο [Die griechische Handelskompanie von Sibiu in Transsylvanien 1636–1848. Organisation und Recht], Thessaloniki 1994; Olga Cicanci: »Le statut juridique et le régime de fonctionnement de la compagnie de commerce de Braşov«, in: Revue des Études Sud-Est Européennes 17 (1979), S. 241–255; Olga Cicanci: »Les statuts et les règlements de fonctionnement des compagnies grecques de Transylvanie (1636–1746). La compagnie de Sibiu«, in: Revue des Études Sud-Est Européennes 14 (1976), S. 477–496. 41 AHG, G 1, Fasz. 1, Schreiben der Gemeinde und Bruderschaft der Kapelle zum Hl. Georg an die zur Untersuchung der Rechnungen der Kapelle eingesetzte Hofkommission, 9. 12. 1771; ediert bei Rans- mayr: Untertanen, S. 381–383. 42 AHG, G 32, Fasz. 23, Stiftbrief Demeter Sava, 25. 7. 1785. Mit ›Er- lösung eines Sklaven‹ war der Freikauf von Griechisch-Orthodoxen aus osmanischer Gefangenschaft gemeint. 29 Im Protokoll der Sitzung vom 24. 1. 1815 wurde auch schriftlich fest- gehalten, dass die Dodekas für die Erledigung vorkommender Han- delsangelegenheiten zuständig sei; Efstratiadis: Ο εν Βιέννη ναός του Aγίου Γεωργίου, S. 183–184. A D I N I S T O R Y 2 / 2 0 1 7 43 Es handelt sich um die Stiftungen von Demeter Sava, von Pano Zot- to für die Schule und Heiratsgut für ein armes Mädchen in Përmet (Stiftbrief 1792, 44.500 Gulden), von Georg Haggi Nicola zur »nötigen Instruktion armer Kinder und verwaister Mädchen« in Melnik (Stift- brief 1797, 7820 Gulden), von Panno Cazzaro für Arme in Ioannina (Stiftbrief 1799, 6500 Gulden), von Johann Consta für die Schule und für Arme in Metsovo (Stiftbrief 1801, 10.600 Gulden), von Iliades Manasses für die Schule und Arme in Melnik (Stiftbrief 1806, 3500 Gulden), von Georg Consta für die Erziehung unmündiger Kinder, für Arme in Ioannina, für die Schule und Arme sowie für Seelenmessen in Kapesovo (Stiftbrief 1808, 10.300 Gulden) sowie von Kyro Nicolitz für Arme und Seelenmessen in Dojran (Testament 1844). 30 Ransmayr: Untertanen, S. 50. 31 Michael Ursinus: »Zur Diskussion um ›millet‹ im Osmanischen Reich«, in: Südost-Forschungen. Internationale Zeitschrift für Geschichte, Kultur und Landeskunde Südosteuropas 48 (1989), S. 195–207. 32 Maria A. Stassinopoulou: »Endowments as Instruments of Integra- tion and Memory in an Urban Environment: The Panadi Building in Vienna«, in: Olga Katsiardi-Hering / Maria A. Stassinopoulou (Hg.): Across the Danube. Southeastern Europeans and Their Travelling Identities (17th – 19th C.), Leiden 2017, S. 171–190, hier S. 174–175. 44 Mit Ausnahme der Stiftbriefe von Panno Cazzaro (1799) und Kyro Nicolitz (1844) enthalten alle Stiftungen für Auswärtige der Gemein- den zum Hl. Georg und zur Hl. Dreifaltigkeit einen solchen Passus. 33 In diesem Jahr wurde die Metropolie von Siebenbürgen mit Sitz in Hermannstadt (Sibiu) gegründet; vgl. Ekkehard Kraft: »Autokephalie«, in: Edgar Hösch et al. (Hg.): Lexikon zur Geschichte Südosteuropas, Wien 2004, S. 77. 45 ÖStA, AVA, AK AGO, Kt. 9, Schreiben von Reichsrat Karl Ignaz von Dvorak, 18. 10. 1804. 34 Es handelt sich dabei um eine Gottesdienststätte nach den Vorga- ben des Toleranzpatents Josephs II. von 1781, das heißt eine Kirche ohne Glockenturm und Eingangsportal, bei der die Außenfassade einem gewöhnlichen Zinshaus gleichen musste; vgl. Peter F. Barton: »›Das‹ Toleranzpatent von 1781. Edition der wichtigsten Fassungen«, in: Peter F. Barton (Hg.): Im Zeichen der Toleranz. Aufsätze zur To- leranzgesetzgebung des 18. Jahrhunderts in den Reichen Joseph II., ihren Voraussetzungen und ihren Folgen. Eine Festschrift, Wien 1981, S. 152–202, hier S. 168–170. 46 Georg Johann von Karajan stiftete in seinem Testament von 1811 für die Griechische Schule und für Arme in Kozani jeweils 2000 Gulden. Je 500 Gulden gingen an die Gemeinde zur Hl. A D I N I S T O R Y 2 / 2 0 1 7 14 Texte bei Noradounghian: Recueil, Bd. 1, S. 208–227. Zu den Verträ- gen vgl. Harald Heppner / Daniela Schanes: »The Impact of the Trea ty of Passarowitz on the Habsburg Monarchy«, in: Charles Ingrao / Nikola Samardžić / Jovan Pešalj (Hg.): The Peace of Passarowitz, 24 Plöchl: Griechen, S. 30–31. 25 Die Abschaffung der Bruderschaften und der Einzug ihres Vermögens sowie der Stiftungen erfolgten mit der Verordnung vom 9. 8. 1783: Sammlung der Kaiserlich-Königlichen Landesfürstlichen Gesetze und 25 Die Abschaffung der Bruderschaften und der Einzug ihres Vermögens sowie der Stiftungen erfolgten mit der Verordnung vom 9. 8. 1783: Sammlung der Kaiserlich-Königlichen Landesfürstlichen Gesetze und 25 Die Abschaffung der Bruderschaften und der Einzug ihres Vermögens sowie der Stiftungen erfolgten mit der Verordnung vom 9. 8. 1783: Sammlung der Kaiserlich-Königlichen Landesfürstlichen Gesetze und 105 105 A D I N I S T O R Y 2 / 2 0 1 7 Festschrift für Werner Ogris zum 75. Geburtstag, Wien 2010, S. 459–476, hier S. 474. 78 Joseph Hartl (1760–1822), ab 1799 Edler von Luchsenstein, war 1802 einer der Hauptinitiatoren der Pottendorfer Spinnfabrik; vgl. Constant von Wurzbach: Biographisches Lexikon des Kaiserthums Österreich, enthaltend die Lebensskizzen der denkwürdigen Personen, welche seit 1750 in den österreichischen Kronländern geboren wurden oder darin gelebt und gewirkt haben, Bd. 7, Wien 1861, S. 405–406, s. v. Hartl Edler von Luchsenstein, Joseph. Die Pottendorfer Spinnfabrik ist auch in Verbindung mit der Stiftung von Zenobius Constantin Popp von Böhmstetten für die Krankenkasse der Arbeiter ebenjener Fabrik (Stiftbrief 1867) relevant. Ab 1811 war Hartl außerdem Unter- direktor der k. k. oktroyierten Kommerzial-, Leih- und Wechselbank, die auch Geschäfte mit griechischen Handelsleuten machte; vgl. Herbert Matis: Die Schwarzenberg-Bank. Kapitalbildung und Industriefinan- zierung in den habsburgischen Erblanden 1787–1830, Wien 2005, S. 210. Die Wohltätigkeits-Hofkommission wurde durch Entschlie- ßung vom 20. 9. 1784 unter dem Vorsitz des Grafen Buquoy ins Le- ben gerufen und war zuständig für das Stiftungs- und Armenwesen in Wien; vgl. Gabriele Schneider: »Zu den Anfängen der staatlichen Stiftungsaufsicht in Österreich«, in: Thomas Olechowski / Christian Neschwara / Alina Lengauer (Hg.): Grundlagen der österreichischen Rechtskultur. Festschrift für Werner Ogris zum 75. Geburtstag, Wien 2010, S. 459–476, hier S. 474. 57 Die Stiftungen und Legate der Wiener Griechen wurden katalogisiert und als Liste auf der Projekthomepage des FWF-Projekts AP2714021 publiziert: Stefano Saracino / Nathalie Patricia Soursos / Maria A. Stassinopoulou: »Liste der Stifterinnen und Stifter der griechi- schen Gemeinden zum Hl. Georg und zur Hl. Dreifaltigkeit in Wien (1769–1918)«, in: Soziales Engagement in den Wiener griechischen Gemeinden (18. – 20. Jh.), online unter: http://wienergriechen.uni- vie.ac.at/die-stiftungen-der-wiener-griechen/liste-der-stifterinnen- und-stifter/ (11.4.2017). opp von Böhmstetten für die Krankenkasse der Arbeiter ebenjener 58 Reichs-Gesetz-Blatt für das Kaiserthum Oesterreich (1867), S. 394– 396, hier S. 396. 59 Ransmayr: Untertanen, S. 225; Plöchl: Griechen, S. 80. 60 Text der Geschäftsordnung bei Plöchl: Griechen, S. 147–152, hier S. 147. 61 Zitiert nach Plöchl: Griechen, S. 149. Die Gemeinde zum Hl. Georg orientierte sich mit dieser Geschäftsordnung inhaltlich an derjeni- gen der Gemeinde zur Hl. Dreifaltigkeit aus dem Jahr 1901. 79 62 AHG, G 29, Fasz. 3, Testaments-Auszug von Demeter Betly, 29. 3. 1838. 79 NÖLA, NÖReg, C-Akten, C 21 (de 1796), Kt. 424, Nr. 3431, Note des Joseph Hartl; zitiert nach Ransmayr: Untertanen, S. 407. Zum Ver- mögen der Wiener Griechen (1750–1850) anhand des jeweiligen Ver- lassenschaftsvermögens vgl. A D I N I S T O R Y 2 / 2 0 1 7 Vassiliki Seirinidou: »Greek Migration in Vienna (18th – First Half of the 19th Century): A Success Story?«, in: Olga Katsiardi-Hering / Maria A. Stassinopoulou (Hg.): Across the Danube. Southeastern Europeans and Their Travelling Identities (17th – 19th C.), Leiden 2017, S. 113–134. 63 AHG, G 29, Fasz. 2, Schreiben ohne Datum. 64 Man nahm an, das Legat für Juliana Betly nur über eine österreichi- sche Verwaltung gewährleisten zu können: ÖStA, AVA, Neuer Kultus, Akatholisch Griechisch-orientalisch [NK AGO], Kt. 27, A23–25, Schrei ben vom 10. 6. 1914. 65 Rudolf Herrmann von Herrnritt: Das österreichische Stiftungsrecht. Mit Berücksichtigung der ausländischen Gesetzgebung und mit Be- nützung amtlicher Quellen dargestellt, Wien 1896, S. 182. 80 NÖLA, NÖReg, C-Akten, C 21 (de 1796), Kt. 424, Nr. 3431, Note des Joseph Hartl; zitiert nach Ransmayr: Untertanen, S. 407. 66 ÖStA, AVA, NK AGO, Kt. 27, A23–25, Schreiben des Ministeriums für Kultus und Unterricht, 5. 11. 1912; vgl. Ransmayr: Untertanen, S. 232. 81 Ransmayr: Untertanen, S. 101; Text des Privilegiums bei Plöchl: Griechen, S. 137–140. 82 Ransmayr: Untertanen, S. 125–134, 457–474. 67 Ransmayr: Untertanen, S. 183–190. 67 Ransmayr: Untertanen, S. 183–190. 68 Ransmayr: Untertanen, S. 225–234. 83 Ransmayr: Untertanen, S. 465: »Dass der Ausschuß die von denen Stiftbriefen verfallene Instruction, dann die Kirchen-Capitall-Inter- essen, endlich auch die mit Testamenten, und anderen dazu gehörigen Documenten versehene Legate, Vermächtnisse, fromme Stiftungs- Gelder, und Stiftbriefs-Capitalien gehörig gegen Quittungen einkas- siren. Das von den Stiftbriefen einkassirte Geld, es sey Interessen oder Capitall, muß der Ausschuss ebenfalls gegen Quittungen, nach dem Inhalte der Stiftbriefe, auszahlen, vorhero aber die für die Kirche bestimmte Provision, welche sie den Epitropen zu übergeben ha- ben, abziehen. Jenes von kirchen Capitalls-Interessen, von Legaten, Vermächtnissen, frommen Stiftungen x. eingegangene Geld, muß der Ausschuß, wenn dies Gelder zu Bestreitung der Kirchenausga- ben nothwendig sind, denen Epitropen gegen Quittung übergeben, außerdem muß der Ausschuß diese Gelder alsogleich pragmatika- lisch sicherstellen, und die darüber erhaltene Urkunde ad cassam deponiren. Die auszustellenden Quittungen über Geldempfänge 69 Staatsgesetzblatt für die Republik Österreich (1920), S. 995–1245, hier S. 1077. 70 AHG, G 29, Fasz. 3, Beschluss des Bezirksgerichts Innere Stadt Wien, 9. 2. 1921. 71 Ransmayr: Untertanen, S. 65–75. 72 Für einen ausführlichen Überblick vgl. Hannelore Burger: »Paßwe- sen und Staatsbürgerschaft«, in: Waltraud Heindl et al. (Hg.): Grenze und Staat. Paßwesen, Staatsbürgerschaft, Heimatrecht und Frem- dengesetzgebung in der österreichischen Monarchie 1750–1867, Wien 2000, S. 1–172. 73 Vesque von Püttlingen: Behandlung, S. 240. A D I N I S T O R Y 2 / 2 0 1 7 Dreifaltigkeit und an die Gemeinde zum Hl. Georg; Album der Familie Karajan, Privatbe- sitz Angelina Fritzsche, Testament Georg Johann von Karajan, 29. 1. 1811. 47 Markus Darvar vermachte in seinem Testament von 1823 der Ge- meinde zum Hl. Georg ein Legat von 200 Gulden Conventionsmün- ze, der Gemeinde zur Hl. Dreifaltigkeit und der Griechischen Natio- nalschule je 400 Gulden; Wiener Stadt- und Landesarchiv [WStLA], Zivilgericht [ZG], A 10 Testamente, 541/1829, Testament Marcus Darvar, 12. 1. 1823. 35 Der Antrag der Gemeinde zum Heiligen Georg wurde am 5. 5. 1804 vom Kaiser abgewiesen: ÖStA, Allgemeines Verwaltungsarchiv [AVA], Alter Kultus, Akatholisch Griechisch-Orthodox [AK AGO], Kt. 4, Vor- trag der vereinigten Hofkanzlei wegen eines Hausankaufs der nicht unierten Griechen zu ihrer Kirche, 7. 12. 1803. 48 AHG, G 6, Fasz. 16, Schreiben der niederösterreichischen Landesre- gierung an den Ausschuss der Gemeinde zum Hl. Georg, 15. 6. 1825. 36 Martin P. Schennach: »Der ›Österreicher‹ als Rechtskonstrukt? Zur Formierung einer österreichischen Staatsbürgerschaft in der ersten Hälfte des 19. Jahrhunderts«, in: Zeitschrift für Neuere Rechtsge- schichte 33 (2011), S. 152–176, hier S. 165–166; Johann Vesque von Püttlingen: Die gesetzliche Behandlung der Ausländer in Oester- reich nach den daselbst gültigen Civilrechts-, Straf-, Commerzial-, Militär- und Polizei-Normen, nebst einer einleitenden Abhandlung über die österreichische Staatsbürgerschaft, Wien 1842, S. 63. 49 Hofkanzleidekret mit dem Titel »Bewilligung für die türkischen Un- terthanen griechischer Religion in Wien zum eigenthümlichen Besit- ze des Hauses, wo ihre Kirche sich befindet und zur Annahme von Legaten und Geschenken für diese Kirche«, 11. 1. 1834, in: Samm- lung der Gesetze für das Erzherzogthum Oesterreich unter der Ens, Bd. 16: Jahr 1834, Wien 1837, S. 52–53. 50 AHD, G 40, Fasz. 5, Stiftbrief Demeter Theocharides, 5. 11. 184 51 Hofkanzleidekret vom 30. 10. 1832; vgl. Vesque von Püttlingen: Be- handlung, S. 232. 37 Efstratiadis: Ο εν Βιέννη ναός του Aγίου Γεωργίου, S. 199; vgl. AHG, G 6, Fasz. 17, Schreiben der niederösterreichischen Landesregie- rung an die Vorsteher der Gemeinde zum Hl. Georg, 7. 12. 1833. 52 Reichs-Gesetz-Blatt für das Kaiserthum Oesterreich (1856), S. 579– 582. 38 Ransmayr: Untertanen, S. 232–233. 53 Vesque von Püttlingen: Behandlung, S. 263. A D I N I S T O R Y 2 / 2 0 1 7 Danube. Southeastern Europeans and Their Travelling Identities (17th – 19th C.), Leiden 2017, S. 135–170, hier S. 147–157. 54 Ransmayr: Untertanen, S. 279–280; Polychronis K. Enepekides: Grie- chische Handelsgesellschaften und Kaufleute in Wien aus dem Jahre 1766 (ein Konskriptionsbuch). Aus den Beständen des Wiener Haus-, Hof- und Staatsarchivs, Thessaloniki 1959. 75 Text bei Plöchl: Griechen, S. 137–140. 76 Ransmayr: »Greek Presence«, S. 155; Ransmayr: Untertanen, S. 96– 100. 55 Ransmayr: Untertanen, S. 225. 56 Als Legate gelten hier auch die Widmungen laut den Testamenten von Demeter Betly (1838, für drei Kirchen in Kastoria), Soterios Antoniades (1839, für das Gefängnis und die griechische Schule in Plovdiv), Kyro Nicolitz (1844, für Arme und Seelenmessen in Doj ran), Constantin Tsatsapa (1844, für Schulen und Arme in Kastoria), Anastasios Pallatides (1845/48, für Melnik), Thomas Konstantin Chucha (1853, für eine jährliche Seelenmesse in Siatista sowie für die Heiratsausstattung zweier armer Mädchen), da bei diesen kein Stiftbrief gefunden werden konnte. Einzig für die Stiftung von Georg Dumovits für Arme in Brașov wurde ein Stiftbrief (1866) ausgestellt. 77 NÖLA, Niederösterreichische Regierung [NÖReg], C-Akten, C 21 (de 1796), Kt. 424, Nr. 3431, Note des k. k. Hofagenten Joseph Hartl bezüglich der Bestätigung der Privilegien für die griechisch nicht unierte Gemeinde zur Hl. Dreifaltigkeit, 12. 3. 1793; ediert bei Rans- mayr: Untertanen, S. 403–409. 78 Joseph Hartl (1760–1822), ab 1799 Edler von Luchsenstein, war 1802 einer der Hauptinitiatoren der Pottendorfer Spinnfabrik; vgl. Constant von Wurzbach: Biographisches Lexikon des Kaiserthums Österreich, enthaltend die Lebensskizzen der denkwürdigen Personen, welche seit 1750 in den österreichischen Kronländern geboren wurden oder darin gelebt und gewirkt haben, Bd. 7, Wien 1861, S. 405–406, s. v. Hartl Edler von Luchsenstein, Joseph. Die Pottendorfer Spinnfabrik ist auch in Verbindung mit der Stiftung von Zenobius Constantin Popp von Böhmstetten für die Krankenkasse der Arbeiter ebenjener Fabrik (Stiftbrief 1867) relevant. Ab 1811 war Hartl außerdem Unter- direktor der k. k. oktroyierten Kommerzial-, Leih- und Wechselbank, die auch Geschäfte mit griechischen Handelsleuten machte; vgl. Herbert Matis: Die Schwarzenberg-Bank. Kapitalbildung und Industriefinan- zierung in den habsburgischen Erblanden 1787–1830, Wien 2005, S. 210. Die Wohltätigkeits-Hofkommission wurde durch Entschlie- ßung vom 20. 9. 1784 unter dem Vorsitz des Grafen Buquoy ins Le- ben gerufen und war zuständig für das Stiftungs- und Armenwesen in Wien; vgl. Gabriele Schneider: »Zu den Anfängen der staatlichen Stiftungsaufsicht in Österreich«, in: Thomas Olechowski / Christian Neschwara / Alina Lengauer (Hg.): Grundlagen der österreichischen Rechtskultur. A D I N I S T O R Y 2 / 2 0 1 7 86 So wurde dem Ausschuss übertragen, die Verwaltung der gestifteten Häuser durch aus seiner Mitte gewählte Häuser-Administratoren vor- nehmen zu lassen (§§ 25–29). Weiters wurde ihm die Oberaufsicht über den Schul-, Kirchen- und Armenfonds übertragen (§§ 30–35, 134–138). Dabei wurden namentlich die Krankenbett-Stiftungen von Freiherrn von Bellio und Demeter Laso und die Panadi’sche Stiftung für Blinde (§ 35) sowie die Regina Rogotti’sche Stiftung für Arme (§ 138) genannt. Die von der Gemeinde zur Hl. Dreifaltigkeit betriebe- ne griechische Nationalschule wurde gesondert behandelt und den Schulvorstehern (Ephoren) die Verwaltung des Schulfonds und die Administration der Schulstiftungshäuser übertragen (§§ 75–78, 89). 96 Simone Derix: »Haus und Translokalität: Orte der Macht – Orte der Sehnsucht«, in: Joachim Eibach et al. (Hg.): Das Haus in der Geschich- te Europas. Ein Handbuch, Berlin 2015, S. 589–604, hier S. 589. 97 Stassinopoulou: »Endowments«, S. 182–183. 98 Bei der Stiftung von Peter Darvar handelte es sich um eine solche Schulbuchstiftung, die insbesondere »den Druck griechischer Schul- bücher, welche mein Bruder Demeter Nicolaus Darvar verfasst, und in der Schule verwendet werden soll[en]«, veranlasste; WStLA, ZG, F2 Verlassenschaftsabhandlungen, 2583/1838, Stiftbrief Peter Darvar, 25. 4. 1863. Weitere Schulbuchstiftungen und -legate hinterließen Ge- org Simon Sina in der Höhe von 1000 Gulden für Schulbücher, die am Sterbetag der Gattin Katharina Sina zu verteilen waren, und Demeter Theocharides; AHD, G 40, Fasz. 5, Stiftbrief Georg Sina, 31. 8. 1853; WStLA, ZG, A 10 Testamente, 68/1836, Testament Demeter Theochari- des, 2. 1. 1836. Zur Bücherspende von Dr. Johann Nicolides von Pindo vgl. »Die griechische Schule und Kirche zu Wien«, in: Oesterreichische Zeitschrift für Geschichts- und Staatskunde 3 (1837), S. 370–371. 87 Nathalie Patricia Soursos: »Die Stiftungsbetten der Wiener Griechen für das Allgemeine Krankenhaus und das Spital der Barmherzigen Brüder«, in: Virus. Beiträge zur Sozialgeschichte der Medizin 16 (2017), S. 169–191. 88 Es handelt sich um die nach seinem Tod 1900 gegründeten Stiftun- gen von Nicolaus Dumba für den Wiener Männergesangsverein und für den Pensionsfonds der Genossenschaft für bildende Künstler sowie den Pensionsfonds des Journalisten- und Schriftstellervereins Concordia, die Stipendienstiftung für die Gesellschaft der Musik- freunde sowie die Preisstiftungen für die Wiener Künstlergenossen- schaft, die Akademie der bildenden Künste, die Genossenschaft für bildende Künste und das Künstlerhaus in Wien. 99 99 AHG, Matrikenbuch 1777–1843, S. 201, 4. 12. 1811 [alten Stils; 17. 12. 1811]; Wiener Zeitung (14. 3. 1812). Interessant ist die Tatsache, dass sich der Sterbeeintrag in den Matriken der Gemeinde zum Hl. A D I N I S T O R Y 2 / 2 0 1 7 93 WStLA, ZG, A 10 Testamente, 68/1836, Testament Demeter Theocha- rides, 2. 1. 1836; Kodizill desselben, 14. 1. 1836. Theocharides war ab 1784 k. k. Untertan; Edition der Urkunde über die Annahme der Staatsangehörigkeit bei Ransmayr: Untertanen, S. 75. Er starb 1836 unverheiratet und kinderlos. Die Stiftung trug den Namen »Stiftung für griechisch-orthodoxe Arme zur Errettung der Seele und Erinne- rung an Demeter Theocharides«. Zum Verkauf des Hauses wurde im Bericht der Generalversammlung der Gemeinde angegeben, es sei für 983,78 Gulden von Katharina Kermpotich gekauft worden; Griechisch-orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rech- nungsabschlüsse (1887). Das Haus wurde kurz darauf von der Ge- meinde Wien gekauft, abgerissen und durch einen Neubau ersetzt; Paul Harrer: Wien, seine Häuser, Menschen und Kultur, Bd. 4/1, Wien 21954, S. 125. müssen von allen 6. Ausschüssen mit Beidrückung des Gemeinde Sigills (von welchen, da solches aus 6. Stücken zusammengesetzt ist, jeden Ausschußmann eines bekommen muss) unterschrieben seyn«. müssen von allen 6. Ausschüssen mit Beidrückung des Gemeinde Sigills (von welchen, da solches aus 6. Stücken zusammengesetzt ist, jeden Ausschußmann eines bekommen muss) unterschrieben seyn«. müssen von allen 6. Ausschüssen mit Beidrückung des Gemeinde Sigills (von welchen, da solches aus 6. Stücken zusammengesetzt ist, jeden Ausschußmann eines bekommen muss) unterschrieben seyn«. 84 Eine klare Unterscheidung zwischen Stiftungen, Fonds und Spenden ist nicht immer einwandfrei möglich. Im Allgemeinen handelt es sich bei Spenden um einmalige Zahlungen, Stiftungen dagegen hatten eine ewige Zinsausschüttung aus einem angelegten Kapital zum Ziel. 85 Κανονισμός των εργασιών δια την έκκλητον της Ελληνοβλαχικής των Κ.Β. υπηκόων Κοινότητος της Αγ. Τριάδος συντεθειμένος εκ των από του έτους 1786 εν τοις αρχείοις της Κοινότητος εγκατα- τεθειμένων αποφάσεων. Επικεκυρωμένος εν τη γενική συνελεύσει την 8/20. Ιανουαρίου 1861. Geschäftsordnung für den Ausschuss der Gemeinde k. k. Unterthanen griechisch nicht unirten Glaubens- bekenntnisses zur heiligen Dreifaltigkeit in Wien, zusammengestellt aus den in den Acten der Gemeinde seit dem Jahre 1786 niederge- legten Beschlüssen derselben. Genehmigt in der Generalversamm- lung der Gemeinde am 8/20. Jänner 1861, Wien 1861. 94 Ransmayr: Untertanen, S. 190–197. 94 Ransmayr: Untertanen, S. 190–197. 95 Zu den Immobilienstiftungen der Wiener Griechen vgl. Nathalie Patricia Soursos: »Financial Management of Donations, Founda- tions and Endowments in the Greek Communities in Vienna (1800– 1918)«, in: Endowment Studies 2 (2017) [im Druck]. A D I N I S T O R Y 2 / 2 0 1 7 74 Zur räumlichen Verteilung der Wiener Griechen sowie zu den von ihnen bewohnten und besessenen Häusern in Wien vgl. Anna Rans- mayr: »Greek Presence in Habsburg Vienna: Heyday and Decline«, in: Olga Katsiardi-Hering / Maria A. Stassinopoulou (Hg.): Across the 107 107 A D I N I S T O R Y 2 / 2 0 1 7 109 Bereits im Jahr 1919 drohte die Schließung der Schule, da sie aus Brennholzmangel nicht geheizt werden konnte; AHD, S 6, Fasz. 9, Schreiben der Gemeinde zur Hl. Dreifaltigkeit an den Magistrat Wien, 1. 10. 1919. 110 AHD, S 8, Fasz. 1, Schreiben von Zomarides an den Ausschuss der Ge- meinde zur Hl. Dreifaltigkeit, 1. 12. 1920; vgl. Maria A. Stassinopoulou: »Habe nun Philologie studiert, und dann? Philologische Karrieren und Diaspora-Schulen am Beispiel des Eugen Zomarides«, in: Kirillos Ka- terelos et al. (Hg.): Σκεύος εις τιμήν. Festschrift zum 25-jährigen Ju- biläum der Bischofsweihe und 20-jährigen Jubiläum der Inthronisation zum Metropoliten von Austria und Exarchen von Ungarn und Mittel- europa Dr. Michael Staikos, Athina 2011, S. 787–794, hier S. 794. 127 Stammer: Handbuch, S. 283. 128 Kataster der in Niederösterreich verwalteten weltlichen Stiftungen nach dem Stande des Jahres 1893, Wien 1898; Mautner von Mark- hof / Guglia: Die Wiener Stiftungen; Johann Nepomuk von Savageri: Chronologisch-geschichtliche Sammlung aller bestehenden Stiftun- gen, Institute, – öffentlichen Erziehungs- und Unterrichts-Anstalten der k. k. österreichischen Monarchie mit Ausnahme von Italien, Bd. 1, Brno 1832. 111 Ransmayr: Untertanen, S. 204–205 112 Ransmayr: Untertanen, S. 208. 113 Ransmayr: Untertanen, S. 591. 114 Ransmayr: Untertanen, S. 593. 115 Griechisch-orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rech- nungsabschlüsse (1922). 129 Außer den letztwilligen Verfügungen und den Stiftbriefen wurden jedoch viele stiftungsrelevante Akten skartiert, weswegen trotz der guten Quellenlage eine Unterscheidung, bei welchen in einem Testament angeführten Legaten es sich um Stiftungen handelt, oft nicht eindeutig möglich ist. 116 Hierunter fallen die Stiftungen von Johann und Anastas Argyri Vreto für Ioannina (Stiftbrief 1792), mehrere Stiftungen von Panajot Haggi Nico für Ioannina und Kronstadt (Brașov) (Stiftbrief 1792 und 1797), mehrere Stiftungen von Johann Consta für Serres und Metsovo (Stift- brief 1801), die Stiftungen von Christoph Constantin für Argyrokastro (Gjirokastra) (Stiftbrief 1793), Georg Consta für Ioannina (Stiftbrief 1793), Demeter Petru für Kronstadt (Brașov) (Stiftbrief 1794), Deme- ter Pauli für Ioannina (Stiftbrief 1795) und die Stiftungen von Johann Emanuel und Johann Argyri für Kastoria (Stiftbrief 1801 und 1802). 130 »Ausweise« wurden in den Jahren zwischen 1860 und 1871 ange- fertigt. Dem ersten Ausweis von 1861 ging ein Schreiben voraus, in dem die niederösterreichische Statthalterei einen Ausweis über die »Umschreibung der Obligationen der Argieri [Johann Argiri], [Jo- hann] Consta, Demeter Paul[i], [Johann und Anastas] Argiri Vreta und Panagiotti Haggi Nico’schen Stiftungen« verlangte: AHD, G 18, Fasz. A D I N I S T O R Y 2 / 2 0 1 7 104 AHD, S 2, Fasz. 15, Finalausweisung der Legate von Kyriak Polyzou, 27. 4. 1822. 120 Allgemeines bürgerliches Gesetzbuch für die gesammten deutschen Erbländer der Oesterreichischen Monarchie, Bd. 2, Wien 1811, S. 131. 105 AHD, G 28, Bücher-Beschreibung und Zins-Ertrags-Bekenntnis des Hauses Nr. 748 alt/Nr. 21 in der Stadt, 1824–1846 und 1846–1904; AHD, G 31–G 35. 121 Beinhauer: »Stiftungsrecht«, S. 379. 122 Erst durch das Inkrafttreten des Privatstiftungsgesetzes von 1993 wurde die Gründung rechtsfähiger Stiftungen zur Verfolgung jedes erlaubten Zweckes ohne staatliche Aufsicht möglich; Privatstiftungs- gesetz vom 14. 10. 1993, Bundesgesetzblatt für die Republik Öster- reich (1993), Nr. 694/1993. 106 In den Berichten der Generalversammlung der Gemeinde zur Hl. Drei- faltigkeit werden die leerstehenden Wohnungen mehrfach themati- siert. Der Bericht der Generalversammlung vom 19. 3. 1900 enthält zudem die Erklärung: »Der heutige Wohnungszug nach den Periphe- rien aus jenen engen, dumpfen Stadtteilen, in welchen dieses Haus liegt, die unentschiedene Regulierungsfrage für dieselben und andere Umstände verringern die Nachfrage zur Miethung«. Dennoch war man sich gewiss, dass dem Stadtteil ein hoher Zukunftswert innewohne, was vielleicht den verzögerten Verkauf erklärt; Griechisch-orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rechnungsabschlüsse (1900). 123 Vgl. Carl Ferdinand Mautner von Markhof / Eugen Guglia: Die Wie- ner Stiftungen. Ein Handbuch, Wien 1895, S. XII: »Eine wahrhafte Stiftung bildet immer eine sogenannte juristische Person [...]; sie kann klagen und geklagt werden. Sie bedarf hiezu eines Vertre- tungsorganes, dem durch Aushändigung des Stiftungsvermögens die Möglichkeit geboten wird, über dasselbe zu verfügen [...]; sie kann aus einer einzigen Person oder aber auch aus mehreren Per- sonen bestehen«. 107 AHD, G 10, Fasz. 20, Kauf-Vertrag (Haus Sonnenfelsgasse 21), 23. 6. 1908. 124 Schneider: »Zu den Anfängen«, S. 459. 108 AHD, S 6, Fasz. 9, Griechische National-Schule: Entfertigung der deutschen Lehrkräfte, 18. 9. 1920. 125 Schneider: »Zu den Anfängen«, S. 465–467, 474–475. 126 Stammer: Handbuch, S. 281: »Nach § 49 des Wirkungskreises der ehemaligen Bezirksämter und § 35 des Wirkungskreises der ehe- maligen Statthalterei vom 19. Jänner 1853 waren die Bezirksämter bei geistlichen und weltlichen Stiftungen [...] verpflichtet, das Auf- sichtsrecht des Staates zu üben, während die Statthaltereien und Landesregierungen als oberste Stiftungsbehörde darauf zu achten hatten, daß die Stiftungen auf Grund gesetzlicher Vorschriften errichtet, das Stiftungsvermögen gehörig erhoben, sichergestellt und verwaltet und die Stiftungsverbindlichkeiten genau vollzogen wurden«. A D I N I S T O R Y 2 / 2 0 1 7 Georg befindet, Polyzou also offenbar osmanischer Untertan, aber den- noch Besitzer eines Hauses war. 89 Eugen Dusy von Laczkowa stiftete »für arme Mädchen aus dem Civil- stande« (1870), zur »Unterstützung unbemittelter Berufsofficiere des k. u. k. Heeres« (1871) sowie für drei Offiziere ungarischer Regimen- ter des k. u. k. Heeres »behufs Curgebrauch im Bade Füred« (1871). 100 AHD, G 30, Fasz. 1, Abschrift des Testaments von Kyriak Polyzou, 29. 9. 1811. 101 ÖStA, AVA, AK AGO, Kt. 8, Schreiben der niederösterreichischen Landesregierung an die Studienhofkommission, 16. 10. 1811. 90 Athanas Szekeres errichtete eine Stiftung zur Ausstattung armer Mädchen griechisch nicht unirten, katholischen und protestanti- schen Glaubensbekenntnisses (1819); Georg Poppovic stiftete für das k. k. Blindeninstitut (1818); Alexander Graf von Nako stiftete für das Civil-Mädchenpensionat (1825); Maria Karkaleky errichtete eine Heiratsausstattungs-Stiftung (1873). 102 Maria A. Stassinopoulou: Weltgeschichte im Denken eines griechi- schen Aufklärers. Konstantinos Michail Koumas als Historiograph, Frankfurt am Main 1992, S. 51; Fritz Valjavec (Hg.): Bartholomäus Kopitars Briefwechsel. 1. Teil: Kopitars Briefwechsel mit Karl Georg Rumy, München 1942, S. 41–47. 91 AHD, G 40, Fasz. 4, Stiftbrief Juliana Betly, 12. 7. 1858. Zur Stiftung von Juliana Betly sowie zu anderen Stifterinnen unter den Griechisch- Orthodoxen in Wien vgl. Stefano Saracino: »Witwen als Stifterinnen in den Wiener griechischen Gemeinden während des 19. Jahrhun- derts«, in: Archiv für Kulturgeschichte 98 (2016), S. 315–357. 103 1816 war in der in Wien herausgegebenen griechischen Zeitschrift Ermis ο Logios (Ερμής o Λόγιος) ein Brief mit einem Bericht über den Zustand der Griechischen Nationalschule erschienen, in dem – wohl auch in Anbetracht des zu erwartenden Erbes von Polyzou – der Hoffnung Ausdruck verliehen wurde, dass die Schule in Zukunft besser organisiert werden würde; »Επιστολή λογίου των εν Βιέννη προς τον εαυτού φίλον κύριον Φ. – Π. εις την 30. Σεπτεμβριου 1816« [Schreiben eines Wiener Intellektuellen an seinen Freund, Herrn F. P., am 30. September 1816], in: Ερμής ο Λόγιος 20 (15. 10. 1816), S. 355–362, hier S. 356. 92 NÖLA, GSbS, Kt. 95, Auszug (ohne Datum) des Testaments Brutus von Zettiry von 1836; AHD, G 40, Fasz. 5: Stiftbriefe Brutus von Zet- tiry, 20. 2. 1841 und 31. 3. 1895. Zwar kommt in Zettirys Testament der Begriff »Armenfond« nicht vor, doch in der zweiten Fassung des Stiftbriefs von 1895 trägt die Stiftung den Namen »Brutus Edler von Zettiry Stiftung für den Armenfond«. 108 A D I N I S T O R Y 2 / 2 0 1 7 A D I N I S T O R Y 2 / 2 0 1 7 132 Kielmansegg vermisste einen Überblick über die Stiftungen und beklagte insbesondere die Baufälligkeit der Stiftungshäuser, wegen der die Mieteinnahmen zu gering ausfielen; Erich von Kielmansegg: Kaiserhaus, Staatsmänner und Politiker. Aufzeichnungen des k. k. Statthalters, hg. von Walter Goldinger, Wien 1966, S. 317. 133 Ziele der Statthalterei waren: »Erstens, daß die Stiftungen streng nach dem ursprünglichen Willen der Stifter persolviert werden; zweitens: daß Stiftungen, welche von diesen Anordnungen abwei- chen oder bei denen zufolge der geänderten Zeit- und Preisverhält- nisse der stifterische Wille nicht mehr wort- und sinngemäß zur Durchführung gelangen konnte, mit entsprechenden Nachträgen versehen werden, und drittens: daß die Vermögenschaften der ein- zelnen Stiftungen derart sorgfältig verwaltet werden, daß sie bei absolut pupillarmäßiger Sicherheit der Anlage dennoch das größt- mögliche Erträgnis abwerfen«; Die Bauthätigkeit der k. k. niederös- terreichischen Statthalterei als Verwalterin von Stiftungen von 1889 bis 1903, Wien 1904, S. 3. 134 Ferdinand Schmid: »Statistik der in Nieder-Oesterreich verwalteten Stiftungen nach dem Stande vom December 1893«, in: Statistische Monatsschrift 23 (1897), S. 205–301. Darin wurden 54.406 Stiftun- gen in Niederösterreich mit einem Bruttovermögen von 61.085.447 Gulden österreichischer Währung aufgelistet. Pläne, einen dritten Band mit den Stiftungsurkunden herauszugeben, wurden nicht ver- wirklicht. 135 Zur Stiftung von Constantin Panadi für das Blindeninstitut vgl. Stas- sinopoulou: »Endowments«. 136 Als Begründung hierfür nannte man: »Weil die geistlichen Stiftun- gen in ihren Details für das grosse Publicum kaum weiteres Inter- esse haben dürften und etwaige Veröffentlichungen über dieselben auch füglich nur von der geistlichen Behörde veranlasst werden könnten«; Kataster der in Niederösterreich verwalteten weltlichen Stiftungen, S. V. 137 Davon betroffen waren: »Diversi ohne Benennung für den Fond der Kirche«, zwei »Diversi ohne Benennung«, »Brutus Edler von Zettiry für den Schulfond (1836)«. 138 AHD, G 40, Fasz. 2, Stiftbrief Armenfond, 25. 5. 1906. 139 AHG, Kopialbuch 1868–1906, S. 162–163, Schreiben vom 12. 4. 1894. 140 Unter den von der Gemeinde zum Hl. Georg genannten waren nur vier osmanische Untertanen, die Gemeinde zur Hl. Dreifaltigkeit nannte nur habsburgische Untertanen. Die Identifikation der Staats angehörigkeit erfolgte über die Sterbe-Matriken der jeweiligen Ge- meinde. Anastasie von Wimpffen, die Enkelin von Georg Freiherrn von Sina, ist nicht in den Matriken der Gemeinde zur Hl. Dreifaltig- keit verzeichnet und war wohl katholisch. 141 Ransmayr: Untertanen, S. 108. Siehe die Spendenlisten in: AHG, G 4, Fasz. A D I N I S T O R Y 2 / 2 0 1 7 8, Schreiben vom 9. 2. 1860. Insgesamt finden sich in den Ar- chiven der Gemeinde zur Hl. Dreifaltigkeit Ausweise von 1860, 1861, 1865, 1867, 1868, 1869, 1870, 1871. Außerdem wurden in den Jah- ren 1866 bis 1877 Ausweise der Peter Darvar’schen Schulbuchstif- tung angefertigt. 117 Es stifteten Theodor Dumba für Serres (Stiftbrief 1883), Thessaloni- ki (Stiftbrief 1883) und Vlasti (Stiftbrief 1883), Anna Th. Dumba für Kastoria (1892), Theophanie von Vranyi für Dienstboten in Athen (Stiftbrief 1901) und Marie Curti zum Andenken an Demeter M. Curti für Arme in Magarevo (Stiftbrief 1906). 131 »Übersichten« finden sich 1833, 1854, 1856, 1859, 1861 und 1863 unter den Akten der Gemeinde zur Hl. Dreifaltigkeit. Sie waren für den internen Gebrauch bestimmt und wurden von Theodor Ducha- teau für die Vorsteher der Gemeinde zur Hl. Dreifaltigkeit verfasst. Sie folgten einem Auftrag der Sitzung vom 6. 5. 1854, eine tabella- rische Übersicht anzufertigen. Als Basis dienten Duchateau die »im Archiv vorgefundenen Testamente (in Abschrift)«, wobei er in einem Schreiben an die Gemeindevorsteher Lücken im Archiv beklagte. 118 Griechisch-orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rech- nungsabschlüsse (1914). 119 Zur Stiftungsgeschichte in Österreich vgl. Schneider: »Zu den An- fängen«; Stammer: Handbuch; Gunter Beinhauer: »Das österreichi- sche Stiftungsrecht«, in: Österreichische Juristen-Zeitung 27 (1972), S. 378–382; Herrmann von Herrnritt: Stiftungsrecht; Mayrhofer: Handbuch, Bd. 3, S. 73–86. 109 A D I N I S T O R Y 2 / 2 0 1 7 9, Verzeichnis der Spender für den Kirchenbau aus der Ge- meinde zur Hl. Dreifaltigkeit, 1803; AHD, G 55, Kassabuch der Kirche (1786–1799), S. 3. 142 Zu dem vielfältigen Stiftungsverhalten von Constantin Freiherrn von Bellio und Cosmas Liveropulos vgl. Soursos: »Bettenstiftungen«. 143 Betly-Stiftung (7 Groschen), Armenfond (93 Groschen), Schulfond (167,34 Groschen), Kirchenfond (340,28 Schilling); vgl. Griechisch- orientalische Gemeinde zur Heil. Dreifaltigkeit in Wien. Bericht über die ordentliche Generalversammlung und die Rechnungsabschlüsse (1926). 144 Durch das sogenannte Verwaltungsentlastungsgesetz vom 21. 7. 1925: Bundesgesetzblatt für die Republik Österreich (1925), S. 968– 998, hier S. 975. 110 110 A D I N I S T O R Y 2 / 2 0 1 7 Abstract From the late 18th century to the end of the Habsburg Mon- archy in 1918, Vienna’s two Greek Orthodox communities administered a remarkable number of endowments. By founding endowments the benefactors acted between sev- eral spaces and subspaces. The transgression of boundaries by endowments addressed to the benefactors’ hometowns in the Ottoman Empire as well as the instability of these boundaries in the 19th century led to various problems in the interaction with the state authorities. But also endow- ments given to Viennese institutions were sometimes prob- lematic, depending on the benefactors’ character as either Ottoman or Habsburg subjects. In contrast to Ottoman sub- jects, Habsburg subjects could also endow real estate and thus show their integration into the Viennese bourgeoisie. In this article we discuss the legal frameworks for the admin- istration of endowments in the two Greek communities in Vienna as well as its practical realization in interaction with the Habsburg authorities. N a t h a l i e P a t r i c i a S o u r s o s is a research associate at the Department of Byzantine and Modern Greek Studies in Vi- enna. Since 2014 she is working on the project »Social Com- mitment in the Greek Communities in Vienna (18th – 20th Century)«. In the course of her work at the Vienna Doctoral College for »European Historical Dictatorship and Transfor- mation Research« she wrote her dissertation about photog- raphy in the regimes of Benito Mussolini in Italy and Metaxas in Greece (2015). A n n a R a n s m a y r studied Byzantine and Modern Greek Studies and Classical Philology (Ancient Greek) at the Univer- sities of Vienna and Crete. She finished her dissertation on the Greek communities in Habsburg Vienna in 2016. Since 2008 she is head of the Byzantine and Modern Greek Studies Library at the Vienna University Library.
https://openalex.org/W4200441624	https://www.researchsquare.com/article/rs-1142426/latest.pdf	English	null	Incidence and Risk Factors of In-hospital Prosthesis-Related Complications Following Total Shoulder Arthroplasty	Research Square (Research Square)	2,021	cc-by	7,756	Incidence and Risk Factors of In-hospital Prosthesis-Related Complications Following Total Shoulder Arthroplasty Qinfeng Yang Nanfang Hospital Hao Xie Nanfang Hospital Shencai Liu Nanfang Hospital Xuanping Wu Nanfang Hospital Zhanjun Shi Nanfang Hospital Jian Wang (  13392127531@163.com ) Nanfang Hospital Results A total of 34,198 cases were capture from the Nationwide Inpatient Sample database. There were 343 cases of in-hospital prosthesis-related complications after total shoulder arthroplasty and the overall incidence was 1%, with a more than 2.5-fold decrease from 2010 to 2014. Dislocation was the most common category among prosthesis-related complications (0.1%). The occurrence of in-hospital prosthesis-related complications was associated with significantly more total charges and slightly longer length of stay while less usage of Medicare. Risk factors of prosthesis-related complications were identified including younger age (<64 years), female, the native American, hospital in the South, alcohol abuse, depression, uncomplicated diabetes, diabetes with chronic complications, fluid and electrolyte disorders, metastatic cancer, neurological disorders, and renal failure. Interestingly, advanced age (≥65 years) and proprietary hospital were found as protective factors. Furthermore, prosthesis-related complications were associated with aseptic necrosis, rheumatoid arthritis, rotator cuff tear arthropathy, Parkinson’s disease, prior shoulder arthroscopy, and blood transfusion. Conclusions It is of benefit to study risk factors of prosthesis-related complications following total shoulder arthroplasty to ensure the appropriate management and optimize consequences although a relatively low incidence was identified. Methods A retrospective database analysis was performed based on Nationwide Inpatient Sample from 2010 to 2014. Patients who underwent total shoulder arthroplasty were included. Patient demographics, hospital characteristics, length of stay, economic indicators, in-hospital mortality, comorbidities, and peri-operative complications were evaluated. Background The occurrence of prosthesis-related complications after total shoulder arthroplasty is devastating and costly. The purpose was to determine the incidence and risk of in-hospital prosthesis-related complications after total shoulder arthroplasty utilizing a large-scale sample database. Research Article Keywords: Prosthesis-related complications, Total shoulder arthroplasty, Nationwide Inpatient Sample, Incidence, Risk factors, Outcomes Posted Date: December 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1142426/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Prosthesis-related complications, Total shoulder arthroplasty, Nationwide Inpatient Sample, Incidence, Risk factors, Outcomes Posted Date: December 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1142426/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Prosthesis-related complications, Total shoulder arthroplasty, Nationwide Inpatient Sample, Incidence, Risk factors, Outcomes Posted Date: December 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1142426/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. Page 1/24 Page 1/24 Data Source The data source of this study was from the Nationwide Inpatient Sample (NIS) database, which was conducted by the Healthcare Cost and Utilization Project and sponsored by the Agency for Healthcare Research and Quality. On behalf of the largest all-payer database of hospital admissions in the United States, NIS collects a stratified sample from more than 1000 hospitals, of approximately 20% of the hospitalizations in the United States each year [9, 29]. The information, including patient demographics (age, sex, and race), hospital characteristics (type of admission and payer, and bedsize, ownership, teaching status, location, and region of hospital), LOS, total charges, in-hospital mortality, preoperative comorbidities, and diagnostic and procedural codes from International Classification of Diseases (ninth revision) Clinical Modification (ICD-9-CM) were provided by this database. This observational study used deidentified publicly available data, hence it was deemed exempt. Background Total shoulder arthroplasty (TSA) has been developed into a new stage, where TSA is a reliable optional for reducing shoulder pain and restoring function in patients for whom nonoperative management for glenohumeral arthritis has failed [1–3]. In the past decades, the prevalence of TSA and increased from 0.013% in the year 1995 to 0.197% in 2017[4]. Whereas, implant failure and revision total shoulder arthroplasty procedures may occur commonly as a result of prosthesis-related complications (PRCs), Page 2/24 Page 2/24 such as dislocation, periprosthetic joint infection (PJI), periprosthetic fracture (PPF), and mechanical loosening of prosthetic joint [5, 6]. PRCs are catastrophic complications that lead to heavy burdens on the patients as well as healthcare system [4]. Early studies have demonstrated that PRCs are associated with severe pain, disability, longer length of stay (LOS), increased rates of readmission, and higher mortality [7, 8]. Most PRCs will cause revision surgeries. Although great advancements in surgical techniques and implant design these years, the demand for shoulder arthroplasty revision procedures continues to rise [9]. It was estimated that more than 10,000 revision procedures were performed in 2017 in the United State, with a direct medical cost nearly $205 million [4]. The reported incidence of PRCs after TSA are different largely determined by the category and follow-up time of specific adverse events, such as dislocation(0.1%-5.6%), mechanical loosening(0.7%-2.19%), PJI(0.8%-7.27%), and PPF(0.449%-6.99%) [10–16]. Therefore, with the purpose of optimizing postoperative outcomes and reducing the requirement for revision procedures after TSA, it is imperative to determine preoperatively whether patients are at high risk of early PRCs. Numerous risk factors for the above complications have been reported by prior studies [7, 9–14, 16–28]. However, there is no study currently investigating the incidence and risk factors of the in-hospital PRCs after TSA, based on a large-scale national sample. Consequently, the objective of this study was to investigate the incidence and risk factors of in-hospital PRCs following TSA, using a nationwide database. Data Collection Prior to 2010, reverse shoulder arthroplasty (RSA) and anatomical TSA shared a common ICD-9-CM procedural codes (81.80) [22, 30]. That is the 2010 year of the NIS database represents the first year that Page 3/24 Page 3/24 a separate ICD-9-CM procedural code was used to differentiate RSA (81.88) from anatomical TSA (81.80). Besides, data after the year of 2015 were not included as this was when the ICD-10 codes were first used, which may have introduced some unintended heterogeneity [31].Thus, data of this study was limitedly extracted from NIS database from 2010 to 2014. Patients undergoing anatomical TSA were identified according to ICD-9-CM procedural codes of 81.80 (n = 34,268). In-hospital PRCs were defined by ICD-9-CM diagnostic codes including dislocation (996.42), PJI (996.66/996.67/998.5/998.51/998.59), PPF (996.44), mechanical loosening (996.41/996.43/996.45), and other prosthesis-related complications (996.40/996.46/996.47/996.49/996.77/996.78/996.79). Patients who were less than 18 years old, lacked data of age, had osteomyelitis, or had pathologic fracture of humerus were excluded (n=70) (Fig. 1). a separate ICD-9-CM procedural code was used to differentiate RSA (81.88) from anatomical TSA (81.80). Besides, data after the year of 2015 were not included as this was when the ICD-10 codes were first used, which may have introduced some unintended heterogeneity [31].Thus, data of this study was limitedly extracted from NIS database from 2010 to 2014. Patients undergoing anatomical TSA were identified according to ICD-9-CM procedural codes of 81.80 (n = 34,268). In-hospital PRCs were defined by ICD-9-CM diagnostic codes including dislocation (996.42), PJI (996.66/996.67/998.5/998.51/998.59), PPF (996.44), mechanical loosening (996.41/996.43/996.45), and other prosthesis-related complications (996.40/996.46/996.47/996.49/996.77/996.78/996.79). Patients who were less than 18 years old, lacked data of age, had osteomyelitis, or had pathologic fracture of humerus were excluded (n=70) (Fig. 1). The recruited cases were divided into two groups depending on the occurrence of PRCs. Patient demographics, hospital characteristics, and outcome measures (LOS, total charges, and in-hospital mortality) were assessed. The NIS database provides 29 inherent preoperative comorbidities that do not require ICD-9-CM diagnostic codes to obtain, because these inherent comorbidities have their respective variable names (Table 1). Data Collection Additional comorbidities including osteoarthritis, aseptic necrosis (AN), rheumatoid arthritis (RA), rotator cuff tear arthropathy (RCTA), proximal humerus fracture, Parkinson’s disease, sleep apnea, smoking, osteoporosis, and prior shoulder arthroscopy (PSA), were searched by ICD- 9-CM diagnostic code and analyzed at the author’s discretion if they had been determined as significant risk factors for any one of the PRCs in previous studies [1, 11, 12, 14, 16, 32–35]. Perioperative complications during hospitalization were extracted from the database by ICD-9-CM diagnostic code. Medical complications included acute renal failure, acute myocardial infarction, pneumonia, pulmonary embolism, stroke, postoperative delirium, urinary tract infection, deep vein thrombosis, sepsis, postoperative shock, urinary tract infection and blood transfusion [12, 36]. Surgical complications were defined as wound dehiscence, hemorrhage/seroma/hematoma, non-healing surgical wound, and nerve injury of upper limb [13, 37]. An additional file shows this in more details [see Additional file 1]. Page 4/24 Page 4/24 Table 1 Variables entered into the logistic regression analysis. Data Collection Between line 118 and line 119 Variables Categories Specific Variables Patient demographics Age (≤64 years and ≥ 65 years), sex (male and female), race (White, Black, Hispanic, Asian or Pacific Islander, Native American and Other) Hospital characteristics Type of admission (non-elective, elective), bed size of hospital (small, medium, large), control of hospital (collapsed category 1 [government or private], public [government, nonfederal], voluntary [private, not-for-profit], proprietary [private, investor-owned], collapsed category 2 [private]), teaching status of hospital (nonteaching, teaching), location of hospital (rural, urban), region of hospital (northeast, Midwest of north central, south, west), type of payer (Medicare, Medicaid, Private insurance, Self-pay, No charge, Other) Comorbidities AIDS, alcohol abuse, deficiency anemia, rheumatoid diseases, chronic blood loss anemia, congestive heart failure, chronic pulmonary disease, coagulopathy, depression, diabetes (uncomplicated), diabetes (with chronic complications), drug abuse, hypertension, hypothyroidism, liver disease, lymphoma, fluid and electrolyte disorders, metastatic cancer, neurological disorders, obesity, paralysis, peripheral vascular disorders, psychoses, pulmonary circulation disorders, renal failure, solid tumor without metastasis, peptic ulcer disease, valvular disease, weight loss D t A l i Type of admission (non-elective, elective), bed size of hospital (small, medium, large), control of hospital (collapsed category 1 [government or private], public [government, nonfederal], voluntary [private, not-for-profit], proprietary [private, investor-owned], collapsed category 2 [private]), teaching status of hospital (nonteaching, teaching), location of hospital (rural, urban), region of hospital (northeast, Midwest of north central, south, west), type of payer (Medicare, Medicaid, Private insurance, Self-pay, No charge, Other) AIDS, alcohol abuse, deficiency anemia, rheumatoid diseases, chronic blood loss anemia, congestive heart failure, chronic pulmonary disease, coagulopathy, depression, diabetes (uncomplicated), diabetes (with chronic complications), drug abuse, hypertension, hypothyroidism, liver disease, lymphoma, fluid and electrolyte disorders, metastatic cancer, neurological disorders, obesity, paralysis, peripheral vascular disorders, psychoses, pulmonary circulation disorders, renal failure, solid tumor without metastasis, peptic ulcer disease, valvular disease, weight loss Data Analysis All the statistical analyses were performed utilizing the statistical software, R version 3.5.3 (The R Foundation Inc., Auckland, New Zealand). Significant differences between two groups were determined by Wilcoxon rank test for continuous data and chi-square test for categorical data. To identify independent risk factors of PRCs, multivariate logistic regression with the stepwise method was performed. All variables, including demographics, hospital characteristics, type of payer, and comorbidities providing by the NIS, were entered into the regression analysis (Table 1 ). Univariate and multivariate logistic regression models were constructed to evaluate the association of additional comorbidities or perioperative complications with PRCs. An alpha level of P<0.05 represented statistical significance. Similar analysis could be found in prior studies [38, 39]. Incidence of in-hospital PRCs in patients undergoing TSA A total of 34,198 TSAs were collected from the NIS database from 2010 to 2014. Overall, there were 343 cases of in-hospital PRCs with an incidence of 1% (Table 2). It was observed that the incidence of PRCs, including dislocation, PJI, PPF, mechanical loosening and others, decreased from 1.62–0.59%, more than a 2.5-fold reduction during this period (Fig. 2). Dislocation (0.1%) was the most common PRCs after TSA, followed by PJI (0.09%), PPF (0.06%), and mechanical loosening (0.03%) except for the miscellaneous (0.74%) (Fig. 3). Page 5/24 Page 5/24 Table 2 Patient characteristics and outcomes of PRCs after TSA (2010-2014). Between line 151 and 152 Parameter No PRCs PRCs P Total (n = count) 33,855 343 – Total incidence (%) 1 Age (median, years) 68 (62-75) 66 (57-73) < 0.0001 Age group (%) 18-44 1.50 7.00 < 0.0001 45–64 32.73 39.36 65–74 40.74 31.49 ≥ 75 25.03 22.16 Sex (% female) 51.98 57.14 0.06 Race (%) White 90.38 87.09 0.03 Black 4.18 4.30 Hispanic 3.00 4.97 Asian or Pacific Islander 0.38 0.66 Native American 0.36 1.32 Other 1.69 1.66 Type of admission (% elective) 95.17 96.49 0.31 Bed size of hospital (%) Small 20.98 21.18 0.95 Medium 24.36 25.00 Large 54.65 53.82 Control of hospital (%) Collapsed category 1 (government or private) 24.76 36.76 < 0.0001 Public (government, nonfederal) 8.14 8.53 Voluntary (private, not-for-profit) 53.32 41.47 Page 6/24 Parameter No PRCs PRCs P Proprietary (private, investor-owned) 12.01 10.88 Collapsed category 2 (private) 1.78 2.35 Teaching status of hospital (% teaching) 51.43 52.65 0.69 Location of hospital (% urban) 90.63 93.24 0.12 Region of hospital (%) Northeast 14.85 12.24 0.003 Midwest or North Central 27.82 20.70 South 35.40 42.57 West 21.93 24.49 LOS (median, day) 2 (1-2) 2 (1-3) < 0.0001 Total charges (median, $) 48,950 (36,409- 65,546) 56,677 (40,239- 79,593) < 0.0001 Type of payer (%) Medicare 64.08 53.06 0.0005 Medicaid 2.21 2.62 Private insurance 29.62 34.99 Self-pay 0.34 0.58 No charge 0.07 0 Other 3.68 8.75 In-hospital mortality (%) 0.05 0 1 PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; LOS: length of stay Patient demographics of two groups Patient demographics of two groups Patients affected by in-hospital PRCs were 2 years younger (66 years vs 68 years) compared to the nonaffected population (P<0.0001) (Table 2). Consistently, there was significant difference of the age distribution between the two groups. PRCs after TSA were 12.13% more likely to occurrence among patients younger than 64 years (46.36% vs 34.23%, P<0.0001) (Table 2). Meanwhile, a significant difference was detected in races, with the White occupying smaller proportions (87.09% vs 90.38%) while the Native American taking slightly larger proportions (1.32% vs 0.36%) in the PRCs (P=0.03) (Table 2). Page 7/24 Page 7/24 However, no significant difference was observed in genders between two cohorts at our defined level (P=0.06) (Table 2). However, no significant difference was observed in genders between two cohorts at our defined level (P=0.06) (Table 2). Consequences of in-hospital PRCs after TSA There was statistical significance in the median LOS between two cohorts (2 days vs 2 days; P<0.0001) (Table 2). The median LOS was equal, but the interquartile range in the PRCs groups (1 days to 3 days) was still larger than the control group (1 days to 2 days), suggesting that the occurrence of PRCs slightly prolonged hospitalization (Table 2). As expected, PRCs increased medical expenditure despite the slightly longer LOS. There was a significant increase of $7727 in total hospital charges, with the presence of in- hospital PRCs ($56,677 vs $48,950, P<0.0001) (Table 2). With regard to the type of payer, the Medicare was observed to occupy a smaller proportion (53.06% vs 64.08%) while Private insurance (34.99% vs 29.62%) and other type (8.75% vs 3.68%) took a larger proportion in the PRCs group (P=0.0005) (Table 2). Fortunately, patients undergoing TSA had quite low in-hospital mortality which was not affected by the presence of PRCs (Table 2). Hospital characteristics of two groups It was found that the occurrence of PRCs was more likely in government or private hospital (collapsed category) (36.76% vs 24.76%, P<0.0001), however, less likely in voluntary hospital (private, not-for-profit) (41.47% vs 53.32%, P<0.0001) and proprietary hospital (private, investor-owned) (10.88% vs 12.01%, P<0.0001) (Table 2). In terms of the region of hospital, in-hospital PRCs tended to occur in the South and West, whereas less likely in the Northeast and Midwest or North Central (P=0.003) (Table 2). Nevertheless, there was no statistical difference between two groups regarding to type of admission (P=0.31), bedsize of hospital (P=0.95), teaching status of hospital (P=0.69), and location of hospital (P=0.12). Risk factors of in-hospital PRCs after TSA Logistic regression analysis was applied to explore risk factors of PRCs (Table 3), and the following indicators were identified: female (odds ratio [OR]=1.45; 95% confidence interval [CI]=1.13-1.86; P=0.003), the native American (OR=4.20; CI=1.49-11.86; P=0.007), hospital in the South (OR=1.61; CI=1.12-2.31; P=0.01), alcohol abuse (OR=4.36; CI=2.32-8.22; P<0.0001), depression (OR=1.48; CI=1.05-2.10; P=0.03), uncomplicated diabetes (OR=1.67; CI=1.18-2.37; P=0.004), diabetes with chronic complications (OR=2.73; CI=1.36-5.48; P=0.005), fluid and electrolyte disorders (OR=1.91; CI=1.24-2.95; P=0.004), metastatic cancer (OR=16.08; CI=3.43-75.35; P=0.0004), neurological disorders (OR=2.02; CI=1.29-3.15; P=0.002), and renal failure (OR=2.15; CI=1.32-3.51; P=0.002). Interestingly, there were several protective factors of PRCs including advanced age (≥65 years; OR=0.68; CI=0.49-0.93; P=0.02), public hospital (OR=0.54; CI=0.34-0.86; P=0.0091), voluntary hospital (OR=0.42; CI=0.32-0.57; P<0.0001), and proprietary hospital (OR=0.46; CI=0.30-0.71; P=0.0005). Page 8/24 Table 3 Risk factors of PRCs after TSA (2010-2014). Between line 181 and line 182 Variable OR 95% CI P Age ≥65 years 0.68 0.49-0.93 0.02 Female 1.45 1.13-1.86 0.003 Race White Ref. — — Black 0.88 0.49-1.56 0.65 Hispanic 1.58 0.92-2.70 0.10 Asian or Pacific Islander 1.79 0.43-7.41 0.42 Native American 4.20 1.49-11.86 0.007 Other 1.04 0.43-2.55 0.93 Elective admission 1.57 0.84-2.92 0.15 Bed size of hospital Small Ref. — — Medium 0.95 0.67-1.35 0.79 Large 0.94 0.69-1.28 0.71 Control of hospital Collapsed category 1 (government or private) Ref. — — Public (government, nonfederal) 0.54 0.34-0.86 0.009 Voluntary (private, not-for-profit) 0.42 0.32-0.57 <0.0001 Proprietary (private, investor-owned) 0.46 0.30-0.71 0.0005 Collapsed category 2 (private) 1.29 0.39-4.32 0.68 Teaching hospital 0.84 0.64-1.10 0.22 Urban hospital 1.81 0.97-3.41 0.06 Region Northeast Ref. — — PRC th ti l t d li ti TSA t t l h ld th l t OR dd ti CI fid Page 9/24 Variable OR 95% CI P Midwest or North Central 0.76 0.49-1.18 0.22 South 1.61 1.12-2.31 0.01 West 1.44 0.96-2.17 0.08 Type of payer Medicare Ref. — — Medicaid 0.94 0.42-2.11 0.88 Private insurance 1.31 0.94-1.83 0.11 Self-pay 1.67 0.40-7.07 0.48 No-charge 0.0000 0.0000 0.97 Other 2.33 1.43-3.79 0.0007 Alcohol abuse 4.36 2.32-8.22 <0.0001 Depression 1.48 1.05-2.10 0.03 Diabetes, uncomplicated 1.67 1.18-2.37 0.004 Diabetes with chronic complications 2.73 1.36-5.48 0.005 Fluid and electrolyte disorders 1.91 1.24-2.95 0.004 Metastatic cancer 16.08 3.43-75.35 0.0004 Neurological disorders 2.02 1.29-3.15 0.002 Renal failure 2.15 1.32-3.51 0.002 PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. Risk factors of in-hospital PRCs after TSA Additional comorbidities and complications associated with in-hospital PRCs after TSA As for the indications for TSA, patients diagnosed with osteoarthritis were less likely to experience in- hospital PRCs (P=0.0005) (Table 4). Nevertheless, those diagnosed with AN, RA, and RCTA receiving TSA were more likely to experience in-hospital PRCs (P=0.0005) (Table 4). Additionally, PRCs after TSA tended to occur in patients with history of PSA (P=0.03) (Table 4). In multivariate analysis, AN (OR=7.15; CI=4.24- 12.06; P<0.0001), RA (OR=3.08; CI=1.35-6.99; P=0.007), and RCTA (OR=2.21; CI=1.38-3.57; P=0.001) Page 10/24 Page 10/24 Page 10/24 compared with osteoarthritis, Parkinson's disease (OR=3.21; CI=1.30-7.90; P=0.01), and PSA (OR=13.63; CI=3.17-58.70; P=0.0005) were independent risk factors for PRCs after TSA (Table 4). compared with osteoarthritis, Parkinson's disease (OR=3.21; CI=1.30-7.90; P=0.01), and PSA (OR=13.63; CI=3.17-58.70; P=0.0005) were independent risk factors for PRCs after TSA (Table 4). ) p ( ) Table 4 Additional comorbidities associated with PRCs after TSA (2010-2014). Between line 196 and line 197 Comorbidity Univariate Analysis Multivariate Logistic Regression NO PRCs PRCs P OR 95%CI P Indications for TSA Osteoarthritis 30,229 (92.29%) 187 (81.30%) 0.0005 Ref. — — Aseptic necrosis 363 (1.11%) 16 (6.96%) 0.0005 7.15 4.24- 12.06 <0.0001 Rheumatoid arthritis 319 (0.97%) 6 (2.61%) 0.0005 3.08 1.35- 6.99 0.007 Rotator cuff tear arthropathy 1390 (4.24%) 19 (8.26%) 0.0005 2.21 1.38- 3.57 0.001 Proximal humerus fracture 455 (1.39%) 2 (0.87%) 0.0005 0.71 0.17- 2.86 0.63 Other comorbidities Parkinson’s disease 250 (0.74%) 5 (1.46%) 0.11 3.21 1.30- 7.90 0.01 Sleep apnea 4644 (13.72%) 42 (12.24%) 0.48 0.99 0.68- 1.45 0.98 Smoking 7551 (22.3%) 82 (23.91%) 0.52 1.07 0.79- 1.46 0.66 Osteoporosis 1689 (4.99%) 24 (7%) 0.12 0.91 0.49- 1.67 0.76 Prior shoulder arthroscopy 24 (0.07%) 2 (0.58%) 0.03 13.63 3.17- 58.70 0.0005 PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. Univariate analysis showed that patients with in-hospital PRCs were more likely to have either medical PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. Univariate analysis showed that patients with in-hospital PRCs were more likely to have either medical perioperative complications including pneumonia, postoperative delirium, urinary tract infection, sepsis, and blood transfusion or surgical complications merely including wound dehiscence compared with those without PRCs (Table 5). Risk factors of in-hospital PRCs after TSA However, multivariate analysis only found that in-hospital PRCs were independently associated with blood transfusion (OR=2.61; CI=1.38-4.92; P=0.003) among all the perioperative complications. Page 11/24 Page 11/24 Table 5 Complications associated with PRCs after TSA (2010-2014). Between line 207 and line 208 Complications Univariate Analysis Multivariate Logistic Regression No PRCs PRCs P OR 95% CI P Medical complications Acute renal failure 425 (1.26%) 8 (2.33%) 0.08 1.00 0.45- 2.19 0.99 Acute myocardial infarction 157 (0.46%) 2 (0.58%) 0.68 0.67 0.16- 2.85 0.58 Pneumonia 127 (0.38%) 4 (1.17%) 0.04 1.47 0.47- 4.54 0.51 Pulmonary embolism 37 (0.11%) 1 (0.29%) 0.32 1.89 0.23- 15.36 0.55 Stroke 207 (0.61%) 5 (1.46%) 0.06 1.74 0.64- 4.70 0.28 Postoperative delirium 183 (0.54%) 5 (1.46%) 0.04 1.57 0.61- 4.07 0.35 Urinary tract infection 588 (1.74%) 16 (4.66%) 0.0001 1.91 0.98- 3.75 0.06 Deep vein thrombosis 33 (0.1%) 1 (0.29%) 0.29 1.14 0.14- 9.45 0.90 Sepsis 18 (0.05%) 2 (0.58%) 0.02 4.99 0.96- 26.10 0.06 Postoperative shock 9 (0.03%) 0 (0%) 1 — — — Continuous invasive mechanical ventilation 61 (0.18%) 1 (0.29%) 0.47 0.49 0.06- 4.24 0.52 Blood transfusion 1238 (3.66%) 40 (11.66%) <0.0001 2.61 1.38- 4.92 0.003 Any medical complicationa 2600 (7.68%) 64 (18.66%) <0.0001 1.27 0.66- 2.44 0.47 Surgical complications Complications Univariate Analysis Multivariate Logistic Regression No PRCs PRCs P OR 95% CI P Wound dehiscence 3 (0.01%) 1 (0.29%) 0.04 0.0000 n/a 0.99 Hemorrhage/seroma/hematoma 90 (0.27%) 1 (0.29%) 0.60 0.0000 n/a 0.99 Non-healing surgical wound 4 (0.01%) 0 (0%) 1 0.0000 n/a 0.99 Nerve injury of upper limb 22 (0.06%) 0 (0%) 1 0.0000 n/a 0.99 Any surgical complicationb 118 (0.35%) 2 (0.58%) 0.34 2073535 n/a 0.99 PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. Any medical complicationa or surgical complicationb: patients with more than one complication are counted only once. PRCs: prosthetic-related complications; TSA: total shoulder arthroplasty; OR: odds ratio; CI: confidence interval. Any medical complicationa or surgical complicationb: patients with more than one complication are counted only once. Any medical complicationa or surgical complicationb: patients with more than one complication are counted only once. Discussion The present study provides a large-scale as well as health-economic analysis of in-hospital PRCs after TSA. With more attention on improving surgical techniques and component design, the incidence of PRCs following TSA decreased from 2010 to 2014 with an exceeding 2.5-fold reduction (Fig. 1). An overall incidence of 1% of in-hospital PRCs after TSA was firstly identified in this study because previous studies mainly focused on the specific categories of PRCs. Interestingly, this overall incidence of in-hospital PRCs following TSA was lower than that following total hip arthroplasty (THA) (1.96%) while higher than that following total knee arthroplasty (TKA) (0.69%) [38, 39]. It was found that the dislocation of prosthetic joint was the most common PRCs, followed by PJI, PPF, and mechanical loosening (Fig. 2), which were to a large extent consistent with the previous studies on implant failure after shoulder arthroplasty [12, 14, 15, 35]. Coincidentally, the prior study conducted by our authors found the same results that dislocation was the most common PRCs, followed by PJI, PPF and mechanical loosening after THA [39]. This consistency between TSA and THA reflect the similarity of these two procedures. A previous literature reported that male was a significant risk factor for revision shoulder arthroplasty, nonetheless in this study, female was identified as a risk factor of PRCs in logistic regression analysis [6]. This disparity may be due to that our study was the analysis of in-hospital PRCs after TSA which was an early stage or even unsure to require revision. Besides, female patients undergoing shoulder arthroplasty were associated with receiving blood transfusion which was at high risk of PRCs in this study [22]. Furthermore, female was reported to be susceptible to suffer from depression which was also a risk Page 13/24 Page 13/24 factor of PRCs in our results [40]. Patients with PRCs were 2 years younger than those without. Besides, from the perspective of age distribution, patients younger than 64 years accounted for a greater proportion in the PRCs group. In addition, in logistic regression analysis, advanced age (≥65 years) was identified as a protective factor of PRCs. On the contrary, younger age (<64 years) could statistically be determined as a risk factor of PRCs. To a great extent, this is consistent with previous studies which identified younger age as an independent predictor of PJI, failure or revision after shoulder arthroplasty [6, 10, 11, 14, 17, 24, 28, 30]. Discussion The etiology underlying this finding is unclear, but this can be used to educate patients, inform surgeons when counseling younger patients regarding their risks, and serve as an impetus for further investigation [24]. Reasons for the Native American patients more likely and risky to experience PRCs are unknown and likely multifactorial. It is possibly that these minority populations have lower levels of cultural and healthy literacy. Therefore, these patients tend to receive lower-quality care or have difficulty understanding and complying with postoperative instructions [41, 42]. Compared with the Northeast region of the United States, hospital in the South was associated with an increased likelihood of PRCs, similar to our prior findings discussing about the PRCs following THA [39]. Combined with the univariate analysis and multivariate analysis, patients in voluntary hospital were less likely and risky to experience PRCs. However, the reasons for these two hospital characteristic remain unclear and require further research. Both the median LOS was 2 days no matter whether the in-hospital PRCs occurred, which is in line with prior studies that also found an mean LOS of approximate 2 days in patients undergoing TSA [29, 33]. Although the interquartile range of LOS presented statistical significance between two groups, this small difference was not obvious and might not be clinically important or meaningful [40]. Luckily, the occurrence of in-hospital PRCs after TSA did not incur patients to death in this study, totally unlike to our previous findings in terms of THA and TKA, suggesting that TSA is safer as well as less traumatic to individuals compared with the other two procedures [38, 39]. Even so, the presence of PRCs still increased total charge during hospitalization, due to the costly treatments and cares of these complications [8, 10]. To moderate these costly events and further reduce the need for revision, preoperative identification of patients at increased risk of in-hospital PRCs after TSA is essential [43, 44]. Logistic regression was performed and several risk factors of in-hospital PRCs following TSA were identified (Table 3, 4, and 5). Similar to our prior study on PRCs after THA, patients with alcohol abuse were at high risk of PRCs after TSA, probably because excessive alcohol consumption is associated with weakened immune system and mechanisms, impaired phagocytic function, or induced cytokine abnormalities which may have a relationship with PJI [26, 39]. Discussion Depression has previously been reported to be associated with a series of adverse outcomes, such as PJI, sepsis, wound complications, return to the operating room for irrigation and debridement, extended LOS, readmission, revision surgery, and greater health care utilization after TSA, in-hospital PRCs after THA and TKA [38–40, 45]. Although the relationship between depression and in-hospital PRCs after TSA may seem obscure, imbalance of immune system inducing PJI likely explain this. Psychological distress produces a systemic state of inflammation leading to dysregulation of the immune system and a resultant susceptible host state [45]. Additionally, depression itself may influence Page 14/24 Page 14/24 T-cell phenotype, and antidepressant medications have been shown to have negative immunoregulatory effects, further causing immune susceptibility [40]. Diabetic patients with vulnerable defenses against bacteria, or impaired wound healing because microangiopathic changes could reduce the tissue concentrations of antibiotics as well as cause local tissue ischemia, consequently are susceptible to PJI, and in-hospital PRCs following TSA, THA and TKA [38, 39, 46]. In concordance with our previous results, metastatic cancer in this study conferred the highest OR value (Table 3). This severe comorbidity possibly predisposes patients to PJI because of immunosuppressive conditions [38, 39]. Patients with either neurological disorders or Parkinson disease were at increased risk of PRCs likely because of the increased tone of the shoulder girdle musculature, the difficulties with rehabilitation, and stretching of the rotator cuff-capsule arthrotomy site, particularly the rotator interval [14]. Besides, patients with Parkinson’s disease lack complete volitional muscular control and have asynchronous motor function, they appear to place the shoulder at high risk for instability [14, 39]. Additionally, constant tremors may result in implant loosening, which further increases the dislocation risk [14]. Intriguingly, fluid and electrolyte disorders not only increase the odds of PRCs after THA and TKA, but also had an increased risk of PRCs after TSA [38, 39]. Renal failure has been reported as a significant risks factor for surgical site infection and revision following TSA [6, 8]. Patients with these two comorbidities to some degree may reflect the weakened status and insufficient immune function, and hence surgeons should be more cognizant of perioperative nephrotoxic medications, intraoperative hypotensive anesthesia, and postoperative fluid management in this complex patient population [47]. As expected, patients with indications for TSA such as AN, RA, or RCTA had higher odds of PRCs compared with osteoarthritis. Discussion Consistently, AN has been associated with a significantly increased risk for postoperative infection, dislocation, fracture, and revision surgery after TSA [16]. Despite our authors previously reported that RA was associated with in-hospital PRCs following THA, it was firstly found in this study that patients RA undergoing TSA were risky to experience in-hospital PRCs [39]. Corticosteroid therapy, alcohol abuse, immunosuppressive conditions, or posttraumatic poor soft tissue bed may partially explain the association of in-hospital PRCs with AN and RA [8, 16, 17, 26, 39]. Patients with RCTA commonly underwent prior soft tissue mobilization and rotator cuff manipulation, which are thought to affect prosthetic implant stabilization and may play a role in the increased dislocation, infection, and bleeding rates [28]. Arthroscopic intervention for shoulder osteoarthritis has been used as a measure to temporize pain or mechanical symptoms in order to delay joint arthroplasty and expedite time to return to recreational activities and physically demanding jobs [17]. However, it was found that patients with history of PSA were risky to PRCs after TSA, similar to our prior finding that prior knee arthroscopy conferred high risk of PRCs after TKA [38]. Furthermore, PSA has also been reported to be associated with a higher risk of infection after shoulder arthroplasty [17]. On the basis of this association, surgeons should proceed with increased caution before performing an arthroplasty procedure in a patient with a history of PSA, and consider a lower threshold to rule out infection as well as the perioperative usage of antibiotic [17]. It has Page 15/24 Page 15/24 Page 15/24 been shown that blood transfusion confers increased risk of PJI and mechanical complications within 2 years after shoulder arthroplasty and is associated with in-hospital PRCs after THA or TKA [22, 38, 39]. Consistent with previous literatures, blood transfusion was the only one factor in this study significantly associated with in-hospital PRCs following TSA among the perioperative complications. Specifically, allogeneic blood transfusion may have an immunomodulatory effect that may lower the threshold for PJI through several mechanisms [22, 38]. The main strengths of the current study include its both large-scale sample and national representativeness with the power to investigate rare events, and the utilization of multivariable regression modeling to reduce confounding [31, 33, 38]. However, several limitations still require mention, mainly inherent to the use of the NIS database. Discussion First, misclassification or discrepancy in the process of coding and documentation may be produced as with any large administrative database [16, 17, 33, 38, 39]. Second, only in-hospital information of each patient is recorded, meaning any complication or adverse outcome that occurs after discharge such as readmission, functional status, and long-term follow-up can not be obtained in this database. This limitation may cause underestimating the incidence of PRCs [8, 9, 26, 29, 33, 38, 39]. Furthermore, only variables provided by the NIS database could be assessed. There are other potential procedural and component characteristics that possibly affect PRCs were unable to capture via the NIS database, such as surgical approach, length of surgery, type of anesthesia, amount of blood loss, cemented or uncemented components, and implant design [7–9, 15, 35, 38, 39, 47]. Conclusion In-hospital PRCs are disastrous and costly complications occurring after TSA with a total incidence of 1%. The annual incidence of PRCs decreased from 2010 to 2014 with a surpassing 2.5-fold reduction. Dislocation was the most common PRCs, followed by PJI, PPF, and mechanical loosening. Several risk factors of PRCs after TSA were identified in our study including younger age (<64 years), female, the native American, hospital in the South, alcohol abuse, depression, uncomplicated diabetes, diabetes with chronic complications, fluid and electrolyte disorders, metastatic cancer, neurological disorders, and rena failure. Advanced age (≥65 years) and proprietary hospital were found as protective factors. In addition, PRCs were associated with other indications for TSA (AN, RA, and RCTA), Parkinson’s disease, prior shoulder arthroscopy, and blood transfusion. Patients with PRCs after TSA demonstrated significantly more total charges and slightly longer LOS while less usage of Medicare. Abbreviations PRCs: prosthesis-related complications TSA: total shoulder arthroplasty NIS: Nationwide Inpatient Sample NIS: Nationwide Inpatient Sample Page 16/24 Page 16/24 LOS: length of stay Consent for publication Not applicable. Availability of data and materials The datasets generated during and analyzed during the current study are not publicly available due to Data Use Agreement and more information can be found in AHRQ HCUP (www.hcup-us.ahrq.gov).They are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. This observational study used deidentified publicly available data, hence there was no requirement for consent to participate and it was deemed exempt by the ethics committee. All methods were performed in accordance with the relevant guidelines and regulations. Competing interests The authors declare that they have no competing interest. Authors' contributions QY contributed to the study design, data acquisition and analysis, interpretation of results, and writing and revising the manuscript. HX and ZS contributed to the study design, interpretation of results, and reviewing the manuscript. SL and XW contributed to data acquisition, data analysis, and reviewing of the manuscript. JW contributed to the study design, interpretation of results, and reviewing the manuscript. All authors read and approved the final manuscript. Funding Page 17/24 This research did not receive any specific grant from funding agencies in the public, commercial, or not- for-profit sectors. This research did not receive any specific grant from funding agencies in the public, commercial, or not- for-profit sectors. Acknowledgements We express our sincere gratitude to Goodwill Hessian Health Technology Co. Ltd. (100007, Beijing, China.) for providing consultation and guidance on statistical analysis in this study. References 1. Norris TR, Iannotti JP. Functional outcome after shoulder arthroplasty for primary osteoarthritis: A multicenter study. J Shoulder Elbow Surg. 2002;11:130–5. 1. Norris TR, Iannotti JP. Functional outcome after shoulder arthroplasty for primary osteoarthritis: A multicenter study. J Shoulder Elbow Surg. 2002;11:130–5. 2. Orfaly RM, Rockwood CA, Esenyel CZ, Wirth MA. A prospective functional outcome study of shoulder arthroplasty for osteoarthritis with an intact rotator cuff. J Shoulder Elbow Surg. 2003;12:214. 3. Bohsali KI, Wirth MA, Rockwood CA. Current concepts review: Complications of total shoulder arthroplasty. 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This shows that dislocation (0.1%) was the most common PRCs followed by periprosthetic joint infection (0.09%), periprosthetic fracture (0.06%), and mechanical loosening (0.03%) except for the miscellaneous (0.74%). Incidence of specific categories among prosthesis-related complications following total shoulder arthroplasty. This shows that dislocation (0.1%) was the most common PRCs followed by periprosthetic joint infection (0.09%), periprosthetic fracture (0.06%), and mechanical loosening (0.03%) except for the miscellaneous (0.74%). Additionalfile1.doc Figure 1 Figure 1 Process to screening data. Page 22/24 Figure 2 Annual incidence of in-hospital prosthesis-related complications following total shoulder arthroplasty. This indicates that the incidence of in-hospital PRCs which include periprosthetic joint infection, mechanical loosening, dislocation, periprosthetic fracture, and others, decreases from 2010 (1.62%) to 2014 (0.59%). Figure 2 Annual incidence of in-hospital prosthesis-related complications following total shoulder arthroplasty. This indicates that the incidence of in-hospital PRCs which include periprosthetic joint infection, mechanical loosening, dislocation, periprosthetic fracture, and others, decreases from 2010 (1.62%) to 2014 (0.59%). Page 23/24 Page 23/24 Page 23/24 Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 24/24
https://openalex.org/W2039815297	https://bmcsurg.biomedcentral.com/counter/pdf/10.1186/1471-2482-10-26	English	null	Sepsis induced changes of adipokines and cytokines - septic patients compared to morbidly obese patients	BMC surgery	2,010	cc-by	7,394	RESEARCH ARTICLE Open Access Open Access © 2010 Hillenbrand et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: Andreas.Hillenbrand@uniklinik-ulm.de † Contributed equally 1Department of General-, Visceral-, and Transplantation Surgery, University Hospital of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany Full list of author information is available at the end of the article Abstract Background: Hyperglycemia and insulin resistance frequently occur in critically ill and in morbidly obese (MO) patients. Both conditions are associated with altered serum levels of cytokines and adipokines. In addition, obesity related alterations in adipokine expression contribute to insulin resistance in metabolic syndrome. In this study we examined the serum adipocytokine profile in critically ill patients, MO patients, and healthy blood donors. Methods: 33 patients who fulfilled the clinical criteria for severe sepsis or septic shock (SP) were prospectively enrolled in this study. A multiplex analysis was performed to evaluate plasma levels of adiponectin, resistin, leptin, active PAI-1, MCP-1, IL-1 alpha, IL-6, IL-8, IL-10, and TNF-alpha in 33 critically ill patients, 37 MO patients and 60 healthy blood donors (BD). Results: In SP, adiponectin was significantly lowered and resistin, active PAI-1, MCP-1, IL-1 alpha, IL-6, IL-8, IL-10, and TNF-alpha were significantly elevated compared to BD. Leptin levels were unchanged. In MO, adiponectin and IL-8 were significantly lowered, leptin, active PAI-1, MCP-1, IL-1 alpha, IL-6, and IL-10 significantly elevated, whereas resistin was unaltered. In SP, adiponectin correlated negatively with BMI, SAPS II and SOFA scores, while resistin correlated positively with SAPS II and SOFA scores and leptin correlated positively with the BMI. Adiponectin was approximately equally diminished in SP and MO compared to BD. With the exception of active PAI-1, cytokine levels in SP were clearly higher compared to MO. Conclusion: A comparable adipocytokine profile was determined in critically ill and MO patients. As in MO, SP showed reduced adiponectin levels and elevated MCP-1, active PAI-1, IL-1 alpha, IL-6, and IL-10 levels. Leptin is only elevated in MO, while resistin, IL-8, and TNF-alpha is only elevated in SP. As in MO patients, increased levels of proinflammatory cytokines and altered levels of adipokines may contribute to the development of insulin resistance in critically ill patients. Hillenbrand et al. BMC Surgery 2010, 10:26 Hillenbrand et al. BMC Surgery 2010, 10:26 Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Sepsis induced changes of adipokines and cytokines - septic patients compared to morbidly obese patients Andreas Hillenbrand1, Uwe Knippschild1, Manfred Weiss2, Hubert Schrezenmeier3, Doris Henne-Bruns1, Markus Huber-Lang4†, Anna M Wolf1† Correspondence: Andreas.Hillenbrand@uniklinik-ulm.de Background with an increased production of both pro- and anti- inflammatory cytokines [2]. Cytokines are low-molecu- lar-weight polypeptides or glycoproteins that play an important role in regulating host response to infection, immune responses, inflammation, and trauma. Sepsis is defined as a systemic inflammatory response syndrome to infection, which, when associated with one or more organ system dysfunctions, is considered as severe sepsis. When sepsis is associated with shock, which is refractory to fluid resuscitation, the patient is considered to be in septic shock [1]. This is associated Some cytokines clearly promote inflammation and are called proinflammatory cytokines, whereas other cyto- kines suppress the activity of proinflammatory cytokines and are called anti-inflammatory cytokines. * Correspondence: Andreas.Hillenbrand@uniklinik-ulm.de † Contributed equally 1Department of General-, Visceral-, and Transplantation Surgery, University Hospital of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany Full list of author information is available at the end of the article Tumor necrosis factor (TNF) and interleukin (IL)-1a are proinflammatory cytokines, which can cause fever, Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Page 2 of 9 Several studies have provided evidence that increased blood glucose levels impair morbidity and mortality in critically ill patients and that intensive insulin therapy aimed at maintaining euglycemia markedly improved the outcome of these patients [17]. inflammation, tissue destruction, and, in some cases, shock and death [3]. IL-10, a potent anti-inflammatory cytokine [4], is a key regulator of the immune system and is essential for homeostasis of the immune system [5]. IL-6, secreted by T cells and macrophages to stimulate immune response, is an interleukin that can exhibit both, pro-inflammatory and anti-inflammatory functions. The aetiology of the “insulin resistance syndrome” caused by sepsis or metabolic syndrome is multifactorial, and the mechanisms underlying the intercorrelations among these metabolic conditions are not entirely clear. Although the involvement of adipose tissue hormones in the obesity-induced insulin resistance has been studied, there is only few information about its changes in criti- cally ill patients [18]. Another class of proinflammatory peptides are chemo- kines that selectively recruit monocytes, neutrophils, and lymphocytes and facilitate the passage from the circula- tion into the tissues. The prototypical chemokine is the neutrophil chemoattractant IL-8 [6]. Monocyte che- moattractant protein-1 (MCP-1) is another key chemo- kine that regulates migration and infiltration of monocytes and macrophages [7]. Background Therefore, the present study was performed to deter- mine a serum adipocytokine profile in critically ill patients which was subsequently compared with the profile of morbidly obese patients and a healthy control group. Further, we investigated if concentrations of adi- pocytokines are shifted in the same direction in critically ill septic patients as in morbidly obese patients com- pared to healthy controls. If so, which group has higher resultant changes? These results might increase our knowledge whether underlying mechanisms for insulin resistance are comparable in both groups of patients. Many of the above mentioned cytokines such as IL-6 or IL-8 are not only synthesized by blood cells, but also by adipose tissue [8]. Particularly IL-6 could provide a potential link between fatty tissue and systemic inflam- mation [9]. In contrast with classical views, adipose tissue not only provides a depot for fat storage but has been increas- ingly recognized as an important endocrine organ, man- ufacturing adipokines, bioactive molecules including adiponectin, leptin, and resistin, and proinflammatory cytokines [10]. Below, the adipose tissue derived hor- mones adiponectin, leptin, and resistin are termed adipokines. Statistical analysis All values were expressed as median with range. When comparing data between the three groups, i. e., SP, MO and BD, statistical analysis was performed using the Mann-Whitney U test. Analysis was performed with WinSTAT, Version 2009.1 (R. FitchSoftware). The Spearman rank-order correlation coefficient was calculated for correlation analysis. R indicates the corre- lation coefficient. A value of p < 0.05 was considered statistically significant. Correlation coefficient values between 0.3 and 0.7 (-0.3 and -0.7) indicate a moderate, values between 0.7 and 1.0 (-0.7 and -1.0) indicate a strong positive (negative) linear relationship. No adjust- ment for multiple testing was done. Patients and controls Septic patients BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Body weight and height for calculation of body mass index (BMI) of septic patients was self reported (or esti- mated if no self-report was possible). well) was added to the wells and then incubated on a plate shaker in the dark at room temperature. There- after, samples were washed three times with 100 μl wash buffer. Fifty microliters of strepavidin-PE (16 μg/ ml in assay buffer) was added to each well, and incuba- tion continued on a plate shaker at room temperature for the first 10 min. Unbound analytes were filtered through the wells using the vacuum manifold. The bound beads were washed three times with 100 μl wash buffer. After the last wash step, 125 μl of assay buffer was added to each well and the plate was placed on a plate shaker set at 500 rpm for 1 min and then for 3 min at a reduced speed of 300 rpm. Finally, samples were analyzed on the Luminex system in accordance with the manufactures’ instruction. As far as reaching the lowest traceable values, the sensitivity of the method was 0.3 ng/ml for leptin and 9.2 pg/ml for IL-1a. Cytokine and adipocytes-derived hormones measurement and reagents Multiplex analysis kits for IL-1a, IL-6, IL-8, IL-10, TNFa, MCP-1, active PAI-1, leptin, resistin, adiponectin were obtained from Millipore, Hamburg, Germany. In brief, the appropriate cytokine standards and samples, diluted in plasma dilution buffer, were added to wells of a filtered plate. The samples were incubated with 50 μl of the antibody -coupled microsphere set on a plate sha- ker in the dark at room temperature for 30 min. The samples were washed three times with 100 μl wash puf- fer. Freshly diluted secondary detection antibody (25 μl/ Morbidly obese patients and controls 37 consecutive patients operated for morbid obesity (MO) at the author’s hospital and 60 healthy blood donors (BD) were enrolled as the group of morbidly obese patients and as the control group, respectively. Blood samples from patients diagnosed with MO were collected preoperatively at 7 a.m. in fasting state. Sam- ples of BD were taken at various times in no fasting state (10 ml venous blood, collected in a chilled syringe with EDTA). Anthropometric data (age and BMI) of patients of all three groups are listed in table 1. The study was performed with the permission of the independent local ethics committee of the University of Ulm (approvals 112/2003 and 73/2009). An informed consent of all patients was necessary. If the patient was not capable of making decisions because of sedation or altered mental state, the informed consent was obtained after recovery. Exclusion criteria were: age < 18 years, pregnancy, rapidly progressing underlying disease, HIV/ AIDS, cardiogenic shock as the primary underlying dis- ease, underlying hematologic disease, or cytotoxic ther- apy given within the previous 6 months. Patients and controls Septic patients Whereas adiponectin and resistin are involved in glu- cose metabolism and exhibit anti-inflammatory features, leptin plays an important role in feeding behavior and exhibits proinflammatory features [11]. Plasminogen activator inhibitor-1 (PAI-1), secreted by adipose tissue has been shown to be an important inhibitor of fibrinolysis. 33 patients in intensive care who fulfilled the clinical criteria for severe sepsis or septic shock were prospec- tively enrolled in this study. The criteria for severe sep- sis and septic shock were in accordance with those defined by Bone [19]. The Simplified Acute Physiology Score (SAPS II) and Sequential Organ Failure Assess- ment score (SOFA) without Glasgow coma scale (GCS) were used to define the severity of disease and organ dysfunctions, respectively [20-22]. The underlying causes of sepsis were intestinal perforation (e.g. perforated ulcer, perforated diverticular disease; n = 11), inflamma- tion (e.g. necrotizing pancreatitis, necrotizing fasciitis; n = 11), postoperative complication (e.g. infections, fis- tula, anastomosis-insufficiency, n = 7), ischemia (n = 2), and trauma (n = 2). The 33 septic patients (SP) had median (range) SAPS II and SOFA scores of 40 (17-60) and 9 (3-14), respectively. Seven patients had pre-exist- ing type II diabetes. The expressions of these adipokines and cytokines are changed in obese persons, since obesity is associated with the appearance of a chronic, low inflammatory state due to changes in adipocyte and macrophage func- tion [12]. Obesity leads to lowered levels of the anti- inflammatory adipokine adiponectin in circulation [13]. Adiponectin is known to be a regulator of insulin sensi- tivity and glucose metabolism. Furthermore, hypoadipo- nectinemia has been shown to be associated with insulin resistance and hyperinsulinemia [14]. Insulin resistance is one key feature of “metabolic syndrome”, character- ized by a distinct combination of metabolic disorders such as elevated triglycerides, reduced high densitiy lipo- proteins, and/or enhanced fasting plasma glucose levels [15]. As obesity and metabolic syndrome, severe sepsis and septic shock are linked to increased blood glucose levels and insulin resistance even without a previous his- tory of diabetes [16]. Blood samples were collected one day after diagnosis of severe sepsis or septic shock at 5 a.m. in the fasting state (10 ml venous blood, collected in a chilled syringe with EDTA). Serum was obtained by centrifugation (1800×g for 15 min), and the samples were subsequently stored in aliquots at -80°C until further analysis. Page 3 of 9 Hillenbrand et al. Results Adipocytokine levels are clearly changed in septic and obese patients. For a better overview, serum concentra- tions of adiponectin, leptin, resistin, TNF-a, MCP-1, active PAI-1, IL-1a, IL-6, IL-8, IL-10 are shown in table 2 and figs. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Table 1 Gender specific anthropometric data (age and BMI) of patients with severe sepsis or septic shock (SP), morbidly obesity (MO), and healthy control group (BD) Age [years] (median; range) BMI [kg/m2] (median; range) SP (n = 33) 66 years (30 - 87) 26 kg/m2 (18 - 37) Female patients (n = 12) 81 years (42 - 87) 26 kg/m2 (20 - 32) Male patients (n = 21) 65 years (31 - 85) 26 kg/m2 (18 - 37) MO (n = 37) 45 years (17 - 59) 52 kg/m2 (33 - 78) Female patients (n = 24) 46 years (23 - 59) 52 kg/m2 (35 - 78) Male patients (n = 13) 42 years (17 - 59) 49 kg/m2 (33 - 70) Control group (n = 60) 45 years (19 - 71) Not ascertained Female control (n = 30) 44 years (19 - 60) Not ascertained Male control (n = 30) 46 years (19 - 71) Not ascertained Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Page 4 of 9 Table 2 Gender specific adiponectin and leptin levels and resistin, MCP-1, active PAI-1, TNF-a, IL-1a, IL-6, IL-8, and IL-10 levels of septic patients (SP), morbidly obese (MO) and controls (BD) Parameters (normal values) SP (n = 33) (Median; range) MO (n = 37) (Median; range) BD (n = 60) (Median; range) Change SP vs. BD Change MO vs. Adipokines (Adiponectin, Resistin, and Leptin) BD, all other cytokines in MO were significantly ele- vated. With the exception of active PAI-1, cytokine levels in SP were clearly higher compared to MO. In SP, adiponectin was lowered and resistin elevated compared to BD. Further, adiponectin correlated nega- tively with the SAPS II (r = -0.30; p = 0.043) and SOFA scores (r = -0.31; p = 0.042), while resistin correlated positively (SAPS II: r = 0.35; p = 0.023; SOFA: r = 0.30; p = 0.045). Leptin levels were unchanged in SP com- pared to BD. BMI correlated negatively with adiponectin (r = 0.32; p = 0.036) and positively with the leptin levels (r = 0.64; p = < 0.001) in SP. In MO, gender specific adiponectin was lowered and leptin elevated compared to BD. Resistin was unaltered. Adiponectin was equally diminished in SP and MO compared to BD. Again, in SP there were positive correlations of active PAI-1, IL-6, IL-8, MCP-1, and IL-10 with SAPS II and SOFA scores. IL-1a and TNF-a showed no correlation with SAPS II and SOFA scores. P-values and correlation coefficients with SAPS II and SOFA scores are: active PAI-1 (p = 0.032, r = 0.33; p = 0.021. r = 0.34), IL-6 (p = 0.006, r = 0.45; p = 0.030, r = 0.39), IL-8 (p = 0.032, r = 0.31; p = 0.012, r = 0.41), MCP-1 (p = 0.082, r= 0.29; p = 0.043, r = 0.33), IL-10 (p = 0.040, r = 0.28; p = 0.074, r = 0.26), IL-1a (p = 0.118, r = -0.21; p = 0.389, r = -0.05) and TNF-a (p = 0.252, r = 0.13; p = 0.419, r = 0.04). As in BD and MO, there was a profound gender dif- ference in adiponectin levels between male and female SP (male median levels: 9.3 μg/ml vs. female median levels: 13.2 μg/ml; p = 0.017). Results BD Adiponectin (μg/ml) 10.3 (2.9 - 47.6) 9.4 (3.8 - 57.8) 14.7 (5.7 - 54.0) ↓ p = < 0.01 ↓ p = < 0.01 Adiponectin male (μg/ml) 9.5 (2.9 - 32.6) 8.3 (3.8 - 12.3) 11.8 (5.7 - 30.7) ↓ p = 0.04 ↓ p = < 0.01 Adiponectin female (μg/ml) 13.2 (5.3 - 47.6) 12.4 (5.3 - 57.8) 22.5 (9.8 - 54.0) ↓ p = 0.02 ↓ p = < 0.01 Leptin (ng/ml) 6.3 (0.3 - 80.7) 45.0 (1.7 - 97.5) 10.8 (0.3 - 58.5) no significant change ↑ p = < 0.01 Leptin male (ng/ml) (< 7 ng/ml) 6.3 (0.3 - 24.7) 34.2 (1.7 - 90.9) 4.6 (0.3 - 19.1) no significant change ↑ p = < 0.01 Leptin female (ng/ml) (< 18 ng/ml) 8.2 (0.9 - 80.7) 47.3 (16.1 - 97.5) 15.8 (0.7 - 58.5) no significant change ↑ p = < 0.01 Resistin (ng/ml) 78.1 (13.4 - 52.3) 9.9 (3.7 - 83.6) 10.8 (5.2 - 24.9) ↑ p = < 0.01 no significant change MCP-1 (pg/ml) 381.5 (72.3 - 887.3) 108 (39.6 - 293) 43.9 (12.1 - 150) ↑ p = < 0.01 ↑ p = < 0.01 Active PAI-1 (ng/ml) 35.9 (11.5 - 160.3) 57.5 (3.7 - 176.7) 6.0 (2.4 - 31.3) ↑ p = < 0.01 ↑ p = < 0.01 TNF-a (pg/ml) 13.1 (2.4 - 65.5) 4.0 (2.4 - 26.8) 3.9 (1.2 - 10.9) ↑ p = < 0.01 no significant change IL-1a (pg/ml) 68.6 (9.2 - 203.3) 59.4 (9.2 - 210.0) 11.0 (9.2 - 168) ↑ p = 0.04 ↑ p = 0.01 IL-6 (pg/ml) 163.6 (14.4 - 10000) 2.9 (0.9 - 64.2) 0.7 (0.4 - 568.6) ↑ p = < 0.01 ↑ p = < 0.01 IL-8 (pg/ml) 59.4 (4.6 - 1335.6) 3.7 (0.3 - 38.8) 9.8 (0.9 - 126.7) ↑ p = < 0.01 ↓ p = < 0.01 IL-10 (pg/ml) 26.7 (3.9 - 276.7) 2.5 (2.4 - 8.6) 1.2 (0.9 - 1038) ↑ p = < 0.01 ↑ p = 0.01 ↓↑: Significant increase/decrease; Table 2 Gender specific adiponectin and leptin levels and resistin, MCP-1, active PAI-1, TNF-a, IL-1a, IL-6, IL-8, and IL-10 levels of septic patients (SP), morbidly obese (MO) and controls (BD) Adipokines (Adiponectin, Resistin, and Leptin) Age dependency of values Since SP were significantly older compared to MO and BD patients, it is necessary to consider an age depen- dency of values. Cytokines (TNFa, MCP-1, active PAI-1 and interleukins) Cytokines (TNFa, MCP-1, active PAI-1 and interleukins) All determined cytokines had significantly higher values in SP compared to BD. In MO, IL-8 was the only assessed value that showed a significant fall compared to Cytokines (TNFa, MCP-1, active PAI-1 and interleukins) In BD, female adiponectin showed certain correlation with age (r = 0.34; p = 0.032; male patients: r = -0.26; Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Hillenbrand et al. BMC Surgery 2010, 10:26 Page 5 of 9 Figure 1 Gender specific serum concentrations of adiponectin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). The top and bottom of the rectangle represent the 25th and 75th percentile. The line within the rectangle represents the median. The whiskers extend from the 5th percentile to the 95th percentile. Figure 3 Serum concentrations of resistin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 1 Gender specific serum concentrations of adiponectin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). The top and bottom of the rectangle represent the 25th and 75th percentile. The line within the rectangle represents the median. The whiskers extend from the 5th percentile to the 95th percentile. Figure 3 Serum concentrations of resistin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). control group of healthy BD. Moreover, most cytokine concentrations were greater in sepsis patients than in the morbidly obese patients. p = 0.085). There was no correlation with patients’ age in BD concerning female/male leptin, resistin, active PAI-1, MCP-1, IL-1a, IL-6, IL-8; IL-10, and TNF-a. Values of MO and SP showed no correlation with age. p = 0.085). There was no correlation with patients’ age in BD concerning female/male leptin, resistin, active PAI-1, MCP-1, IL-1a, IL-6, IL-8; IL-10, and TNF-a. Values of MO and SP showed no correlation with age. Seven patients of the sepsis group had preexisting type 2 diabetes. These seven patients showed no difference in adiponectin, resistin, leptin, active PAI-1, MCP-1, IL-1a, IL-6, IL-8, IL-10, and TNF-alpha levels. Adipokines Our results revealed that the only known adipose tissue derived factor with major insulin-sensitizing and anti- inflammatory properties, adiponectin, is equally dimin- ished in SP and MO. Adiponectin is the most abundant protein produced by adipose tissue and its blood con- centration is much higher than the concentration of other known hormones. Normal serum adiponectin levels are higher in women compared to men. Adipo- nectin attenuates inflammatory actions on several levels. It suppresses function of mature macrophages and inhi- bits growth of macrophage precursors [23]. Further, adiponectin attenuates the production of TNF-a and IL-6 production in macrophages and induces that of IL-10 [24]. Thus, the lower levels of adiponectin in the Seven patients of the sepsis group had preexisting type 2 diabetes. These seven patients showed no difference in adiponectin, resistin, leptin, active PAI-1, MCP-1, IL-1a, IL-6, IL-8, IL-10, and TNF-alpha levels. Discussion In this study we determined serum levels of classical cytokines and adipokines in septic and morbidly obese patients and compared them to healthy blood donors. The most important finding of this study is that both, SP and MO have similarly shifted values compared to a Figure 2 Gender specific serum concentrations of leptin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 2 Gender specific serum concentrations of leptin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 4 Serum concentrations of MCP-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 4 Serum concentrations of MCP-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 2 Gender specific serum concentrations of leptin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 2 Gender specific serum concentrations of leptin in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 4 Serum concentrations of MCP-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Hillenbrand et al. BMC Surgery 2010, 10:26 Page 6 of 9 Figure 5 Serum concentrations of active PAI-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 7 Serum concentrations of IL-1a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 5 Serum concentrations of active PAI-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 7 Serum concentrations of IL-1a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 7 Serum concentrations of IL-1a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 5 Serum concentrations of active PAI-1 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). present study in the septic and morbid obese patients in comparison to healthy controls might have contributed to the higher levels of IL-6 and TNF-a. Otherwise, adi- ponectin has an insulin sensitizing effect. Adiponectin levels decrease in parallel with the progression of insulin resistance and type 2 diabetes [25], and the antidiabetic drug thiazolidinedione increases adiponectin levels [26]. Given the anti-inflammatory effects of adiponectin, it is plausible that lowered adiponectin levels may predispose to sepsis-related proinflammatory complications in states of obesity, diabetes, and insulin resistance. Cytokines Figure 9 Serum concentrations of IL-8 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). y Several studies suggest that TNF-a and IL-6 are both involved in obesity-related insulin resistance and that TNF-a is one of the most important mediators of inflammation [43]. TNF-a is not secreted by adipocytes but by infiltrating macrophages in adipose tissue, whereas adipose tissue is a significant source of IL-6 [44]. Expression and secretion of TNF-a increases with obesity and correlates positively with body mass index [45]. TNF-a and IL-6 are known to promote lipolysis and the secretion of free fatty acids, which contributes to an increase in hepatic glucose production and insulin resistance [46]. On a cellular level, TNF-a is a potent inhibitor of the insulin-stimulated tyrosine phosphoryla- tions on the beta-chain of the insulin receptor and insu- lin receptor substrate-1, suggesting that TNF-a may play a crucial role in the systemic insulin resistance of non insulin dependent diabetes mellitus [47]. In analogy, the elevated IL-6 levels in our septic and morbidly obese patients may contribute to insulin resistance. In contrast to the morbidly obese patients, in addition, the elevated TNF-a levels in our septic patients might play a role in insulin resistance. Since pro-inflammatory cytokines were higher in SP than in MO, this could have led to the higher resistin levels in SP than in MO in our study. Otherwise, the higher resistin levels in SP compared to MO may have contributed to the higher pro-inflammatory cytokines in SP compared to MO. In our SP and MO patients, as well as in prior studies of septic patients [35,36], resistin did not correlate to obesity measured by BMI which suggests that in circumstances of critical illness the release of resistin by macrophages plays a superior role compared with the secretion from adipocytes. This could possibly also explain why there was no difference in resistin levels of patients with preexisting diabetes mellitus. Leptin was the only measured adipokine in the present study that was marginally reduced in SP compared to BD. The MO group had significantly elevated levels. In the present study, normal leptin serum levels were higher in women compared to men. In the obese, Human fat cells are also known to produce proinflam- matory IL-8 which was increased in insulin-resistant subjects [48]. Surprisingly, we found reduced levels of IL-8 in MO compared to BD. Discussion On the other hand, the reduced adiponectin levels in our sepsis patients might support insulin resistance. Several other studies also showed lower adiponektin levels in septic patients [27,28]. obesity and causing insulin resistance in mice [30]. In contrast to mice, resistin in humans is mainly derived from macrophages rather than adipocytes [31]. In humans, resistin is a protein with proinflammatory properties. Its secretion is stimulated by inflammatory processes, glucocorticoids and lipopolysaccharides (LPS), whereas TNF-a and b-adrenergic stimulation act as inhibitors [32]. Resistin has been shown to increase transcriptional events leading to an increased expression of several pro-inflammatory cytokines including IL-1, IL-6, IL-12, and TNF-a [33]. In a posi- tive feedback loop, resistin can be up-regulated by interleukins, and also by microbial antigens such as LPS [34]. In accordance with these reports, we found significantly higher resistin levels in SP, but not in MO, compared to healthy BD supporting the hypoth- esis that in humans resistin is predominantly a compo- nent of the systemic inflammatory response to infection. Besides adiponectin, resistin has been reported to participate in the inflammatory response [29]. Resistin was originally discovered in mice as an adipocytes derived hormone. It was found to be increased in Figure 6 Serum concentrations of TNF-a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 8 Serum concentrations of IL-6 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 6 Serum concentrations of TNF-a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 8 Serum concentrations of IL-6 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 8 Serum concentrations of IL-6 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 8 Serum concentrations of IL-6 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Figure 6 Serum concentrations of TNF-a in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Page 7 of 9 Page 7 of 9 increased fat storage will lead to enhanced leptin levels [37]. Leptin can affect glucose metabolism and increases insulin sensitivity. Obese humans are often insulin- and leptin-resistant [38]. The role of leptin in sepsis and sep- tic shock is controversial. Discussion Earlier reports suggested that high lepin levels are associated with increased survival in sepsis and septic shock [39,40], several other reports -such as our study- fail to show a correlation between leptin and sepsis [41]. Our minor changes of leptin serum levels in SP are in line with a reported temporal decrease in leptin after operative stress followed by a subsequent rise slightly above the initial levels [42]. Figure 9 Serum concentrations of IL-8 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Received: 14 February 2010 Accepted: 9 September 2010 Published: 9 September 2010 Received: 14 February 2010 Accepted: 9 September 2010 Published: 9 September 2010 Author details 1 f 1Department of General-, Visceral-, and Transplantation Surgery, University Hospital of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany. 2Department of Anesthesiology University Hospital of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany. 3Institute of Clinical Tranfusion Medicine and Immunogenetics, Helmholtzstr. 10; D-89081 Ulm; Germany. 4Department of Traumatology, Hand and Reconstructive Surgery, University Hospital of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany. 18. Haluzik M, Parizkova J, Haluzik MM: Adiponectin and its role in the obesity-induced insulin resistance and related complications. Physiol Res 2004, 53:123-129. 19. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis: the ACCP/SCCM Consensus Conference Committee: American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992, 101:1644-1655. References Deshmane SL, Kremlev S, Amini S, Sawaya BE: Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 2009, 29:313-26. 7. Deshmane SL, Kremlev S, Amini S, Sawaya BE: Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 2009, 29:313-26. 8. Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW: Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 2004, 145:2273-82. Cytokines Serum levels of MO patients (median: 3.7 pg/ml) match reported levels in the obese. Serum level of BD seems (median: 9.8 pg/ml) markedly elevated compared to reported levels of approximately 3.2 pg/ml [49]. Insofar, reported IL-8 levels should be interpreted with care. Nevertheless, the higher IL-8 levels in septic patients compared to the morbidly obese patients underline the infection induced origin of IL-8 in our study. Figure 10 Serum concentrations of IL-10 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). The presented study has several limitations. In the healthy control group, there was no investigation of hid- den or not yet clinically manifest side diagnoses. Conse- quently, there is no information about type 2 diabetes or other chronic inflammatory diseases without clinical manifestation. Moreover, the control group was Figure 10 Serum concentrations of IL-10 in septic (SP) and morbidly obese (MO) patients and healthy controls (BD). Page 8 of 9 Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Page 8 of 9 Acknowledgements h k D Acknowledgements We thank Dr. M. Templin, NMI, Tübingen, Germany for analyzing the cytokines. g We thank Dr. M. Templin, NMI, Tübingen, Germany for analyzing the cytokines. 16. Van Cromphaut SJ, Vanhorebeek I, Van den Berghe G: Glucose metabolism and insulin resistance in sepsis. Curr Pharm Des 2008, 14:1887-99. 17. Van den Berghe G, Wilmer A, Hermans G, Meersseman W, Wouters PJ, Milants I, Van Wijngaerden E, Bobbaers H, Bouillon R: Intensive insulin therapy in the medical ICU. N Engl J 2006, 354:449-461. Conclusion In conclusion, we could determine a sepsis specific adi- pocytokine profile in critically ill patients. In comparison with levels in a healthy control group, we observed a marked reduction of adiponectin and a profound increase of resistin, active PAI-1, MCP-1, IL-1a, IL-6, IL-8, IL-10, and TNF-a in septic patients. As in SP, MO showed reduced adiponectin levels and elevated MCP-1, active PAI-1, IL-1a, IL-6, and IL-10 levels. Leptin is only elevated in MO, while resistin, IL-8, and TNF-a is only elevated in SP. Taken together, the finding of increased levels of proinflammatory cytokines and altered levels of adipokines underlines its possible con- tribution in the development of insulin resistance in cri- tically ill patients with sepsis. 9. Fontana L, Eagon JC, Trujillo ME, Scherer PE, Klein S: Visceral fat adipokine secretion is associated with systemic inflammation in obese humans. Diabetes 2007, 56:1010-3. 10. Libby P, Okamoto Y, Rocha VZ, Folco E: Inflammation in atherosclerosis: transition from theory to practice. Circ J 2010, 74:213-20. 11. Mattioli B, Straface E, Matarrese P, Quaranta MG, Giordani L, Malorni W, Viora M: Leptin as an immunological adjuvant: enhanced migratory and CD8+ T cell stimulatory capacity of human dendritic cells exposed to leptin. Faseb J 2008, 22:2012-2022. 12. Ross R: Atherosclerosis - an inflammatory disease. N Engl J Med 1999, 340:115-126. 13. Fonseca-Alaniz MH, Takada J, Alonso-Vale MI, Lima FB: Adipose tissue as an endocrine organ: from theory to practice. J Pediatr (Rio J) 2007, 83(Suppl 5):192-203. 14. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA: Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 2001, 86:1930-5. 15. Wolf AM, Buffington C, Beisiegel U: Comparison of metabolic risk factors between severely and very severely obese patients. Obesity 2006, 14:2177-83. References 1. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis: the ACCP/SCCM Consensus Conference Committee: American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992, 101:1644-55. 1. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis: the ACCP/SCCM Consensus Conference Committee: American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992, 101:1644-55. 2. Cavaillon JM, Adib-Conquy M, Fitting C, Adrie C, Payen D: Cytokine cascade in sepsis. Scand J Infect Dis 2003, 35:535-44. 2. Cavaillon JM, Adib Conquy M, Fitting C, Adrie C, Payen D: Cytokine cascade in sepsis. Scand J Infect Dis 2003, 35:535-44. 3. Dinarello CA: Proinflammatory Cytokines. Chest 2000, 118:503-508. 4. Opal SM, DePalo VA: Anti-inflammatory cytokines. Chest 2000, 117:1162-72 5. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009, 9:447-53. 2. Cavaillon JM, Adib Conquy M, Fitting C, Adrie C, Payen D: Cytokine cascade in sepsis. Scand J Infect Dis 2003, 35:535-44. 3. Dinarello CA: Proinflammatory Cytokines. Chest 2000, 118:503-508. 4. Opal SM, DePalo VA: Anti-inflammatory cytokines. Chest 2000, 117:1162-72 5. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009, 9:447-53. 3. Dinarello CA: Proinflammatory Cytokines. Chest 2000, 118:503-508. 4. Opal SM, DePalo VA: Anti-inflammatory cytokines. Chest 2000, 117:1162-72 5. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009, 9:447-53. 5. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009, 9:447-53. It is also important to note that many other factors in addition to proinflammatory cytokines such as cortisol, catecholamines, growth hormone, and other stress- related factors can significantly contribute to the devel- opment of insulin resistance in critically ill patients [50]. 6. Waugh DJ, Wilson C: The interleukin-8 pathway in cancer. Clin Cancer Res 2008, 14:6735-41. 6. Waugh DJ, Wilson C: The interleukin-8 pathway in cancer. Clin Cancer Res 2008, 14:6735-41. 7. Competing interests significantly younger than the sepsis group and blood samples were not taken in fasting state in the control group. However, only leptin showed a significant corre- lation with the blood donor’s age in the control group, whereas resistin, IL-1a, IL-6, and IL-8 did not. Thus, the differences between these values in the septic patients group and control group may be less striking. Further, no weight/height for BMI calculation was ascer- tained in BD group. Another crucial points are the small number and the heterogeneousness of the septic group including patients with different cause of sepsis. How- ever, this reflects the typical clinical situation and case mix in intensive care units. Received: 14 February 2010 Accepted: 9 September 2010 Published: 9 September 2010 Pre-publication history h bl h 29. Nagaev I, Bokarewa M, Tarkowski A, Smith U: Human resistin is a systemic immune-derived proinflammatory cytokine targeting both leukocytes and adipocytes. PLoS ONE 2006, 1:e31. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2482/10/26/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2482/10/26/prepub doi:10.1186/1471-2482-10-26 Cite this article as: Hillenbrand et al.: Sepsis induced changes of adipokines and cytokines - septic patients compared to morbidly obese patients. BMC Surgery 2010 10:26. doi:10.1186/1471-2482-10-26 Cite this article as: Hillenbrand et al.: Sepsis induced changes of adipokines and cytokines - septic patients compared to morbidly obese patients. BMC Surgery 2010 10:26. 30. Steppan CM, Lazar MA: Resistin and obesity-associated insulin resistance. Trends Endocrinol Metab 2002, 13:18-23. 31. Patel L, Buckels AC, Kinghorn IJ, Murdock PR, Holbrook JD, Plumpton C, Macphee CH, Smith SA: Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators. Biochem Biophys Res Commun 2003, 300:472-6. 32. Fonseca-Alaniz MH, Takada J, Alonso-Vale MI, Lima FB: Adipose tissue as an endocrine organ: from theory to practice. J Pediatr (Rio J) 2007, 83(Suppl 5):S192-203. 33. Silswal N, Singh AK, Aruna B, Mukhopadhyay S, Ghosh S, Ehtesham NZ: Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway. Biochem Biophys Res Commun 2005, 334:1092-101. y 34. Lu SC, Shieh WY, Chen CY, Hsu SC, Chen HL: Lipopolysaccharide increases resistin gene expression in vivo and in vitro. FEBS Lett 2002, 530:158-62. 35. Sundén-Cullberg J, Nyström T, Lee ML, Mullins GE, Tokics L, Andersson J, Norrby-Teglund A, Treutiger CJ: Pronounced elevation of resistin correlates with severity of disease in severe sepsis and septic shock. Crit Care Med 2007, 35:1536-42. 36. Koch A, Gressner OA, Sanson E, Tacke F, Trautwein C: Serum resistin levels in critically ill patients are associated with inflammation, organ dysfunction and metabolism and may predict survival of non-septic patients. Crit Care 2009, 13:R95. 37. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995, 269:543-6. 38. van den Hoek AM, Teusink B, Voshol PJ, Havekes LM, Romijn JA, Pijl H: Leptin deficiency per se dictates body composition and insulin action in ob/ob mice. J Neuroendocrinol 2008, 20:120-7. 39. Authors’ contributions Rotter V, Nagaev I, Smith U: Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-alpha, overexpressed in human fat cells from insulin-resistant subjects. J Biol Chem 2003, 278:45777-84. 25. Spranger J, Kroke A, Möhlig M, Bergmann MM, Ristow M, Boeing H, Pfeiffer AF: Adiponectin and protection against type 2 diabetes mellitus. Lancet 2003, 361:226-8. 26. Yu JG, Javorschi S, Hevener AL, Kruszynska YT, Norman RA, Sinha M, Olefsky JM: The effect of thiazolidinediones on plasma adiponectin levels in normal, obese, and type 2 diabetic subjects. Diabetes 2002, 51:2968-74. 49. Kremen J, Dolinkova M, Krajickova J, Blaha J, Anderlova K, Lacinova Z, Haluzikova D, Bosanska L, Vokurka M, Svacina S, Haluzik M: Increased subcutaneous and epicardial adipose tissue production of proinflammatory cytokines in cardiac surgery patients: possible role in postoperative insulin resistance. J Clin Endocrinol Metab 2006, 91:4620-7. 27. Venkatesh B, Hickman I, Nisbet J, Cohen J, Prins J: Changes in serum adiponectin concentrations in critical illness: a preliminary investigation. Crit Care 2009, 13:R105. 50. Van den Berghe G: Neuroendocrine pathobiology of chronic critical illness. Crit Care Clin 2002, 18:509-528. 50. Van den Berghe G: Neuroendocrine pathobiology of chronic critical illness. Crit Care Clin 2002, 18:509-528. 28. Langouche L, Vander Perre S, Frystyk J, Flyvbjerg A, Hansen TK, Van den Berghe G: Adiponectin, retinol-binding protein 4, and leptin in protracted critical illness of pulmonary origin. Crit Care 2009, 13:R112. Authors’ contributions 20. Le Gall JR, Klar J, Lemeshow S, Saulnier F, Alberti C, Artigas A, Teres D: A New Simplified Acute Physiology Score (SAPS II) Based on a European/ North American Multicenter Study. JAMA 1993, 270:2957-2963. All authors have contributed substantially to the submitted work and have read and revised the manuscript and approved the final version. In particular AH participated in the design of the study, data acquisition, analysis and drafting of the manuscript. UK participated in data acquisition, analysis and drafting of the manuscript. MW assessed and recorded sepsis scores and participated in the drafting of the manuscript, HS took blood samples of control group, DHB initialized the work, provided laboratory results and gave approval for submission. AMW and MHL provided general idea, initialized the work, took blood samples, provided laboratory results and got ethical approval. 21. Teasdale G, Jennett B: Assessment of coma and impaired consciousness. A practical scale. Lancet 1974, 2:81-84. 22. Janssens U, Dujardin R, Graf J, Lepper W, Ortlepp J, Merx M, Zarse M, Reffelmann T, Hanrath P: Value of SOFA (Sequential Organ Failure Assessment) score and total maximum SOFA score in 812 patients with acute cardiovascular disorders. Critical Care 2001, 5(Suppl 1):P225. Page 9 of 9 Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 23. Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, Kihara S, Funahashi T, Tenner AJ, Tomiyama Y, Matsuzawa Y: Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2000, 96:1723-32. 45. Sethi JK, Hotamisligil GS: The role of TNF alpha in adipocyte metabolism. Semin Cell Dev Biol 1999, 10:19-29. 46. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM: Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 1995, 95:2409-15. 24. Wulster-Radcliffe MC, Ajuwon KM, Wang J, Christian JA, Spurlock ME: Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun 2004, 316:924-9. 47. Hotamisligil GS, Spiegelman BM: Tumor necrosis factor alpha: a key component of the obesity-diabetes link. Diabetes 1994, 43:1271-8. 47. Hotamisligil GS, Spiegelman BM: Tumor necrosis factor alpha: a key component of the obesity-diabetes link. Diabetes 1994, 43:1271-8. 48. Hillenbrand et al. BMC Surgery 2010, 10:26 http://www.biomedcentral.com/1471-2482/10/26 Pre-publication history h bl h Bornstein SR, Licinio J, Tauchnitz R, Engelmann L, Negrão AB, Gold P, Chrousos GP: Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm, in cortisol and leptin secretion. J Clin Endocrinol Metab 1998, 83:280-3. 40. Maruna P, Gurlich R, Fraska R, Haluzik M: Serum Leptin Levels in Septic Men Correlate Well with C-Reactive Protein (CRP) and TNF-alpha but not with BMI. Physil Res 2001, 50:589-594. 40. Maruna P, Gurlich R, Fraska R, Haluzik M: Serum Leptin Levels in Septic Men Correlate Well with C-Reactive Protein (CRP) and TNF-alpha but not with BMI. Physil Res 2001, 50:589-594. 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: y 41. Vachharajani V: Influence of obesity on sepsis. Pathophysiology 2008, 15:123-34. 41. Vachharajani V: Influence of obesity on sepsis. 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https://openalex.org/W4321350995	https://jfin-swufe.springeropen.com/counter/pdf/10.1186/s40854-023-00458-6	English	null	Adverse selection, loan access and default behavior in the Chilean consumer debt market	Financial innovation	2,023	cc-by	17,322	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate- rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. Abstract Why do households use different types of loans? Which factors cause borrowers to default? Using a comprehensive survey dataset from Chile, I estimate a partial infor- mation model of consumer debt access, lender choice, loan amount and default. The model consists of a first-stage multinomial logit that explains the choice across the five loan types, plus the options of no access to debt due to credit constraints and a no wish for consumer debt. In the second and third stages, the model assumes a log-linear regression of the debt amount and a logit regression of the default behav- ior, accounting for the loan type selection probability. Identification is obtained using factors measured at different time periods for the default and the loan type choices. I find that households choose different lenders based on income, education and labor risks. Higher income and education decrease the probability of credit constraints, while increasing bank lending and debt amounts. Unemployment risk and household size increase the chances of all the loan types; however, unemployment decreases the debt amount. Age and wage volatility reduce the probability of all loans. Default decreases with income, education and age, whereas it increases with indebtedness, unemploy- ment, household size, health shocks, and paying previous loans. Counterfactual exer- cises demonstrate that pension reform, higher requirements for borrowers’ capacities, and financial literacy programs could substantially reduce default risk. Financial literacy could greatly reduce arrears, households with credit constraints, the number of debtors and the aggregate debt amounts, especially for non-bank lending. Financial Innovation Financial Innovation Madeira Financial Innovation (2023) 9:49 https://doi.org/10.1186/s40854-023-00458-6 Open Access Open Access Adverse selection, loan access and default behavior in the Chilean consumer debt market Carlos Madeira* Correspondence: carlosmadeira2009@u. northwestern.edu Central Bank of Chile, Santiago, Chile Introduction Household debt has consistently increased in recent decades, both in emerging econo- mies and developed countries (Edelberg 2006), with strong financial innovation offering consumers a wide range of loan products (Agarwal et al. 2020). Many consumers can access credit from a variety of sources, such as credit cards, auto loans, or education loans, and with motives as diverse as health, vacations, purchase of durable goods, or a renegotiation of previous debts. However, the question of lender choice remains under- studied, especially in developing countries where consumer credit is widely used, often at high interest rates (Madeira 2019b) and high default risk (Madeira 2018). This paper examines the access to consumer credit, choice of lender, loan amount decision and repayment behavior of families in Chile. Consumer debt is defined as unsecured loans requested for consumption (unlike mortgages which are secured by real properties). Consumer loans are particularly relevant in Chile since over 60% of the households have some consumer debt. Using data from the Chilean Household Finance Survey (in Spanish, Encuesta Financiera de Hogares; EFH), I estimate an econometric model in which families choose among a variety of lender types according to their earn- ings, labor risk, demographics, and unobserved factors. I find that families are sorted among different lenders according to their observable characteristics.1 Furthermore, household debt levels, income, and labor market risk significantly impact default behav- ior. As expected, lenders offer higher loan amounts to households with better observable characteristics, but unobservable risks (known to borrowers but not to lenders, such as debt motives) make riskier borrowers borrow and default more. The loan market in Chile has many providers, which represent imperfect substitutes for consumers. These loan providers have access to different customer information and are subject to different regulations, which affects their loan terms and the ability to target borrowers. Loan screening costs, asymmetric information, brand preferences, marketing, search, travel costs, and tied products, can create substantial frictions for customers, so loan choice and repayment are modeled as a differentiated product choice (Hensher et al. 2015). The model of loan choice and repayment behavior has three main components: (i) the categorical choice between having no wish for debt, wanting debt but having no access, and five lender types; (ii) the loan amount decision; and (iii) the categorical outcome of whether the household defaulted or not on their debt. 1 Families are sorted among different lender types, either through self-selection according to their preferences or through screening (that is, selection from the lenders), most likely through a mix of both channels. In general terms, sorting is a methodology which groups individuals and solves a problem through a separating equilibrium. Sorting is especially useful in credit market problems, due to moral hazard and adverse selection (Jaffee and Stiglitz 1990). Highlights Chilean borrowers present heterogeneous adverse selection across lender types. No Debt Access decreases with income, age, education, but it increases with risk. Default is associated with income, unemployment, indebtedness and demographics. Paying past loans and health needs are associated with indebtedness and default. Financial literacy programs may be a powerful policy to improve the debt market. Keywords: Consumer credit, Default risk, Unemployment, Financial literacy, Adverse selection, Credit constraints Page 2 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Introduction The five lender types correspond to: Banks, Banks and Retail Stores, Retail Stores, Union Credit (i.e., loans provided by credit and labor unions), and Other Loans (including auto loans, educational debt, pawn shops and some informal lending). Banks and Retail Stores are the two major lenders in Chile, therefore using both lenders is treated as a separate choice from the option of using only one type of lender. Other types of lenders represent a small proportion of the population and therefore are not modeled. The option of “No Access to Debt” represents families with credit constraints. These are families who applied for consumer loans but were denied credit and those who wished to apply for credit but did not do so because Madeira Financial Innovation (2023) 9:49 Page 3 of 29 they expected to be refused. “No wish for Loans” represents the outside option for all agents, comprising the families who report not having consumer debt and no interest in applying for loans. Debt choices are affected by both observable and unobservable factors. The observables include income, education, labor income risk (measured by unemployment risk and wage volatility), the motives behind the loans, and demographic characteristics such as the age of the household head and household size. Unobservable factors, such as random-effects, include idiosyncratic tastes of households or bargaining conditions that affects preferences for all types of debt and banking debt. The lender type choice, modeled as a Mixed Multi- nomial Logit Model, is estimated by Simulated Maximum Likelihood (Hensher et al. 2015). The subsequent loan amount decision and the default behavior are estimated as second- step regressions, with the inverse Mill’s ratio of the lender type choice included as addi- tional control variables (Vella 1998). I demonstrate that households with different income, education, debt, unemployment risk and wage volatility are sorted among different lenders. Banks resort more to credit scoring and customer specific interest rates, therefore selecting the households of highest income and education and with the lowest unemployment rates among loan applicants. Bank plus Retail and Other debt borrowers are younger and have larger families, therefore they have the largest amount of debt. Their income is slightly lower and their unemploy- ment risk a bit higher than those of the Bank-only borrowers. Literature review This study is related to a growing literature of empirical models of household finance. It relates to the study of borrower heterogeneity and its impact on credit constraints, loan choice and default behavior (Einav et al. 2012). It is especially related to empiri- cal studies of household budgeting behavior (Habibah et al. 2018; Hamakhan 2020), default choices (Tang et al. 2020) and micro-founded applications of financial policies (Ahmed et al. 2018). It is also related to the studies that use household finance sur- veys to inform on credit frictions and policy (Eurosystem 2009; Gallardo and Madeira 2022), with these surveys being particularly important to obtain a complete view of borrowers’ loans because many households rely on a diversity of funding sources, including bank and non-bank lenders (Cull et al. 2019; Madeira 2018). Due to their complexity and cost, complete household finance surveys (comparable to the Survey of Consumer Finances in the USA or the eurozone Household Finance and Consump- tion Survey) are especially scarce in emerging markets and developing countries, hav- ing only started in Chile, China, Uruguay and Mexico in 2007, 2011, 2014 and 2019, respectively. Other studies have followed the goal of classifying consumers or SMEs (Small and Medium Enterprises) in terms of their bankruptcy, credit or fraud risks using large data- sets from a financial services provider such as payment and transactions networks, banks or credit card managers (Li et al. 2022). It is relevant to note that the credit risk literature differs substantially for SMEs and households (which are the final consumers). SMEs default or bankruptcy decisions depend on the accounting profitability, cashflow, or net present value of their investments (see Kou et al. 2014 and references therein). However, households tend to be more influenced by short-term shocks such as unemployment or health (Eurosystem 2009; Gallardo and Madeira 2022). Unlike SMEs, which can decide to implement bankruptcy or exit a market, households in Chile cannot declare bank- ruptcy (similar as in many other countries). Furthermore, in many countries the SMEs can be much larger than households or consumers, with SMEs having up to 250 employ- ees in the European Union and up to 500 employees in the USA (except for very rich households, most of the population tends to be employees, self-employed or owners of micro firms with 4 employees or less). Introduction Households with No Access to Debt have the lowest income and education levels, while experiencing the highest unem- ployment risk and wage volatility, which excludes them from the consumer loan market. The Union Debt borrowers are a case in the middle of the debt and labor income risk profiles, because these households have a low income level but also a low wage volatility. Therefore, these households also have a moderate level of indebtedness. Finally, among the households with access to debt, Retail Store only borrowers show the lowest income and education coupled with the highest unemployment risk. Therefore, such households have the lower debt amounts. Both education and income increase the probability of Banks, Banks-Retail and Other Debts, while decreasing the probability of No Access to Debt. Unemployment risk and household size increase the probability of opting for any loan type, while age and wage volatility decrease the probability of any loans. Unemployment risk has a greater effect on increasing the Retail Store and Other Debt probabilities. Wage volatility increases the prob- ability of No Access to Debt. Loan amounts increase with income, education and debt motivated by Durables, Pay Other Debts and Health needs, consistent with life cycle consumption patterns (Attana- sio and Weber 2010). Loan amounts are negatively associated with unemployment risk, age and the share of household debt financed by a single lender type. The probability of default decreases with income, education and age, while increasing with high indebtedness ratios, unemployment risk, household size, loans motivated by Paying Previous Debts and Health needs. The remainder of this paper is organized as follows. “Past research and credit market insti- tutions in Chile” section summarizes the credit literature and Chile’s regulatory environ- ment. “An empirical model of choice of lender, loan amount and default” section describes the model of loan choice and default. “Data: the Chilean Household Finance Survey (EFH)” Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Page 4 of 29 section summarizes the Chilean Household Finance Survey. “Characterization of borrow- ers’ loan options” section describes the households across loan types. “Results” section pre- sents the results of the model of lender choice, loan amount and default. “Counterfactual simulations of policies” section estimates the counterfactual effect of different policies on households’ choices. Finally, “Conclusions and policy implications” section concludes the study with a summary of the results and policy implications. Literature review This research article instead uses a small survey dataset with a range of variables that inform the factors behind the consumer decisions and which economic theories such as permanent income, precautionary behavior, lifecy- cle motives, or adverse selection, to explain consumer behavior.l Households borrow because of habit formation, life cycle changes, temporary fluc- tuations in income, consumption smoothing, and behavioral biases such as a lack of Madeira Financial Innovation (2023) 9:49 Page 5 of 29 Madeira Financial Innovation financial literacy and temptation (Attanasio and Weber 2010; Agarwal et al. 2020), with excessive debt being associated with psychological and economic distress (Agar- wal et al. 2020). Risk-based interest pricing, loan caps and higher down payments can reduce the adverse selection and moral hazard of borrowers (Einav et al. 2012). Risk- priced credit supply can reduce liquidity constraints and increase product demand, because riskier consumers are more sensitive to credit terms than to car prices (Einav et al. 2012). Finally, lender screening can control the borrowers’ adverse selection and moral hazard by offering different contracts (Jaffee and Stiglitz 1990). In summary, loan markets should present three results (Einav et al. 2012): (i) lenders will offer bet- ter and larger loans to agents with observable characteristics of low risk; (ii) unob- servable high risk characteristics will be associated with both larger loan amounts and default; and (iii) agents with very high risk will be credit constrained. This study is relevant, because it includes a substantial amount of information about the borrower, its loan application, its family and the labor risk factors of its members. The article, therefore, provides a rich perspective of the credit markets in a developing middle income country. Research on consumer preferences between different alternative products and default risk is increasingly relevant in recent years. Technological develop- ment increased the menu of financial products and the available information for credit risk scoring (Thakor 2020), with several banks across the world investing in FinTech to expand their services and products (Kou et al. 2021).hi This article extends the literature in five ways. First, it studies loan choice and default in a middle income country such as Chile (while most studies have been either with advanced economies, see Badarinza et al. (2016), or with low income countries, see Banerjee et al. (2015)). The structure of consumer loan providers in Chile In Chile all lenders have access to a commercial registry of debtors who defaulted on payments.2 However, this registry is limited only to negative events, and therefore, lend- ers’ information sets on the positive characteristics of loan applicants differ substantially (Cohen and Dijkman 2021). Additionally, Chilean banks have access to a common credit registry with information on all loan amounts and debt default within the banking system. However, banks do not observe non-banking loans (Cohen and Dijkman 2021). Banks also make strong use of credit scoring, according to agents’ history of credit and other products, such as direct deposit of wages, automatic bill payment or mortgages. Credit unions (denoted as Savings and Loans’ cooperatives3) and labor unions (denoted as Family Compensation Funds4) are another lender type, regulated as provid- ers of “social credit”. According to the legislation, all Chilean companies must register their workers with one among several Family Compensation Funds, which offer social credit and other services to their affiliates. These labor unions or Family Compensation Funds represent 67.6% of the aggregate “social credit”. Family Compensation Funds are chosen by each employer for all its workers, therefore, workers do not choose their insti- tution directly. Family Compensation Funds benefit from being able to deduct loan pay- ments directly from their clients’ wage payroll, and therefore, face little risk of default. Even when a debtor loses its job, Family Funds may deduct a substantial payment from the workers’ severance pay. Therefore, their risk is limited even in the face of unex- pected unemployment events. Union credit providers must offer the same conditions to all members. Therefore, unions can change interest rates according to loan size and maturity, but are unable to differentiate debtors according to their characteristics such as income. Furthermore, Family Compensation Funds cannot offer other financial ser- vices (for example, insurance or checking accounts) like banks do and cannot sell ordi- nary consumer appliances such as retail stores. This may put unions at a disadvantage in approaching customers if the search frictions and travel costs are significant. Finally, Family Compensation Funds cannot grow their assets through deposits, corporate debt or equity, which limits their size relative to banks and retail stores. Retail stores are another type of credit provider, with a strong brand image and their own credit cards,5 and access their own private databases of customers. 7 The regulation of credit by insurance companies is detailed in several norms of the Chilean Superintendency of Assets and Insurance, such as norms NCG 152 of 2002, NCG 208 of 2007 and NCG 247 of 2009. Literature review Second, it introduces a wider range of loan options, with borrow- ers being able to choose from banks, retail, credit unions or other lenders (such as auto sellers), whereas most previous studies have been limited to specific markets such as the banking sector, auto loans (Einav et al. 2012) and payday loans (Bertrand and Morse 2011), or a specific lender (Einav et al. 2012). Third, it uses a self-reported measured of households that do not wish for debt and it also includes households that expected to be rejected as being credit constrained. For instance, in administrative datasets, such as Einav et al. (2012), the researchers only observe the borrowers who applied for loans and were rejected, but there is no identification for those who did not apply thinking that a rejection was likely. Furthermore, such datasets often include only the loans of the bor- rowers with a single company and it is not measured whether the consumers obtained loans from other sources. Fourth, it measures unobserved preferences for loans by using tools from the applied product choice models in the field of industrial organization (Hensher et al. 2015). Fifth, it uses a more diverse characterization of labor income risk by separating overall risk into different variables such as unemployment risk and wage volatility, with such risks being measured for all the members of the household and not just the borrower. For instance, many studies of default risk only use the unemployment status of the borrower, although the spouse and other household members also present income shocks and risk factors. Also, some past studies such as Ampudia et al. (2016) consider unemployment, but not wage volatility. Page 6 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation 6 See www.dicrep.cl. 3 See the Chilean Government Department of Cooperatives, www.decoop.cl, the General Law of Cooperatives, DFL 5 (2003), www.bcn.cl, and Chapter III.C.2 of the Compendium of Financial Norms of the Central Bank of Chile. 4 These institutions are regulated by the Chilean Superintendency of Social Security. Each Family Compensation Fund is associated with one of the five labor unions registered at the Confederation of Production and Trade. See the General Statute of Family Compensation Funds, articles 29 to 31 of the Law N ◦ 18.833 of 1989. 5 The norms for non-banking credit card providers are detailed in the Chapter III.J.1 of the Compendium of Financial Norms of the Central Bank of Chile. 2 See www.dicom.cl/. 3 See the Chilean Government Department of Cooperatives, www.decoop.cl, the General Law of Cooperatives, DFL 5 (2003), www.bcn.cl, and Chapter III.C.2 of the Compendium of Financial Norms of the Central Bank of Chile. 4 These institutions are regulated by the Chilean Superintendency of Social Security. Each Family Compensation Fund is associated with one of the five labor unions registered at the Confederation of Production and Trade. See the General Statute of Family Compensation Funds, articles 29 to 31 of the Law N ◦ 18.833 of 1989. 5 The norms for non-banking credit card providers are detailed in the Chapter III.J.1 of the Compendium of Financial Norms of the Central Bank of Chile. 6 See www.dicrep.cl. 7 The regulation of credit by insurance companies is detailed in several norms of the Chilean Superintendency of Assets and Insurance, such as norms NCG 152 of 2002, NCG 208 of 2007 and NCG 247 of 2009. 2 See www.dicom.cl/. 2 See www.dicom.cl/. 3 See the Chilean Government Department of Cooperatives, www.decoop.cl, the General Law of Cooperatives, DFL 5 (2003), www.bcn.cl, and Chapter III.C.2 of the Compendium of Financial Norms of the Central Bank of Chile. 4 These institutions are regulated by the Chilean Superintendency of Social Security. Each Family Compensation Fund is associated with one of the five labor unions registered at the Confederation of Production and Trade See the General 10 Some consumers may have more than one debt type, say debt at Banks and Other Loans (for example, an educational loan), but except for retail store credit (which reaches around 7 million people in Chile) there are few observations with such interactions and such options represent a negligible amount of the consumer debt. 8 The aggregate amount of other loans (such as automotive and informal lending) is not entirely known, since credits of smaller and unregulated institutions do not need to be registered for statistical purposes. 9 Families with no consumer loans are classified in two categories: “No Access to Debt” and “No wish to apply for Con- sumer Debt”. “No Access to Debt” represents families who applied for credit but were denied and the ones who did not apply for credit because they expected to be refused. “No wish for Debt” is the outside option for all agents, comprising the families who report no consumer debt and no interest in applying for loans. The structure of consumer loan providers in Chile Retailers pro- vide few cash advances, but their credit cards are widely accepted by several stores, util- ity companies and merchants. Finally, there are lenders with more specific goals, such as auto loans at car dealers, education loans, pawn shops6, and consumer loans provided by insurance companies.7 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation (2023) 9:49 Page 7 of 29 Madeira Financial Innovation In Chile, during 2006, banks represented 62.1% of the total consumer credit, while social credit institutions represented 13.1% and retail stores 24.8% of the market, respec- tively.8 However, the market presence in terms of the number of customers differs from the aggregate loan amounts. There are 3.5 million debtors with banking loans, while social institutions and retail stores reach around 2.5 million and 7 million customers, respectively. Therefore, retail stores are actually the largest provider of small consumer loans and reach the widest number of customers. Over the last half-decade, the market size for each type of lender has differed substantially. The aggregate amount of consumer loans in banks at the end of 2013 was 233% as large as at the beginning of 2006 (Madeira 2019c). Aggregate consumer credit by social institutions in 2013 was 245% the level of 2006, but retail store credit grew only 57% during the same period. Financial time series from the Central Bank of Chile show that, from the end of 2013 until the end of 2019, the real value of consumer loans from banks, retail stores and unions, education, vehi- cle sellers and other debts grew 4.2%, 4.4%, 10.2%, 5.5% and 1.4%, respectively. How- ever, following the Covid pandemic in 2020 and 2021, the consumer credit for banks and retail-unions dropped by 11.3% and 4.4%, respectively, whereas the other lender types reported almost zero growth. 8 The aggregate amount of other loans (such as automotive and informal lending) is not entirely known, since credits of smaller and unregulated institutions do not need to be registered for statistical purposes. 9 Families with no consumer loans are classified in two categories: “No Access to Debt” and “No wish to apply for Con- sumer Debt”. “No Access to Debt” represents families who applied for credit but were denied and the ones who did not apply for credit because they expected to be refused. “No wish for Debt” is the outside option for all agents, comprising the families who report no consumer debt and no interest in applying for loans. 10 Some consumers may have more than one debt type, say debt at Banks and Other Loans (for example, an educational loan), but except for retail store credit (which reaches around 7 million people in Chile) there are few observations with such interactions and such options represent a negligible amount of the consumer debt. An empirical model of choice of lender, loan amount and default An empirical model of choice of lender, loan amount and default The consumer choice model considers three endogenous variables: (i) a categorical choice between having no debt, wanting debt but being credit constrained,9 and five lender types; (ii) the loan amount decision; and (iii) a categorical outcome of whether the household defaulted on its debt. The five lender types correspond to: Banks, Banks and Retail Stores, Retail Stores, Union Credit, and Other Loans (which includes mainly auto loans, educational debt, pawn shops and informal lending). For simplicity, I clas- sify the observed lender choice of each household as the one corresponding to the larg- est loan amount reported by each family.10 Banks and Retail Stores are the two major lenders in Chile, therefore, using both lenders with positive debt is treated as a separate choice. After considering the households’ largest loan amount among these 5 options, the remaining debt amount is quite negligible. For at least 75% of the borrowers, the choice among these 5 options represents 100% of its consumer debt. Therefore, there is little practical interest to increase the model by adding more options that mix specific lenders such as Banks with relatively rare options of “Other Loans” such as education or pawn loans (which represent a small number of debtors and share of the consumer debt). Let Ui,b,t denote the utility of household i from the option b in period t, with b ∈{ 1 “Bank”, 2 “Bank & Retail”, 3 “Retail”, 4 “Union”, 5 “Other Loans”, 6 “No Access”} , with the utility of the outside option, “No wish for Debt”, standardized Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Page 8 of 29 as zero, Ui,0,t = 0 . Consumer chooses the option Yi,t = b with the highest utility ( Ui,b,t ≥max(Ui,0,t, Ui,1,t, .., Ui,B,t) ) and then a loan-amount Li,t , which are affected by observable characteristics, xi,t0 , plus unobservable preferences for each loan type b, εi,b,t , and loan-amount, ζi,t . The observed characteristics xi,t0 correspond to a previous period t0 < t , because the debt choice was made in the past and not at the exact time of the survey interview. The utility of each loan type be a linear function of the observables and the error term is: (1) Ui,b,t = αb,t + βbxi,t0 + εi,b,t. (1) Ui,b,t = αb,t + βbxi,t0 + εi,b,t. An empirical model of choice of lender, loan amount and default Sometimes, it is difficult to interpret the coefficients of a multivariate choice model (Hensher et al. 2015), due to the agents’ B + 1 possible alternatives. For a given choice b, βb > 0 implies that the probability of option b relative to option 0 is increasing in x. However, there could be another option c which has a larger coefficient than b, implying x decreases the chance of b being chosen relative to option c. Therefore, in the multivari- ate case βb > 0 only unambiguously increases the probability that b is chosen with larger x if βb ≥max(β1, . . . , βB). The unobserved terms εi,b,t may include a diverse set of factors, such as idiosyncratic preferences, geographical distance, marketing influence, the burden for loan applicants to provide the information and legal documents requested by certain lenders, contrac- tual costs of loans such as penalty charges or insurance fees, and negative events during the loan bargaining process.11 McFadden and Train 2000 show that under mild regular- ity conditions, any discrete choice model derived from random utility maximization has choice probabilities that can be approximated as closely as possible by a Mixed Multi- nomial Logit model. Therefore, the unobserved tastes εi,b,t are specified as the sum of an independent extreme valued component and normal heteroscedastic random-effects that are correlated over different choices (McFadden and Train 2000): (2) εi,b,t = 1(1 ≤b ≤5)ηi,1,t + 1(1 ≤b ≤2)ηi,2,t + ˜εi,b,t, (2) with 1(.) being the indicator function, ˜εi,b,t ∼EV (0, 1) and ηi,a,t ∼N(0, σηa) . ηi,1,t is a random factor denoting agent i’s taste for any type of loan, while ηi,2,t is a random factor denoting agent i’s taste for both the Bank and Bank plus Retail options. This model is then estimated using Simulated Maximum Likelihood with 250 Hammersley integration points (Hensher et al. 2015). After estimating the loan type choice probabilities, the corresponding inverse Mill’s ratio ( f (x) Pr(Yi,t=b\|xi,t0) = Pr(Yi,t = b \| xi,t0) in the Multinomial Logit) is used in the second step regression to correct for the loan type selection in the total consumer debt amount12 model: (3) ln(Li,t) = πt + δxi,t + B−1 b=1 θb1(Yi,t = b) Pr(Yi,t = b \| xi,t0) + ζi,t. (3) 11 Hensher et al. (2015) give further interpretation about unobserved preferences of multiple choice models. An empirical model of choice of lender, loan amount and default 12 Considering the total consumer debt helps account better for the default risks in the final stage of the model. Madeira Financial Innovation (2023) 9:49 Page 9 of 29 Madeira Financial Innovation These second step equations are identified semi-parametrically, with no need to specify the distribution of the unobservable factor ζi,t (Vella 1998). All that is required is at least one variable that affects loan choice but not the default (which is valid because xi,t0 dif- fers from xi,t and zi,t due to the lagged variables), and the assumption that the unob- servables are uncorrelated with the vector variables xi,t after accounting for the loan type selection probability, 1(Yi,t = b) Pr(Y = b \| xi,t0). The decision to default at time t, Di,t ∈{0, 1} , is then given by whether the latent propensity to default is positive, di,t > 0 , expressed by a linear function of the observable characteristics, zi,t , the inverse Mill’s ratio for the loan type decision ( 1(Yi,t = b) Pr(Yi,t = b \| xi,t0) ), and an unobserved shock νi,t (which, for simplicity, is assumed to be extreme-value distributed, ˜νi,t ∼EV (0, 1)): (4) di,t = µt + zi,t + B−1 b=1 γb1(Yi,t = b) Pr(Yi,t = b \| xi,t0) + νi,t. (4) The empirical variable measure of debt default is determined by whether the household is in arrears for any of its consumer loans, with arrears implying that the household is late on the payments of at least one of its consumer loans for one or more months. Some households can have multiple loans with the same lender or with different lenders. How- ever, an event of arrears is considered to be a serious sign of delinquency of the bor- rower, with many borrowers ending up defaulting on their other unsecured consumer loans later on (Madeira 2018). For this reason, it is appropriate to treat any loan arrears as a delinquency event of the borrower. Finally, treating the consumer delinquency at the borrower level (a dummy for arrears in any of its consumer loans) is more appropri- ate econometrically, because the loan by loan decisions of the same borrower cannot be treated as statistically independent since the same factors affect all loans of the borrower. f The observable variables that explain default, zi,t , differ from the vector explaining loan types, xi,t . An empirical model of choice of lender, loan amount and default This is because loans have a maturity of several periods; therefore, the decision of loan choice and amounts happens before the repayment period, with loan choice being affected by the income and unemployment risk in the past year and default behavior by the current variables. The explanatory variables for explaining the lender type choice include demograph- ics, income and job risks (Attanasio and Weber 2010; Agarwal et al. 2020). Permanent income influences both household loan demand and its credit worthiness (Attanasio and Weber 2010; Agarwal et al. 2020). Age and household size account for life cycle compo- nents that affect the demand for durable goods and other expenditures (Attanasio and Weber 2010). Unemployment risk and wage volatility account for the large uninsurable shocks that can affect loan demand when the loan choice is made (Attanasio and Weber 2010) and delinquency behavior at the time of the survey measurement (Agarwal et al. 2020). Education proxies for financial literacy (Attanasio and Weber 2010; Badarinza et al. 2016), whereas the region proxies for differential access to lenders outside the capi- tal and the survey wave dummies account for aggregate shocks (Wooldridge 2010). The loan amount also includes the selection Mill’s ratios (Vella 1998) to account for borrowers’ self-selection with each lender and the debt motives known by the borrower but unseen by the lender. Finally, the final default behavior also controls for measures of Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Page 10 of 29 borrower liquidity and solvency (Einav et al. 2012), such as the debt service to monthly income ratio (DSIR) and the consumer debt to the annual permanent income ratio (CDPIR). Similar results to those in this parametric partial information model are confirmed by the analysis of: (i) the households’ mean statistics in section 5 of this article; (ii) the non-parametric probability density function (PDF) and cumulative distribution func- tion (CDF) distributions of households’ loan levels and labor risks13; (iii) a parametric full information model (see Madeira 2019c); iv) different time periods, such as just the 2007–2011 waves (Madeira 2019c). 13 These results are available as figures from the author upon request. Similar figures are also available for the waves between 2007 and 2011 in the earliest draft of this article (Madeira 2019c). 4 The study uses all the waves as a pooled cross-section to increase sample size and efficiency (Wooldridge 2010). Data: the Chilean Household Finance Survey (EFH) (3) The family Madeira Financial Innovation (2023) 9:49 Page 11 of 29 Madeira Financial Innovation would be unable to repay the loan. (4) The family would be rejected for the loan requests or the loan would not be granted. (5) Other reasons.”. The last question can separate the families with “No Wish for Consumer Debt” (those that report the options 1 and 2 for either no need for credit or disliking loans) and those with “No Access to Debt” (report- ing wishing to have debt, but not making loan requests due to options 3 and 4 of being unable to repay the loans or likely to be refused for loans even if a request was made). Families are also classified as “No Access to Debt” if they made loan requests, but all of them were refused. I represent the size of each household using two measures. The first is the total number of household members ni . Other measures for the household size can account for scale economies in terms of the consumption of joint goods within the household. Therefore, I also use the OECD-modified scale for the household size (OECD 2008), which assigns a value of 1 to the household head, 0.5 for each additional adult member (above age 15) and 0.3 for each child: neOECD i = 1 + 0.5(adultsi −1) + 0.3(childreni). The EFH survey collects detailed information on the income, education, age and other characteristics of each household member, but has limited data on some aspects such as income volatility or employment stability. For this reason I estimate the income and employment risks of the EFH workers based on the mean statistics for workers with the same characteristics in another dataset. Using the quarterly Chilean Employment Survey, which covers 35,000 households, I obtain two measures of labor risk for the period 1990–2017 (Madeira 2015): the unem- ployment rate ( uk,t = Pr(Uk,t = 1 \| t, xk) ) and the labor income volatility even if no job is lost, σζ,t(xk) = E (Yk,t −E[Yk,t \| Yk,t−1, xk])2 \| Vk,t, xk , with Vk,t = (t, Uk,t = Uk,t−1, Yk,t) . Data: the Chilean Household Finance Survey (EFH) This study uses data from the Chilean Household Finance Survey (in Spanish, Encuesta Financiera de Hogares, hence on EFH), which is a representative cross-sectional survey with detailed information on households’ assets, debts, income and financial behav- ior. The EFH survey is broadly comparable to similar surveys in the United States and Europe (Eurosystem 2009). The EFH covered a total of 21,319 urban households from 2007 to 2017. The 2007, 2011, 2014 and 2017 waves were implemented at the national level, including all the regions of Chile, with an interval of three or four years between each wave, having interviewed 3828, 4059, 4502 and 4549 households, respectively. The 2008, 2009 and 2010 waves were implemented in the capital city of Santiago (which has over 40% of the national population); therefore, those waves only interviewed 1154, 1190 and 2037 households, respectively.14 All sample statistics in the article use expansion factors and are representative. The EFH has a particularly detailed focus of the loans and debt commitments of each household. It asks for the largest three debts that each household has for each type of loan among a total of 13 categories: Banking Credit Card Debt, Banking Line of Credit, Banking or Financial Agency Consumer Credit Loan, Retail Store Credit Card, Retail Store Consumer Loan, Auto Loans, Union Credit, Education Loans, Loans from rela- tives, Loans from usurers, Pawn shops, Grocery and Shopping on credit (i.e., store tabs), and Other Debts. Therefore, the survey may ask up to a total of 39 current debts of the household, although obviously few agents report having debts with all possible catego- ries of loans. Although the survey indicates which household member signed each loan, this article’s analysis follows the standard that considers that all household members share expenses and are co-responsible for debts (Eurosystem 2009; Attanasio and Weber 2010). In addition to asking for the details (debt amount, monthly loan payment, maturity, interest rate) of each loan, the EFH also inquires about its credit applications over the previous year: (i) “Has the family made any loan request over the previous 12 months?”, (ii) “How many requests for loans has the family made?”, (iii) “How many loan requests have been rejected?”, and (iv) “What is the main reason why your family did not make any loan requests? Options: (1) No need for credit. (2) Dislikes loans. Data: the Chilean Household Finance Survey (EFH) The vector xk creates 540 mutually exclusive groups, given by xk = {Santiago Metropolitan city or Outside, Industrial Activity (primary, secondary, tertiary sectors), Gender, Age (3 brackets, ≤35 , 35 −54 , ≥55 ), Education (less than secondary schooling, secondary or technical education, college), and Household Income quintile}. Using these labor risk measures, I calculate the expected income ¯Pi,t of each EFH household i as the sum of their non-labor income, ai , and its expected labor income, Pi,t : ¯Pi,t = ai + Pi,t , where Pi,t = kPk,t is the sum of the expected labor income of each household member k. Pk,t = Wk,t(1 −uk,t) + Wk,tRk,t(uk,t) is the worker k’s average labor income in the employed and unemployed states. The employment risk and labor income volatility of each household are then given by a weighted average of the rates of each member (using as weights their labor income relative to the total household labor income): ¯ui,t = k Pk,t Pi,t uk,t and ¯σi,t = k Pk,t Pi,t σζ,t(xk). Characterization of borrowers’ loan options Borrowers according their type of lender Table 1 illustrates the proportion of households that chose each of the five lender types, plus those with either No Consumer Debt (because the family does not want debt) and No Access to Debt (if the family applied for loans, but was refused). Retail Stores are the most popular choice among households, representing more than 40% of the popula- tion, with 26% being Retail Store only users and 14% being users of both Bank and Retail Store Loans. Banks are the second largest lender in terms of the number of borrowers, Madeira Financial Innovation (2023) 9:49 Page 12 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation reaching 23.4% of the households, with 9.4% being Bank only and 14% being Bank plus Retail borrowers. Finally, Union lenders and Other Debts reach, respectively, 6.1% and 6.4% of the households. Over the last ten years (that is, from the first wave in 2007 until the last wave in 2017), the users of Bank debt only and Other debts increased signifi- cantly in each year. Union borrowers fluctuated substantially over the years, increasing from 3.8% in 2007 to 8.1% in 2011, before falling in 2014 and increasing again to 7.2% in 2017. Retail Store debt only and No Access consumers decreased significantly over the last ten years, with Retail Store only declining mostly from 2007 to 2011 and again from 2014 to 2017, while the fall in No Access households was moderate from 2011 to 2014 and then had a stronger drop from 2014 to 2017. Households choosing Retail Stores only (that is, without bank loans) decreased over time from 31.9% in 2007 to just 19.4% in 2017. However, borrowers choosing loans at Banks only increased from 6.5% in 2007 to 10.9% in 2017. Other debts also increased steadily, from 4.6% in 2007 to 10% in 2017. The proportion of households without a wish for consumer debt represents 27% of the Chilean population, while those with No Access to Debt represent 11% of the popula- tion. Over time, the fraction of household with No Access to Debt steadily decreased from 12.7% in 2007 to 8.9% in 2017. The consumers with No Wish for Debt fluctuated over time, increasing between 2007 and 2011, then dropping in 2014 and increasing again recently in 2017 to a higher level of 30.4%. There is limited information in the EFH about the loan interest rates. 15 One reason why Education and Other Loans changed interest rate conditions so dramatically between periods is that public education and a debt free college education was one of the major political flags of the Chilean government of Michelle Bachelet, which started in March of 2014. The government promised a debt free college education, which reduced demand for education loans. This government agenda was materialized by the laws number 21040 (November, 2017), 21091 (May, 2018) and the regulatory decree 333 (November, 2019). 16 Education and Other loans were the cheapest loan product before 2011, but this could have been explained due to strong government subsidization of education loans and an implicit public guarantee against borrower default. Characterization of borrowers’ loan options Borrowers according their type of lender The survey asks about the loan’s interest rates, but less than half the respondents report to remember this information. Furthermore, it is not possible to include such information in the mul- tiple choice model, because the survey does not ask the interest rates and maturities for the loan options not chosen by the consumers. Table 2 shows the distribution of inter- est rates reported by the borrowers from the waves between 2007–2011 and from 2014 to 2017. The analysis is separate into two periods, because a 2013 legislation in Chile reduced the maximum interest rate for consumer loans from 52% to just 37% (Madeira 2019b). Looking at the top interest rates (as expressed by the percentile 75), all lenders reduced their interest rates between 2007–2011 and 2014–2017, although only the Bank and Auto lenders reduced their median interest rates. Retail, Union and Auto lenders also reduced their loan maturity (in terms of median and mean) between 2007–2011 and 2014–2017. Median maturity fell by 12 months for Union and Auto Lenders. The worst fall in maturity was for Retail, which dropped their median maturities by 24 months. Banks kept similar median maturities before and after the 2013 interest rate, although the mean was lower by 8 months. Education lenders, however, increased their maturity substantially, perhaps due to government interest subsidization and guarantees against individual borrowers’ default. Before 2011 the most expensive consumer loans were those obtained from Retail Stores and Auto Lenders, while those for Education and Other Loans were the most expensive loan product after 2014.15 Prior to 2011, Bank loans were the second cheapest Page 13 of 29 Madeira Financial Innovation (2023) 9: Madeira Financial Innovation Table 1 Fraction of households (in %) choosing each type of debt over the years Type of debtor All waves 2007 2011 2014 2017 Bank 9.4 6.5 8.4 10.7 10.9 Bank + Retail 14.0 14.1 11.9 16.8 13.2 Retail Store 25.8 31.9 25.9 26.8 19.4 Union Debt 6.1 3.8 8.1 5.7 7.2 Other Debts 6.4 4.6 5.1 5.8 10.0 No Wish for Debt 27.0 26.4 27.9 23.3 30.4 No Access to Debt 11.3 12.7 12.6 11.0 8.9 Table 1 Fraction of households (in %) choosing each type of debt over the years loan product jointly with Union debt,16 while being the cheapest loan product since 2014. Characterization of borrowers’ loan options Borrowers according their type of lender In terms of maturity, Bank and Auto consumer loans present the longest maturi- ties for the borrowers, although other lenders also offer maturities close to four years. This table, however, only accounts for installment loans; therefore, it does not include many of the loans with banks and retail stores which are in terms of revolving debt, being renewed monthly and not presenting a maturity. I summarize the overall household indebtedness in terms of three variables: (i) the debt service to monthly income ratio (DSIR=DSi,t Yi,t ), with the debt service including the loan amortization plus all the fees and interest to be paid in a given month; (ii) the con- sumer debt amount to annual permanent income ratio (CDPIR= Li,t 12 × ¯Pi,t ); and (iii) the log of the real value of the consumer debt. The debt service ratio (DSIR) is a measure of the liquidity constraints faced by households at the end of the month (Madeira 2019b), while the consumer debt amount to the annual permanent income ratio (CDPIR) is a solvency measure that households may face in terms of their total debt amount. The consumer debt (in real terms) can be viewed as an overall measure of indebtedness. Table 3 illustrates that both the Bank-Retail and the Other Debt borrowers are the most indebted in terms of any of the three measures (DSIR, CDPIR, and real debt amount). The data also demonstrate a substantial difference between Bank only and Bank plus Retail borrowers, with Bank plus Retail borrowers showing a much higher indebtedness with almost 50% higher consumer debt whether in level (in Table 3, exp(4.5)/exp(4.1)−1=49.1%) or CDPIR income ratio (30% for Bank plus Retail compared to 21.4% for Bank only borrowers). Bank plus Retail borrowers also present a much higher loan payment service, with a DSIR of 42.6% for Bank plus Retail compared to 29.7% for Bank only borrowers. Retail Store only and Union debt borrowers present the lowest indebtedness levels, whether in terms of debt service (DSIR), consumer debt to income (CDPIR) or in level (consumer debt in real terms). Retail Store only borrowers are the least indebted, with a consumer debt to income ratio (CDPIR) of just 10% (much lower than the 16.5% of CDPIR of Union debt borrowers) and a real consumer debt which is just one third of the level of the Union borrowers. Characterization of borrowers’ loan options Borrowers according their type of lender Page 14 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 2 Distribution of the interest rate and maturity of each loan type (before and after the 2013 interest rate ceiling law) Loan type Interest rate (%) Maturity (months) Percentiles, Number of observations (N) Median (P50), Mean, Observations (N) 2007–2011 2014–2017 2007–2011 2014–2017 P25 P50 P75 N P25 P50 P75 N Median Mean N Median Mean N Bank 9 20 40 511 13 18 26 355 36 47 1795 36 39 1426 Retail Store 9 24 42 95 16 23 29 79 36 44 463 12 22 541 Union Debt 13 19 27 200 19 24 27 138 36 45 984 24 33 909 Auto 18 30 39 66 13 21 29 31 48 48 305 36 36 337 Education, Other 9 10 27 77 13 26 27 205 24 37 292 84 104 415 Table 2 Distribution of the interest rate and maturity of each loan type (before and after the 2013 interest rate ceiling law) Loan type Interest rate (%) Maturity (months) Percentiles, Number of observations (N) Median (P50), Mean, Observations (N) 2007–2011 2014–2017 2007–2011 2014–2017 P25 P50 P75 N P25 P50 P75 N Median Mean N Median Mean N Bank 9 20 40 511 13 18 26 355 36 47 1795 36 39 1426 Retail Store 9 24 42 95 16 23 29 79 36 44 463 12 22 541 Union Debt 13 19 27 200 19 24 27 138 36 45 984 24 33 909 Auto 18 30 39 66 13 21 29 31 48 48 305 36 36 337 Education, Other 9 10 27 77 13 26 27 205 24 37 292 84 104 415 Page 15 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 3 Debt service to monthly income ratio (DSIR), Consumer debt to annual permanent income ratio (CDPIR), and Real consumer debt (in log): average values per borrower Table 3 Debt service to monthly income ratio (DSIR), Consumer debt to annual permanent income ratio (CDPIR), and Real consumer debt (in log): average values per borrower Average values for borrowers with positive debt Type of debtor Debt service to income (in %) Consumer debt to annual permanent income (in %) Consumer debt (real value in UF) (in log) All waves 2007 2011 2014 2017 Bank 29.7 21.4 4.1 3.8 3.8 4.0 4.4 Bank + Retail 42.6 30.0 4.5 4.3 4.3 4.5 4.8 Retail Store 22.4 10.0 2.3 2.5 2.1 2.3 2.3 Union Debt 24.1 16.5 3.4 3.1 3.5 3.3 3.5 Other Debts 31.7 32.6 4.8 4.4 4.7 4.7 5.1 All debtors 29.2 19.3 3.4 3.2 3.2 3.4 3.8 Bank and Retail Store borrowers have the highest indebtedness in terms of the debt service ratio (DSIR), whereas Other Debt borrowers have a higher consumer debt amount to the annual permanent income ratio (CDPIR) and a higher real consumer debt amount. Characterization of borrowers’ loan options Borrowers according their type of lender It is noteworthy that the average real consumer debt per borrower has increased for all debt types, except for the Retail Store only debtors, between 2007 and 2017. Looking at the entire population of borrowers, the strongest real debt increase was between 2014 and 2017, with an especially strong growth for the debtors of Banks, Bank-Retail and Other Debts. The real debt also dropped momentarily for the Union borrowers between 2011 and 2014. Households with both Bank and Retail Store debt have much higher loan amounts than the debtors of Bank and Retail Stores separately, which could be a sign that these are debtors with particularly high needs for liquidity. Similar conclusions can be obtained by analyzing the entire probability density func- tion (pdf) and the cumulative distribution function (cdf) of the consumer loan amounts, the debt service to monthly income ratio, and the consumer debt to annual permanent income ratio (Madeira 2019c). Between 2007 and 2017, the EFH survey asked the following question about loan arrears, “Approximately, in the last 12 months have you ever fallen into arrears or late payments for your loans?”. This variable measures default as a dummy denoting one or more events of arrears in the previous year, whether for the current period’s loans or loans during the previous year. Arrears imply that the borrower has been late with his loan payment for 30 days or more. Since 2010 the survey also asks for each of the cur- rent loans whether the households have been in arrears and for how many months. This allows to build a similar measure of borrower risk for arrears of one month or more for the current loans or a measure of arrears for a failure to repay after three months or more, which is a more common measure of debt risk used at the international level (Madeira 2018, 2019a). Note that this one month arrears measure differs slightly from the previous measure, which includes any arrears event in the previous 12 months, not just for the present period. Note also that these three definitions of loan arrears (“arrears in the past year”, “arrears of the current loans: 1 month or more”, “arrears of the current loans: 3 months or more”) apply to any consumer debt loan, that is, the variable is a dummy with value one if any of the consumer loans is in arrears. Characterization of borrowers’ loan options Borrowers according their type of lender Table 4 illustrates that Bank plus Retail borrowers have the highest risk in all the three measures of arrears, with, respectively, a rate of 27.4% of arrears in the past year, 21.2% Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation (2023) 9:49 Page 16 of 29 Madeira Financial Innovation of current arrears (one month or more), and 7.7% of current arrears (three months or more). Other Debt borrowers show the lowest risk of current arrears, with rates of 8.9% and 2.2% at the horizon of one month and three months, respectively. Across all bor- rowers, the arrears of three months or more are just 25% (that is, 4.2/17.1) of the level of the arrears of one month or more. This shows that most debtors recover a good financial health after being in arrears for one month. Retail Store only borrowers show a high risk of arrears at the horizon of one month with a rate of 19.6%, although a risk of just 2.2% after three months. Bank only, Union and Other debt borrowers show a low risk of arrears both at the horizon of one and three months. Indeed, Bank only borrowers report an arrears rate that is around half the value reported by users of both Bank and Retail credit, demonstrating arrears rates of just 11.6% and 4.2%, respectively, at the horizons of one month and three months or more. In Chile neither Retail Stores or Unions offer heterogeneous interest rates to the cus- tomers. Banks offer customer specific interest rates (Madeira 2019c), therefore, the economic theory predicts that Banks will get the best observable risk types by offering better loan terms, such as lower interest rates, larger loan amounts and longer maturi- ties. Union Debt lenders cannot risk price their offers, but are able to garnish their cli- ents’ wages easily; therefore, this high punishment cost should explain their low arrears rates. However, households with both Bank and Retail Store debt have arrears rates as high as the customers of Retail Stores only. Perhaps this can be explained because such debtors have an unobservable taste for high loan amounts. Households with Other Debts also have high debt and a high value of arrears in the previous year. Perhaps this is because education loans are granted to younger agents, who may be more subject to unemployment risk and unstable income. Labor income risks In addition to their indebtedness level, household risks can also depend on their demographics and labor market profile. Table 5 illustrates a clear sorting pattern of households across different debt types. For the average of all debtors, the household has 3.5 members (or 2.1 members in the OECD household size scale), log permanent income of 3.9, the household head’s education is 12.8 years (just slightly above high school completion) and its age is 47 years. The unemployment and income volatility risk of the average debtor are 3.8% and 30.9%, respectively. g y In terms of income, Bank debtors have the highest income, while showing the low- est unemployment risk. Other Debt households have the highest education and the highest income levels next to Bank and Bank plus Retail borrowers, but also the low- est age and one of the highest values of labor income volatility (only below the No Access households). On the other end, households with No Access to Debt have the lowest income and education, while showing the highest unemployment risk, age and labor income volatility. Next to the No Access households, Retail store borrowers are the ones with the lowest income and education, while showing the highest unemploy- ment risk. In terms of the household size (whether measured as the number of house- hold members or by the OECD modified scale), Bank plus Retail debtors represent the largest households, whereas those with No Wish for Debt and No Access to Debt represent the smallest households. It is relevant to note that Union debt borrowers have one of the lowest income (only above the No Access households), but also the lowest labor income volatility, possibly due to the types of occupations of their work- ers. Similar conclusions can be obtained by analyzing the probability density function (pdf) and the cumulative distribution function (cdf) of the permanent income, labor income volatility, and unemployment risk across different debtor types (Madeira 2019c). Overall, this section portrays a clear picture of the household groups. Households with No Access to Debt have the lowest income and highest unemployment risk. The users of just Bank loans are the ones with the highest income and lowest unemploy- ment rate; however, they experience moderate wage volatility, which creates a demand for debt to smooth consumption. Characterization of borrowers’ loan options Borrowers according their type of lender Additionally, education and auto loans may have lower punishment costs for arrears, because lenders cannot deduct payments and punishment fees from clients’ bank accounts (as Banks do) or their wages (as Union Credit institutions do). However, households report a lower risk of arrears above three months for other debts; therefore, these short term risks do not translate into long term default. Table 4 also reports the share of the consumer loan destined for a given purpose of the household, more specifically “Purchase of Durables or Household Investments”, “Pay previous debts” and “Health needs”. Other motivations are classified as “General Consumption” so that the total motivations sum to 100% of the debt. The average bor- rower dedicates, respectively, 38%, 22%, 11% and 5% of its consumer debt to General Consumption, Durables, Pay Previous Debts, and Health Expenses. Other debt bor- rowers are mostly related to Durable goods purchases, with 56% of its debt dedicated to this purpose. All the other borrower types have General Consumption as the larg- est reason for their consumer indebtedness. Households with Bank, Bank plus Retail, and Union Debt are more likely to have motivations of “Pay previous debts”, dedicat- ing between 17 and 20% of their consumer debt to such purpose. Union borrowers have the highest share of Health Expenses, with a rate of 13.4%. In terms of the share of loan motives, Bank only borrowers are very similar to the Bank plus Retail bor- rowers, which makes sense, because the consumer debt loans of the Retail Stores are much lower than the Banks. For instance, Table 3 lists that the average Retail Store only borrower has a debt of just 16.5% of the level of Bank only borrowers. Characterization of borrowers’ loan options Borrowers according their type of lender Page 17 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 4 Consumer loan arrears (in %) for each debtor type (all waves) and shares of each loan motive as a fraction of the total consumer debt (in %): averages across borrowers (all waves) Table 4 Consumer loan arrears (in %) for each debtor type (all waves) and shares of each loan motive as a fraction of the total consumer debt (in %): averages across borrowers (all waves) motive as a fraction of the total consumer debt (in %): averages across borrowers (all waves) Type of debtor Arrears in past year Arrears of the current loans Shares of loan motives (1 month or more) (1 month or more) (3 months or more) Durables Pay past debts Health expenses 2007–2017 2010–2017 2010–2017 Bank 16.7 11.6 4.2 34.2 16.7 5.3 Bank + Retail 27.4 21.2 7.7 24.8 19.0 5.7 Retail Store 22.4 19.6 2.2 5.3 4.5 3.6 Union Debt 21.0 16.1 6.1 28.1 20.4 13.4 Other Debts 25.2 8.9 2.2 55.9 4.4 1.6 All debtors 22.8 17.1 4.2 21.6 11.2 5.1 Labor income risks Bank plus Retail and Other Debts have a slightly lower income than the Bank only borrowers, but they are subject to a considerably higher Page 18 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 5 Demographic characteristics of the households according to their debt type: average values per borrower The OECD equivalence scale for household size corresponds to a value of 1 for the household head plus 0.5 for each additional adult and 0.3 for each child, in order to account for economies of scale in the sharing of home goods (OECD 2008) Type of debtor Permanent Income in UF (log) Unem- ployment risk (%) Labor income volatility (%) Education (in years) Age (years) Number of household members Household size (OECD scale) Bank 4.2 4.1 30.9 14.1 46.5 3.3 2.0 Bank + Retail 4.1 4.4 28.8 13.5 46.0 3.6 2.2 Retail Store 3.6 5.3 31.0 11.7 48.2 3.4 2.1 Union Debt 3.7 4.8 27.6 12.1 52.7 3.4 2.0 Other Debts 4.1 4.8 38.6 14.3 42.3 3.5 2.1 No Wish for Debt 3.8 4.5 33.3 12.5 52.6 2.9 1.8 No Access to Debt 3.5 5.4 41.9 11.0 53.0 3.0 1.9 All debtors 3.9 4.8 30.9 12.8 47.3 3.5 2.1 The OECD equivalence scale for household size corresponds to a value of 1 for the household head plus 0.5 for each additional adult and 0.3 for each child, in order to account for economies of scale in the sharing of home goods (OECD 2008) unemployment risk and, therefore, have a higher indebtedness level than Bank only bor- rowers. The Union Debt borrowers are in the middle of the debt and labor income risk profiles, because these households have a low income level but also a low wage volatil- ity and a moderate level of unemployment risk. Therefore, these households also have a moderate level of indebtedness. Finally, Retail Store only borrowers among the house- holds with access to debt have the lowest income and education coupled with the high- est unemployment risk; therefore, such households also have the lower debt amounts. The role of demographics, income profile and unobserved preferences This section discusses the results from the consumer loan choice and default model exposed in “An empirical model of choice of lender, loan amount and default” section. The model requires some variables that affect loan choice, but not default. Consumer loans typically have a maturity of 12–48 months, therefore, it is reasonable to assume that the labor market conditions influencing loan choice happened 4 quarters or more before the current period. The model also controls for the education, age, region, house- hold size and debt motivations: xi,t =    ln(¯Pi,t−4), ¯ui,t−4, ¯σi,t−4, years of education and age of household head, dummies by year, dummy for residence region, OECD household size scale.   . xi,t =    ln(¯Pi,t−4), ¯ui,t−4, ¯σi,t−4, years of education and age of household head, dummies by year, dummy for residence region, OECD household size scale.   ln(¯Pi,t−4), ¯ui,t−4, ¯σi,t−4, Similarly, I assume that the vector zi,t that explains loan arrears or default at time t includes some variables that do not necessarily affect loan choice, such as the Debt service to monthly income ratio (DSIR) and the Consumer debt to annual permanent income ratio (CDPIR): Page 19 of 29 (2023) 9:49 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation zi,t =              DSIRi,t, CDPIRi,t, ln(¯Pi,t), ¯ui,t, ¯σi,t, years of education and age of household head, dummies for each year, dummy for residence region, OECD household equivalence size scale, Share of debt justiﬁed by “Durables”, “Pay Previous Debts” and “Health”. zi,t =              DSIRi,t, CDPIRi,t, ln(Pi,t), ¯ui,t, ¯σi,t, years of education and age of household head, dummies for each year, dummy for residence region, OECD household equivalence size scale, Share of debt justiﬁed by “Durables”, “Pay Previous Debts” and “Health”. Table 6 shows the estimates for the coefficients of loan choice, βb , from the Mixed Multinomial Logit Model. The coefficients for both the lagged household expected income ( ln(¯Pi,t−4) ) and years of education have a positive impact on choosing the Bank, Bank plus Retail, and Other Debts options, while having a negative effect on the No Access option. Both the income and unemployment coefficients have its most positive impact for the option of Bank loans. The role of demographics, income profile and unobserved preferences This implies that higher income and edu- cation unambiguously increase the option of a Bank loan, while decreasing the option of No-Access. The coefficient of lagged unemployment ( ¯ui,t−4 ) increases the probabil- ity of all the loan options relative to No Debt, especially for the Other Debt and Retail only options. This demonstrates that unemployment risk is a factor for choosing loans under a motivation of consumption smoothing (Attanasio and Weber 2010). It also represents that Retail Store and Other Debt borrowers are more subject to unemploy- ment risk (Agarwal et al. 2020). Wage volatility decreases the probability of choosing any of the the debt alternatives, but it increases the probability of No Access, a sign of a precautionary savings motive to avoid debt (Attanasio and Weber 2010). Older households are less likely to choose any debt option, which makes sense due to their savings needs at the end of life (Attanasio and Weber 2010). The age effect is lower for the Union and No Access alternatives. Larger households (measured by the OECD scale) are more likely to choose all kinds of loan options, although this has a small effect on the No Access alternative. This demonstrates that household size can be positively related to expenditure shocks, whereas age is negatively related to con- sumption shocks. The Santiago capital region dummy is not statistically significant for any alternative, which demonstrates that the Chilean lenders have an adequate presence across the entire country. Finally, the probability of Bank only, Union and Other Debts increased in 2017, whereas the probability of No Access decreased in the same year. This demonstrates that the Chilean households decreased their credit con- straints and improved their loan options in recent years. Table 7 shows the results for the choice of loan amount and arrears. Note that the expected income ( ln(¯Pi,t−4),), unemployment risk ( ¯ui,t−4 ) and wage volatility ( ¯σi,t−4 ) that affect the loan amount decision have a lag of 4 quarters, whereas the variables affecting default correspond to the current period t. Income, education, and loan motivations of “Durables”, “Pay previous debts” and “Health” are positively related to loan amounts. Unemployment risk and age are both associated with lower loan amounts. Wage volatility, household size and the region dummy have small coeffi- cients, which are statistically insignificant. The role of demographics, income profile and unobserved preferences After controlling for observables, the loan amounts fell in 2010/2011 and 2014, which is consistent with the effects of the 2010 earthquake and the interest rate ceiling law of 2013 (Madeira 2019b). The coefficients for the Odds Ratios of selection indicate that the probability of making another choice besides the chosen alternative has a positive effect on the loan amount for the Bank Page 20 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 6 Coefficients for the utility of each type of debt (Mixed Multinomial Logit Model) Robust Huber-White Standard-errors in () , , Denote 1%, 5%, 10% statistical significance Control variables 1 = Bank 2 = Bank + Retail 3 = Retail Store 4 = Union Debt 5 = Other Debt 6 = No Access Age − 0.107 (0.0161) − 0.109* (0.0161) − 0.102* (0.0160) − 0.0766* (0.0159) − 0.123*** (0.0163) − 0.00340* (0.00183) OECD equivalent household size 2.253* (0.346) 2.508* (0.347) 2.608* (0.348) 2.643* (0.350) 2.534* (0.349) 0.340* (0.0460) Income: ln(¯Pi,t−4) 1.094* (0.183) 0.806* (0.182) − 0.0273 (0.178) − 0.0636 (0.184) 0.958* (0.186) − 0.757* (0.0591) Unemploy- ment ¯ui,t−4 15.93* (2.214) 16.29* (2.186) 18.13* (2.144) 17.25* (2.286) 20.33* (2.264) 1.873* (0.694) Wage volatil- ity ¯σi,t−4 − 3.636* (0.577) − 3.788* (0.577) − 3.484* (0.573) − 3.796* (0.581) − 3.523* (0.576) 0.252* (0.0810) Education (years) 0.123* (0.0389) 0.0935 (0.0387) − 0.00713 (0.0386) 0.0591 (0.0402) 0.110* (0.0400) − 0.0763* (0.0118) Santiago capi- tal region 0.206 (0.217) 0.214 (0.215) 0.174 (0.213) 0.205 (0.221) 0.279 (0.221) 0.0467 (0.0601) 2008/2009 − 0.662* (0.384) − 1.039* (0.380) − 0.830 (0.377) − 0.805 (0.401) − 1.121* (0.397) 0.191* (0.106) 2010/2011 − 0.656 (0.302) − 1.092* (0.299) − 0.475 (0.296) 0.246 (0.312) − 0.636 (0.306) 0.530* (0.0908) 2014 0.433 (0.353) 0.453 (0.351) 1.221* (0.353) 1.796* (0.371) 0.551 (0.359) 0.810* (0.112) 2017 1.011* (0.324) 0.281 (0.321) − 0.122 (0.322) 1.029* (0.338) 1.334* (0.331) − 0.505* (0.100) Constant − 8.319* (0.515) − 3.676 (0) 7.217*** (1.899) 3.556* (1.981) − 8.251* (1.989) 9.179* (0.655) Observations: 21,319 households. McFadden’s Pseudo R-squared: 0.071 Table 6 Coefficients for the utility of each type of debt (Mixed Multinomial Logit Model) plus Retail and the Other Debts, while having a negative effect on the loan amount for the Bank, Retail and Union Debt borrowers. The role of demographics, income profile and unobserved preferences This demonstrates that the borrow- ers of Bank-Retail and Other Debts have a strong preference for higher loan amounts, whereas the Bank, Union and, especially, the Retail Store borrowers prefer lower loan amounts. Default behavior is negatively associated with age, income, education, and the share of the largest debt type in terms of the consumer debt (which makes sense, because it is easier to renegotiate debt with a single lender than with many lenders). Arrears are posi- tively associated with larger households, unemployment risk, Debt service to monthly income (DSIR), Consumer debt to annual permanent income (CDPIR), the motives of “Pay previous debts” and “Health needs”. Wage volatility and the region dummy have small coefficients, which are statistically insignificant. In the case of Arrears in the past year, there is a negative effect of the selection probability of not having a Bank, Union Debt and Other Debt alternative. However, although with the same sign, these effects are not significant for the Ordered Logit model of current Arrears above 1 and 3 months. The role of demographics, income profile and unobserved preferences This could be because these selection probabilities affect the short term liquidity of being in arrears or not, but their effect is perhaps less powerful for determining more Page 21 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 7 Coefficients for the loan amount (in log) and propensity for arrears Robust Huber-White Standard-errors in () * Denote 1% 5% 10% statistical significance Control variables Consumer debt (in log) OLS Arrears longer than 1 and 3 months Ordered Logit Arrears in the past year Logit Age − 0.002 (0.0009) − 0.0249* (0.00254) − 0.0224* (0.00193) OECD equivalent household size 0.00188 (0.0176) 0.502* (0.0506) 0.398* (0.0376) Income: ln(¯Pi,t) 0.439* (0.0211) − 0.452* (0.0705) − 0.327* (0.0486) Unemployment ¯ui,t − 0.622 (0.296) 3.201* (0.844) 2.304* (0.642) Wage volatility ¯σi,t − 0.0220 (0.0359) 0.0680 (0.0846) − 0.0568 (0.0708) Education (years) 0.0308* (0.00473) − 0.0985* (0.0138) − 0.0686* (0.0104) Santiago capital region 0.00903 (0.0252) − 0.102 (0.0689) − 0.0637 (0.0536) 2008/2009 0.0375 (0.0485) 0.368* (0.115) 2010 / 2011 − 0.277* (0.0354) 0.711* (0.0867) 2014 − 0.307* (0.0406) − 0.367* (0.0966) 0.404* (0.0984) 2017 0.0646 (0.0407) 0.0249 (0.0973) 1.252* (0.0948) Share of loan for “Durables” 0.906* (0.0354) − 0.182* (0.109) 0.154 (0.0769) Share of loan for “Pay previous debts” 0.969* (0.0419) 0.431* (0.118) 0.531* (0.0872) Share of loan for health needs 0.416* (0.0588) 0.355 (0.150) 0.522* (0.116) 1(Yi,t = 1) Pr(Yi,t = 1 \| xi,t) − 0.470* (0.138) − 0.359 (0.404) − 1.107* (0.302) 1(Yi,t = 2) Pr(Yi,t = 2 \| xi,t) 0.278 (0.137) 0.423 (0.390) − 0.374 (0.299) 1(Yi,t = 3) Pr(Yi,t = 3 \| xi,t) − 1.930* (0.157) 0.402 (0.464) − 0.471 (0.349) 1(Yi,t = 4) Pr(Yi,t = 4 \| xi,t) − 1.098* (0.130) − 0.0540 (0.390) − 0.704 (0.289) 1(Yi,t = 5) Pr(Yi,t = 5 \| xi,t) 0.382* (0.131) − 0.245 (0.390) − 0.511* (0.288) Ratio of Debt Service to Income, DSIRi,t 1.267* (0.148) 0.998* (0.122) Ratio of Debt to Income, CDPIRi,t 0.797* (0.164) 0.751* (0.130) Share of the largest loan type of the total consumer debt − 0.951* (0.0462) − 0.578* (0.131) − 0.358* (0.100) Constant 8.449* (0.286) 5.732* (0.956) 3.870* (0.654) Cutoff 2 1.624* (0.953) Observations 12,075 9071 12,075 R-squared or Pseudo R2 0.498 0.106 0.104 Table 7 Coefficients for the loan amount (in log) and propensity for arrears serious arrears above three months. Counterfactual simulations of policies I now use the pooled EFH (2007–2017) sample and the estimated models to develop four counterfactual exercises to analyze their effect on the debtor type choices Pr(Yi,t = b \| Xi,t0) , consumer loan amounts E[ln(Li,t) \| Zi,t0] , and arrears (past arrears, arrears above 1 or 3 months), Pr(Di,t ≥1 \| Zi,t) . I then obtain the aggregate consumer debt across all households ( aggDt = i(5 b=1 Pr(Yi,t = b \| Xi,t0)) exp(E[ln(Li,t) \| Zi,t0]) ), which accounts for each borrower having some positive probability of obtaining con- sumer debt, and the average debt per borrower with a positive debt amount, E[ln(Li,t) \| Zi,t0] . The exercise also computes the average borrower risk ( Pr(Di,t ≥1 \| Zi,t) ), the risk across all households which takes into account that some households have no debt ( 5 b=1 Pr(Yi,t = b \| Xi,t0) Pr(Di,t ≥1 \| Zi,t) ), and the portfolio risk which also accounts for households having different amounts of debt ( i(5 b=1 Pr(Yi,t=b\|Xi,t0)) exp(E[ln(Li,t)\|Zi,t0]) Pr(Di,t≥1\|Zi,t) aggDt ). The first counterfactual exercise is the Baseline, showing the average values of the vari- ables obtained with the current exogenous variables observed in the EFH sample, Xi,t0 , Zi,t0 , Zi,t. The second counterfactual exercise accounts for a large increase of the public pen- sions on January of 2022 (Law 21419), with a minimum noncontributory public pen- sion of 185,000 pesos (roughly, 210 USD). This law, implemented owing to the social discontent caused by the Covid pandemic and the Social Explosion (Madeira 2022), provided all the retirees in households within the lowest nine deciles of income (there- fore, almost universal coverage) with a monthly solidarity pension of 185,000 pesos for retirees with pensions below 630,000 pesos, with a decreasing linear amount until the benefit reaches zero pesos for pensions equal to or greater than one million pesos: Bk,t = b11(˜pk,t ≤b2) + b1(1 −˜pk,t−b2 b3−b2 )1(b2 < ˜pk,t < b3) , with b1 = 185, 000 , b2 = 630, 000 , and b3 = 1, 000, 000 , with ˜pk,t being the current contributory pen- sion income of the k-th member of household i at time t. The households’ new income level is: ln(¯Pi,t + 1(¯Pi,t ≤DY9) k Bk,t1(agek,t ≥65)) . The same adjustment is applied to ln(¯Pi,t−4) . The role of demographics, income profile and unobserved preferences The years 2010–2011 show a higher propensity of Arrears, which could have been due to the Chilean mega-earthquake of 2010. In terms of the estimated heteroscedasticity of unobserved tastes for choosing differ- ent debt alternatives, the results demonstrate that the unobserved factor for the prefer- ence for all debts has a big unobserved standard-deviation and it has a significant effect on choices. However, the unobserved factor for bank loans has a small standard-devia- tion and it is statistically insignificant. These results are available from the author upon request and are reported with higher detail in the full information model estimated in the previous version of this article (Madeira 2019c). This demonstrates that the consum- ers with debt (choices 1 to 5) are different in some unobservable way from those with No Page 22 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Access and No Wish for Debt, perhaps because of their preferences, previous credit his- tory or a better overall impression these households make with lenders. Access and No Wish for Debt, perhaps because of their preferences, previous credit his- tory or a better overall impression these households make with lenders. Counterfactual simulations of policies This Finan- cial literacy program consists in obtaining new values for the outcomes of interest ( Pr(Yi,t = b \| Xi,t0), E[ln(Li,t) \| Zi,t0], Pr(Di,t ≥1 \| Zi,t) ) by considering that: (i) all households increase their financial knowledge by the equivalent to one year of educa- tion, educationi(new) = 1 + educationi ; (ii) the coefficient for years of education for getting No Access to Credit becomes twice as strong, βeducation b=B = 2 ˆβeducation b=B 19; (iii) the coefficients for the impact of education for choosing each loan alternative become less positive, owing to borrowers higher awareness of the median interest rate charged by those lenders, βeducation b = ˆβeducation b −ib for b = 1, . . . , 5 , with i1 = 0.15 , i2 = 0.195 , i3 = 0.24 , i4 = 0.22 , i5 = 0.24.20 Table 8 shows the probabilities of each type of debt under the different counterfac- tual policies. The Baseline and the data statistics in Table 1 are quite close; therefore, the model is reliable for counterfactual analysis. Relative to the Baseline, all the counterfac- tuals increase the probability of the Bank debt type and decrease the probability of the Retail Store only debt type, with both effects being stronger in the case of the Financial Literacy program. The Bank plus Retail and Union debt types are highest for the Income test scenario, while being lowest for the Financial Literacy program. The Other Debts option is highest for the Pension law policy, while being lowest for the Financial Liter- acy program. Both the Pension Law and the Financial Literacy policies decrease the No Access and increase the No Wish for Debt option, with both effects especially strong in the case of the Financial Literacy program. The Pension law and Income test increase the Bank plus Retail option, while the Financial literacy decreases it substantially. Overall, the Financial Literacy program shows the largest transformation. The prob- ability differences between the Pension Law and the Baseline for each alternative vary between − 0.5% for the case of No Access to +0.5% for the No Wish for Debt option. The Income test policy has much stronger effects than the Pension Law. Counterfactual simulations of policies The adjustment accounts for households being within the 9-th income decile ( 1(¯Pi,t ≤DY9) ), whether each member is above 65 and entitled to public pensions ( 1(agek,t ≥65) ), and the pension income received by each member ( Bk,t).h The third counterfactual exercise, labeled “Income test for repayment purposes”, aims to simulate a scenario in which regulators require lenders to make stronger assessments of the repayment capacity of the borrowers, as suggested by the IMF-World Bank Finan- cial Sector Assessment (FSA) mission to the Chilean government (Cohen and Dijkman 2021). This exercise is implemented by changing the coefficients βb of the Mixed Multi- nomial Logit model, with the coefficients for unemployment ( βunemployment b ) and wage volatility ( βwage−volatility b ) for each debt alternative with loan access (options 1 to 5) being equal to the minimum across all lender types: βunemployment b = minb′=1,...,5 ˆβunemployment b′ Madeira Financial Innovation (2023) 9:49 Page 23 of 29 Madeira Financial Innovation and βwage−volatility b = minb′=1,...,5 ˆβwage−volatility b′ .17 The reasoning is that the regulator will require the same standards of screening for unemployment and wage volatility as those that are already implemented by the most robust lenders. Furthermore, I impose that the coefficients of unemployment risk and wage volatility have twice as much effect on the probability of household having No Access: βunemployment b=B = 2 ˆβunemployment b=B and βwage−volatility b=B = 2 ˆβwage−volatility b=B .18 Therefore, this exercise demonstrates the potential effects of requiring lenders to make more robust decisions regarding borrowers who may have repayment problems later on. The fourth counterfactual exercise is related to the potential benefits that Chile could achieve if it invested in a large scale financial literacy program. 18 The coefficients for unemployment risk and wage volatility in the “No Access” column of Table 6 become 3.746 and 0.504, respectively. hfi 20 The coefficients for the years of education in Table 6 become − 0.027, − 0.1015, − 0.24713, − 0.1609 and − 0.13, for, respectively, the lender options of 1 “Bank”, 2 “Bank & Retail”, 3 “Retail”, 4 “Union”, 5 “Other Loans”. 19 The coefficient for years of education on the column of “No Access” in Table 6 becomes − 0.1526. 17 The coefficients for the unemployment risk and wage volatility in Table 6 become, respectively, 15.93 and − 3.796 for all the lender options with positive debt (that is, options 1 to 5). 17 The coefficients for the unemployment risk and wage volatility in Table 6 become, respectively, 15.93 and − 3.796 for all the lender options with positive debt (that is, options 1 to 5). 18 The coefficients for unemployment risk and wage volatility in the “No Access” column of Table 6 become 3.746 and 0.504, respectively. 19 The coefficient for years of education on the column of “No Access” in Table 6 becomes − 0.1526. 20 The coefficients for the years of education in Table 6 become − 0.027, − 0.1015, − 0.24713, − 0.1609 and − 0.13, for, respectively, the lender options of 1 “Bank”, 2 “Bank & Retail”, 3 “Retail”, 4 “Union”, 5 “Other Loans”. Counterfactual simulations of policies For the Income test, the differences relative to the Baseline go from − 1.7% in the case of a reduction of the Retail Store only option to an increase of +1.5% in the No Access option. How- ever, the Financial Literacy program has much larger effects. Relative to the Baseline, the Page 24 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 8 Probabilities (in %) for each type of debt choice under different counterfactual policies Table 8 Probabilities (in %) for each type of debt choice under different counterfactual policies Counterfactual Bank Bank + Retail Retail Store Union Debt Other Debts No Wish No Access Baseline 9.6 13.2 26.0 5.9 5.9 28.0 11.3 Pension law of 2022 9.8 13.5 25.6 5.8 6.0 28.5 10.8 Income test 10.1 14.4 24.3 6.3 5.0 27.2 12.8 Financial literacy 16.9 12.6 14.7 4.4 2.9 41.9 6.5 Financial Literacy program increases the Bank only and the No Wish for debt options by, respectively, 7.3% and 13.9%. It also reduces the Retail Store only and the No Access options by, respectively, 11.3% and 4.8%. Furthermore, it decreases the Other Debts, Union and Bank plus Retail options by 3%, 1.5% and 0.6%. Table 9 illustrates the policies’ effects on the average debt probability (the sum of the probabilities for the choices 1 to 5 in Table 8), the aggregate consumer debt across all households and the average debt per borrower, with the debt amounts being standard- ized relative to the baseline (reported as 100%). The Financial Literacy program implies the largest reduction of the debt probability and of the aggregate consumer debt. How- ever, the financial literacy program increases the average debt per borrower, although the number of borrowers decreases, implying an overall drop in the aggregate consumer debt. The Pension Law increases the debt probability, aggregate debt and average debt per borrower, while the Income test decreases all those variables. Again, the Financial Literacy program shows the largest transformations of the consumer debt choices. The Pension law increases aggregate consumer debt by 1%, whereas the Income test reduces aggregate debt by 0.9%. The Financial literacy program reduces the aggregate household consumer debt by 12.4%, more than ten times as much as the Income test policy. Table 10 analyzes the policies’ effect on default risk, as given by the probability of arrears in the previous year, arrears above 1 month or above 3 months. Counterfactual simulations of policies Every policy reduces (even if only slightly) the default risk, regardless of the measure of arrears used or the population (average borrower, all households, and portfolio risk). The Financial Literacy program is the policy with the strongest reduction in default risk. The Pension law differs from the baseline risk measures by at most 0.1% (for instance, it reduces port- folio arrears in the past year by 0.1%), whereas the Income test has a somewhat larger effect with a reduction in risk measures that reaches 0.2% (its effect on the portfolio arrears in the past year, for instance). The Financial Literacy program has a large effect in reducing arrears risk. It reduces the arrears risk in the past year by 1.5%, 2.7% and 2.7%, respectively, across all borrowers, all households and for the debt portfolio. It reduces the current arrears risk at the horizon of one month by 1.2%, 1.9% and 1.7%, respectively, across all borrowers, all households and for the debt portfolio. Finally, for the most standard risk measure which is the arrears at 3 months, the Financial Literacy program reduces risk by 0.4%, 0.5% and 0.5%, respectively, across all borrowers, all households and for the debt portfolio. Overall, the Financial Literacy program would reduce the portfolio risk of arrears above 3 months from a Baseline of 2.5% to just 2%, a reduction of 20% in the overall risk faced by lenders. Page 25 of 29 Madeira Financial Innovation (2023) 9:49 Madeira Financial Innovation Table 9 Effect of different policies on the consumer debt amount (for all households and the average borrower) relative to the baseline (in %) and on the overall probability (in %) of being a consumer debtor Counterfactual Any Debt Probability Consumer debt (aggregate) Consumer debt (average borrower) Baseline 60.6 100 100 Pension law of 2022 60.7 101.0 100.7 Income test 60.0 99.1 99.9 Financial literacy 51.6 87.6 103.5 Table 10 Effect of different policies on the arrears risk (in %) of borrowers, all households and for the consumer debt portfolios Table 10 Effect of different policies on the arrears risk (in %) of borrowers, all households and for the consumer debt portfolios All households’ risk takes into account that households with no debts have zero risk. Counterfactual simulations of policies Portfolio risk takes into account the size of the loans held by each borrower Counterfactual Default definition Borrower risk All households risk Portfolio risk Arrears past year Arrears (months) Arrears past year Arrears (months) Arrears past year Arrears (months) 1 3 1 3 1 3 Baseline 24.1 18.2 4.8 14.3 9.7 2.5 16.7 9.6 2.5 Pension law of 2022 24.0 18.1 4.8 14.3 9.6 2.5 16.6 9.5 2.5 Income test 24.0 18.2 4.8 14.1 9.5 2.4 16.5 9.4 2.4 Financial literacy 22.6 17.0 4.4 11.6 7.8 2.0 14.0 7.9 2.0 All households’ risk takes into account that households with no debts have zero risk. Portfolio risk takes into account the size of the loans held by each borrower Table 11 summarizes the policies’ effect on the aggregate debt and portfolio default risk across debt types. Every policy reduces the default risk for all the debt types, even if only slightly. The Pension law increases the aggregate debt of all the debt types, espe- cially for the Unions. It increases aggregate debt in Unions by 2.1%, whereas its effect on other debt types is between 0.5 and 0.7%. The Income test reduces the aggregate debt of all the debt types, especially for the Retail Store only. The Retail Store only debt is reduced by 4.7%, the Other Debts experience no effect, while the remaining types are reduced by 0.3–1%. The effects of the Pension law and the Income test policies on reduc- ing arrears at either the one or three months horizons are negligible. Finally, the Financial Literacy program decreases the aggregate debt amount across all the debt types, with a particularly strong effect on Retail Store only loans. It also sub- stantially decreases the Bank, Bank plus Retail, Retail and Union Debt default risks, with especially strong effects on the Retail Store only, Bank plus Retail Store, and Union risks. Its strongest effect is on reducing Retail Store only debt by 30%, whereas the other debt forms fall between 8.6% for Bank only and 14% for the Union option. The Financial Lit- eracy program also decreases the risk of arrears substantially. It decreases the portfolio arrears risk at the one month horizon between 0.9% for the Bank only and 2.8% for the Retail Store only options. Conclusions and policy implications This study demonstrates how households’ characteristics affect their choice of lenders, con- sumer debt amounts and default behavior. I find that borrowers’ characteristics differ sub- stantially across lender types. Banks lend to the borrowers of highest income and education and the lowest unemployment rates. Households with No Access to Debt are at the precise opposite, with the lowest income and education and the highest unemployment risk. This makes sense because Banks represent the lender institutions that make the most intensive use of credit scoring and customer specific interest rates, therefore, being able to attract the safest borrowers. Bank plus Retail and Other Debt borrowers have slightly lower income and education than those with Bank loans only; however, they are younger and with larger debts. Retail store borrowers, among those with credit access, have the lowest income and education plus an unemployment risk almost as high as those with No Access to Debt; therefore, they have the lowest consumer debt amounts. Union borrowers have somewhat better conditions, with slightly higher income and education and also larger loans. A multiple choice model demonstrates that education and income increase the prob- ability of Banks, Banks-Retail and Other Debts, while decreasing the probability of No Access to Debt. Unemployment risk and household size increase the probability of opting for all loans (especially Retail Stores and Other Debts), whereas age and wage volatility decrease the probability of all loans. A second step regression demonstrates that loan amounts increase with income and education, while decreasing with unem- ployment risk, age and the share of debt with the main lender. The default probability decreases with income, education and age, while increasing with high indebtedness ratios, unemployment risk, household size, loans motivated by Paying Previous Debts and Health needs. Paying previous debts is a motive that proxies “moral hazard” (since it benefits previous lenders, not the current lender), while Health shocks can be viewed as an adverse selection variable that lenders do not observe. The model can be used to simulate counterfactual results for a variety of policies. As examples, I analyze the recent increase in pensions, higher regulatory requirements from lenders to assess borrowers’ repayment capacity, or a Financial Literacy program. The counterfactuals demonstrate that all of these policies reduce the borrowers’ risk lev- els. Higher public pensions increase both the number of debtors and their debt amounts, whereas the repayment capacity test lowers them. CM: conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writ- ing—original draft, writing—review & editing, visualization, supervision, project administration, funding acquisition. The author read and approved the final manuscript. Counterfactual simulations of policies It also decreases the portfolio arrears risk at the three months horizon between 0.2% for the Bank only and 0.8% for the Retail Store only options.hf This exercise demonstrates that, while all policies affect borrowers’ choices, the Finan- cial Literacy program presents the strongest impact on the debt market, whether in terms of the debt type choice, overall amount of debt, or the loan default. In particular, Madeira Financial Innovation (2023) 9:49 Page 26 of 29 Table 11 Effect of different policies on the loan amount aggregate relative to the baseline (in %) and the portfolio arrears risk (in %) across debtor types Counterfactuals Bank Bank+Retail Retail Store Union Debt Other Debts Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) 1 3 1 3 1 3 1 3 1 3 Baseline 100 6.6 1.5 100 14.0 3.9 100 14.2 3.8 100 12.2 3.2 100 8.6 2.1 Pension law of 2022 100.6 6.6 1.5 100.5 14.0 3.9 100.7 14.1 3.8 102.1 12.1 3.2 100.6 8.6 2.1 Income test 99.7 6.5 1.5 99.0 13.8 3.8 95.3 13.9 3.7 99.5 12.1 3.2 100.0 8.5 2.1 Financial literacy 91.4 5.7 1.3 88.3 11.6 3.2 70.0 11.4 3.0 86.0 10.1 2.6 90.0 7.0 1.7 Table 11 Effect of different policies on the loan amount aggregate relative to the baseline (in %) and the portfolio arrears risk (in %) across debtor types Counterfactuals Bank Bank+Retail Retail Store Union Debt Other Debts Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) Debt aggregate Arrears (months) 1 3 1 3 1 3 1 3 1 3 Baseline 100 6.6 1.5 100 14.0 3.9 100 14.2 3.8 100 12.2 3.2 100 8.6 2.1 Pension law of 2022 100.6 6.6 1.5 100.5 14.0 3.9 100.7 14.1 3.8 102.1 12.1 3.2 100.6 8.6 2.1 Income test 99.7 6.5 1.5 99.0 13.8 3.8 95.3 13.9 3.7 99.5 12.1 3.2 100.0 8.5 2.1 Financial literacy 91.4 5.7 1.3 88.3 11.6 3.2 70.0 11.4 3.0 86.0 10.1 2.6 90.0 7.0 1.7 Madeira Financial Innovation (2023) 9:49 Page 27 of 29 Madeira Financial Innovation this program substantially increases the probability of No Wish for Debt and Bank loans, while decreasing the aggregate loan amounts and the Any Debt, No Access, and the default risk probabilities. Conclusions and policy implications A financial literacy program could have a great impact on reducing the number of debtors, the value of consumer debt and the share of households with No Access to Debt, while increasing the share of borrowers with Banks. It also substantially decreases the arrears risk of all the debt options. Acknowledgements g I thank seminar participants at the Federal Reserve Bank of New York, Deutsche Bundesbank, Federal Reserve Bank of Philadelphia, LACEA, Universidad Carlos III, University of York, Bank of Spain, Bank of England and Central Bank of Chile. I thank Bernardita Piedrabuena for all the great conversations at an early stage of this project. g thank seminar participants at the Federal Reserve Bank of New York, Deutsche Bundesbank, Federal Reserve Bank of Philadelphia, LACEA, Universidad Carlos III, University of York, Bank of Spain, Bank of England and Central Bank of Chile. I hank Bernardita Piedrabuena for all the great conversations at an early stage of this project. Availability of data and materials Availability of data and materials All the codes are freely available at the repository Mendeley data: https://data.mendeley.com/datasets/8hv5d3sm2h/1. The datasets used in this study are freely available from the Chilean Bureau of Official Statistics (INE) and from the Central Bank of Chile after a research project form is filled. Received: 9 August 2021 Accepted: 16 January 2023 Received: 9 August 2021 Accepted: 16 January 2023 Received: 9 August 2021 Accepted: 16 January 2023 Funding g The author received no funding for this project. Funding The author received no funding for this project. Funding The author received no funding for this project. 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https://openalex.org/W2314120933	https://pure.rug.nl/ws/files/57880169/Ablikim_2015_Chinese_Phys._C_39_041001.pdf	English	null	Evidence for e <sup>+</sup> e <sup>−</sup> →γχ <sub>c1,2</sub> at center-of-mass energies from 4.009 to 4.360 GeV	Chinese Physics C, High Energy Physics and Nuclear Physics/Chinese physics C	2,015	cc-by	9,519	University of Groningen Evidence for e(+)e(-)->gamma chi c1,2 at center-of-mass energies from 4.009 to 4.360 GeV Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; Bondarenko, O.; Haddadi, Z.; Kalantar-Nayestanaki, N. Published in: University of Groningen Evidence for e(+)e(-)->gamma chi c1,2 at center-of-mass energies from 4.009 to 4.360 GeV Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; Bondarenko, O.; Haddadi, Z.; Kalantar-Nayestanaki, N. Published in: University of Groningen University of Groningen Evidence for e(+)e(-)->gamma chi c1,2 at center-of-mass energies from 4.009 to 4.360 GeV Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; Bondarenko, O.; Haddadi, Z.; Kalantar-Nayestanaki, N. Evidence for e(+)e(-)->gamma chi c1,2 at center-of-mass energies from 4.009 to 4.360 GeV Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; Bondarenko, O.; Haddadi, Z.; Kalantar-Nayestanaki, N. DOI: 10.1088/1674-1137/39/4/041001 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 Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ablikim, M., Achasov, M. N., Ai, X. C., Albayrak, O., Albrecht, M., Ambrose, D. J., Amoroso, A., Bondarenko, O., Haddadi, Z., Kalantar-Nayestanaki, N., Kavatsyuk, M., Loehner, H., Messchendorp, J. G., Tiemens, M., & BESIII Collaboration (2015). Evidence for e(+)e(-)->gamma chi c1,2 at center-of-mass energies from 4.009 to 4.360 GeV. 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For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Chinese Physics C Related content Precision measurement of the integrated luminosity of the data taken by BESIII at center-of-mass energies between 3.810 GeV and 4.600 GeV Ablikim M., M. N. Achasov, Ai X. C. et al. - Study of tracking efficiency and its systematic uncertainty from J/ pp+ at BESIII* Wen-Long Yuan, Xiao-Cong Ai, Xiao-Bin Ji et al. - Study of cluster reconstruction and track fitting algorithms for CGEM-IT at BESIII* Yue Guo, Liang-Liang Wang, Xu-Dong Ju et al. - Related content Precision measurement of the integrated luminosity of the data taken by BESIII at center-of-mass energies between 3.810 GeV and 4.600 GeV Ablikim M., M. N. Achasov, Ai X. C. et al. - Study of tracking efficiency and its systematic uncertainty from J/ pp+ at BESIII* Wen-Long Yuan, Xiao-Cong Ai, Xiao-Bin Ji et al. - Study of cluster reconstruction and track fitting algorithms for CGEM-IT at BESIII* Yue Guo, Liang-Liang Wang, Xu-Dong Ju et al. - PAPER • OPEN ACCESS This content was downloaded from IP address 129.125.148.244 on 26/04/2018 at 12:08 PAPER • OPEN ACCESS Related content Received 25 October 2014 ∗Supported by National Key Basic Research Program of China (2015CB856700), Joint Funds of National Natural Science Foundation of China (11079008, 11179007, U1232201, U1332201, U1232107), National Natural Science Foundation of China (NSFC) (10935007, 11121092, 11125525, 11235011, 11322544, 11335008), Chinese Academy of Sciences (CAS) Large-Scale Scientiﬁc Facility Program, CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology; German Research Foundation DFG (Collaborative Research Center CRC-1044), Istituto Nazionale di Fisica Nucleare, Italy, Ministry of Development of Turkey (DPT2006K-120470), Russian Foundation for Basic Research (14-07-91152), U. S. Department of En- ergy (DE-FG02-04ER41291, DE-FG02-05ER41374, DE-FG02-94ER40823, DESC0010118), U.S. National Science Foundation, University of Groningen (RuG) and Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0) Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Article funded by SCOAP3 and published under licence by Chinese Physical Society and the Institute of High Energy Physics of the Chinese Academy of Sciences and the Institute of Modern Physics of the Chinese Academy of Sciences and IOP Publishing Ltd Recent citations Measurement ofe+ecJvia initial state radiation at Belle Y. L. Han et al - Study of the XYZ states at the BESIII Chang-Zheng Yuan - This content was downloaded from IP address 129.125.148.244 on 26/04/2018 at 12:08 This content was downloaded from IP address 129.125.148.244 on 26/04/2018 at 12:08 Chinese Physics C Vol. 39, No. 4 (2015) 041001 Evidence for e+e−→γχc1,2 at center-of-mass energies from 4.009 to 4.360 GeV∗ L. He()1 Z. Y. He()29 T. Held3 Y. K. Heng()1 Z. L. Hou( )1 C. Hu()27 H. M. Hu()1 J. F. Hu( )47A T. Hu()1 Y. Hu()1 G. M. Huang()5 G. S. Huang()44 H. P. Huang()49 J. S. Huang()14 X. T. Huang()32 Y. Huang()28 T. Hussain46 Q. Ji()1 Q. P. Ji()29 X. B. Ji( )1 X. L. Ji( )1 L. L. Jiang( )1 L. W. Jiang( )49 X. S. Jiang()1 J. B. Jiao()32 Z. Jiao()16 D. P. Jin()1 S. Jin()1 T. Johansson48 A. Julin42 N. Kalantar-Nayestanaki24 X. L. Kang( )1 X. S. Kang()29 M. Kavatsyuk24 B. C. Ke4 R. Kliemt13 B. Kloss21 O. B. Kolcu39B,d B. Kopf3 M. Kornicer41 W. Kuehn23 A. Kupsc48 W. Lai()1 J. S. Lange23 M. Lara18 P. Larin13 C. H. Li()1 Cheng Li()44 D. M. 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Han( )1 )27 Y. Guo( )1 Y. P. Guo21 Z. Haddadi24 A. Hafner21 S. Han( )49 Y. L. H C. Hu()27 H. M. Hu()1 J. F. Hu( )47A T. Hu()1 Y. Hu()1 G. M. Huang()5 G. S. Huang()44 H. P. Huang()49 J. S. Huang()14 X. T. Huang()32 () () ( ) () () g() G. S. Evidence for e+e−→γχc1,2 at center-of-mass energies from 4.009 to 4.360 GeV∗ M. Ablikim()1 M. N. Achasov8,a X. C. Ai()1 O. Albayrak4 M. Albrecht3 D. J. Ambrose43 A. Amoroso47A,47C F. F. An()1 Q. An()44 J. Z. Bai()1 R. Baldini Ferroli19A Y. Ban()30 D. W. Bennett18 J. V. Bennett4 M. Bertani19A D. Bettoni20A J. M. Bian()42 F. Bianchi47A,47C E. Boger22,h O. Bondarenko24 I. Boyko22 R. A. Briere4 H. Cai()49 X. Cai()1 O. Cakir39A,b A. Calcaterra19A G. F. Cao()1 S. A. Cetin39B J. F. Chang()1 G. Chelkov22,c G. Chen()1 H. S. Chen()1 H. Y. Chen()2 J. C. Chen()1 M. L. Chen()1 S. J. Chen()28 X. Chen()1 X. R. Chen()25 Y. B. Chen()1 H. P. Cheng()16 X. K. Chu()30 G. Cibinetto20A D. Cronin-Hennessy42 H. L. Dai()1 J. P. Dai()33 A. Dbeyssi13 D. Dedovich22 Z. Y. Deng()1 A. Denig21 I. Denysenko22 M. Destefanis47A,47C F. De Mori47A,47C Y. Ding()26 C. Dong()29 J. Dong()1 L. Y. Dong()1 M. Y. Dong()1 S. X. Du()51 P. F. Duan()1 J. Z. Fan()38 J. Fang()1 S. S. Fang()1 X. Fang()44 Y. Fang()1 L. Fava47B,47C F. Feldbauer21 G. Felici19A M. Ablikim()1 M. N. Achasov8,a X. C. Ai()1 O. Albayrak4 M. Albrecht3 D. J. Ambrose43 A. Amoroso47A,47C F. F. An()1 Q. An()44 J. Z. Bai()1 R. Baldini Ferroli19A Y. Ban()30 D. W. Bennett18 J. V. Bennett4 M. Bertani19A D. Bettoni20A J. M. Bian()42 F. Bianchi47A,47C E. Boger22,h O. Bondarenko24 I. Boyko22 R. A. Briere4 H. Cai()49 X. Cai()1 O. Cakir39A,b A. Calcaterra19A G. F. Cao()1 S. A. Cetin39B J. F. Chang()1 G. Chelkov22,c G. Chen()1 H. S. Chen()1 H. Y. Chen()2 J. C. Chen()1 M. L. Chen()1 S. J. Chen()28 X. Chen()1 X. R. Chen()25 Y. B. Chen()1 H. P. Cheng()16 X. K. Chu()30 G. Cibinetto20A D. Cronin-Hennessy42 H. L. Dai()1 J. P. Dai()33 A. Dbeyssi13 D. Dedovich22 Z. Y. Deng()1 A. Denig21 I. Denysenko22 M. Destefanis47A,47C F. De Mori47A,47C Y. Ding()26 C. Dong()29 J. Dong()1 L. Y. Dong()1 M. Y. Dong()1 S. X. Du()51 P. F. Duan()1 J. Z. Fan()38 J. Fang()1 S. S. Fang()1 X. Fang()44 Y. Fang()1 L. Fava47B,47C F. Feldbauer21 G. Felici19A C. Q. Feng()44 E. Fioravanti20A M. Fritsch13,21 C. D. Fu()1 Q. Gao()1 Y. Gao()38 Z. Gao()44 I. Garzia20A K. Goetzen9 W. X. Gong()1 W. Gradl21 M. Greco47A,47C M. H. Gu()1 Y. T. Gu()11 Y. H. Guan()1 A. Q. Guo( )1 L. B. Guo( )27 T. Guo( )27 Y. Guo( )1 Y. P. Guo21 Z. Haddadi24 A. Hafner21 S. Han( )49 Y. L. Han( )1 F. A. Harris41 K. Evidence for e+e−→γχc1,2 at center-of-mass energies from 4.009 to 4.360 GeV∗ Qi( )28 S Qian()1 C F Qiao()40 L Q Qin( )32 N Qin()49 X S Qin( )1 Y Qin( )30 () Q y g( ) g() K. Peters9 J. L. Ping( )27 R. G. Ping()1 R. Poling42 Y. N. Pu()17 M. Qi( )28 S Q ()1 C Q ()40 Q Q ( )32 Q ()49 S Q ( )1 Q ( )30 F. Qiu( )1 K. H. Rashid46 C. F. Redmer21 H. L. Ren()17 M. Ripka21 Z. H. Qin( )1 J. F. Qiu( )1 K. H. Rashid46 C. F. Redmer21 H. L. Ren()17 M. 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Li()1 Jin Li()31 K. Li()12 K. Li()32 P. R. Li()40 T. Li()32 W. D. Li( )1 W. G. Li()1 X. L. Li( )32 X. M. Li()11 X. N. Li()1 X. Q. Li( )29 Z. B. Li()37 H. Liang( )44 Y. F. Liang( )35 Y. T. Liang( )23 G. R. Liao()10 D. X. Lin(Lin)13 B. J. Liu( )1 C. L. Liu4 C. X. Liu( )1 F. H. Liu()34 Fang Liu( )1 Feng Liu( )5 H. B. Liu()11 H. H. Liu()15 041001-1 Chinese Physics C Vol. 39, No. 4 (2015) 041001 H. L. Ma()1 L. L. Ma()32 Q. M. Ma()1 S. Ma()1 T. Ma()1 X. N. Ma()29 X. Y. Ma() F. E. Maas M. Maggiora Q. A. Malik Y. J. Mao() Z. P. Mao() S. Marcello47A,47C J. G. Messchendorp24 J. Min()1 T. J. Min( )1 R. E. Mitchell18 ) gg Q ( ) arcello47A,47C J. G. Messchendorp24 J. Min()1 T. J. Min( )1 R. E. Mitchell1 S. Marcello47A,47C J. G. Messchendorp24 J. Min()1 T. J. Min( )1 R. E. Mitchell18 X. H. Mo() Y. J. Mo() C. Morales Morales K. Moriya N. Yu. Muchnoi H. Muramatsu Y. Nefedov22 F. Nerling13 I. B. Nikolaev8,a Z. Ning()1 S. Nisar7 S. L. Niu( )1 X. Y. Niu( )1 Y. Nefedov22 F. Nerling13 I. B. Nikolaev8,a Z. Ning()1 S. Nisar7 S. L. Niu( )1 X. Y. Niu( )1 Y. Nefedov22 F. Nerling13 I. B. Nikolaev8,a Z. Ning()1 S. Nisar7 S. L. Niu( )1 X. Y. Niu( )1 S. L. Olsen() Q. Ouyang() S. Pacetti P. Patteri M. Pelizaeus H. P. Peng() K. Peters9 J. L. Ping( )27 R. G. Ping()1 R. Poling42 Y. N. Pu()17 M. Evidence for e+e−→γχc1,2 at center-of-mass energies from 4.009 to 4.360 GeV∗ Xie()1 G. F. Xu( )1 L. Xu()1 Q. J. Xu()12 Q. N. Xu( )40 X. P. Xu()36 44 W. B. Yan()44 W. C. Yan()44 Y. H. Yan()17 H. X. Yang( L. Yang( )49 Y. Yang( )5 Y. X. Yang( )10 H. Ye( )1 M. Ye( )1 M. H. Ye( )6 J. H. Yin()1 B. X. Yu( )1 C. X. Yu( )29 H. W. Yu()30 J. S. Yu( )25 C. Z. Yuan( )1 W. L. Yuan()28 Y. Yuan()1 A. Yuncu39B,g A. A. Zafar46 A. Zallo19A 17 1 1 28 1 Y. Zeng()17 B. X. Zhang( )1 B. Y. Zhang( )1 C. Zhang( )28 C. C. Zhang( )1 D. H. Zhang( )1 H. H. Zhang( )37 H. Y. Zhang()1 J. J. Zhang( )1 J. L. Zhang( )1 J. Q. Zhang( )1 J. W. Zhang( )1 J. Y. Zhang( )1 Y. Zeng()17 B. X. Zhang( )1 B. Y. Zhang( )1 C. Zhang( )28 C. C. Zhang( )1 D. H. Zhang( )1 H. H. Zhang( )37 H. Y. Zhang()1 J. J. Zhang( )1 J. L. Zhang( )1 J. Q. Zhang( )1 J. W. Zhang( )1 J. Y. Zhang( )1 J. Z. Zhang( )1 K. Zhang( )1 L. Zhang( )1 S. H. Zhang( )1 X. Y. Zhang( )32 Y. Zhang( )1 Y. H. Zhang( )1 Y. T. Zhang( )44 Z. H. Zhang( )5 J. Z. Zhang( )1 K. Zhang( )1 L. Zhang( )1 S. H. Zhang( )1 X. Y. Zhang( )32 Y. Zhang( )1 Y. H. Zhang( )1 Y. T. Zhang( )44 Z. H. Zhang( )5 Z. P. Zhang( )44 Z. Y. Zhang( )49 G. Zhao()1 J. W. Zhao()1 J. Y. Zhao()1 J. Z. Zhao()1 Lei Zhao()44 Ling Zhao( )1 M. G. Zhao()29 Q. Zhao()1 A. Zhemchugov22,h B. Zheng()45 J. P. Zheng()1 W. J. Zheng()32 Y. H. Zheng()40 B. Zhong( )27 L. Zhou()1 Li Zhou()29 X. Zhou( )49 X. K. Zhou()44 X. R. Zhou( )44 X. Y. Zhou()1 K. Zhu()1 K. J. Zhu()1 S. Zhu()1 A. Zhemchugov22,h B. Zheng()45 J. P. Zheng()1 W. J. Zheng()32 Y. H. Zheng()40 B. Zhong( )27 L. Zhou()1 Li Zhou()29 X. Zhou( )49 X. K. Zhou()44 g g( ) g( ) g( ) g( ) B. Zhong( )27 L. Zhou()1 Li Zhou()29 X. Zhou( )49 X. K. Zhou()44 X. H. H. Liu()1 H. M. Liu( )1 J. Liu()1 J. P. Liu( )49 J. Y. Liu( )1 K. Liu()38 K. Y. Liu()26 L. D. Liu( )30 P. L. Liu()1 Q. Liu()40 S. B. Liu( )44 X. Liu()25 X. X. Liu()40 Y. B. Liu()29 Z. A. Liu()1 Zhiqiang Liu()1 Zhiqing Liu21 H. Loehner24 X. C. Lou( )1,e H. J. Lu()16 J. G. Lu()1 R. Q. Lu()17 Y. Lu()1 Y. P. Lu()1 C. L. Luo()27 M. X. Luo()50 T. Luo41 X. L. Luo( )1 M. Lv()1 X. R. Lyu()40 F. C. Ma()26 H. L. Ma()1 L. L. Ma()32 Q. M. Ma()1 S. Ma()1 T. Ma()1 X. N. Ma()29 Evidence for e+e−→γχc1,2 at center-of-mass energies from 4.009 to 4.360 GeV∗ R. Zhou( )44 X. Y. Zhou()1 K. Zhu()1 K. J. Zhu()1 S. Zhu()1 X. L. Zhu( )38 Y. C. Zhu()44 Y. S. Zhu()1 Z. A. Zhu()1 X. R. Zhou( )44 X. Y. Zhou()1 K. Zhu()1 K. J. Zhu()1 S. Zhu()1 X. L. Zhu( )38 Y. C. Zhu()44 Y. S. Zhu()1 Z. A. Zhu()1 041001-2 041001-2 Chinese Physics C Vol. 39, No. 4 (2015) 041001 J. Zhuang()1 B. S. Zou()1 J. H. Zou()1 (BES collaboration) 1 Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China 2 s, Chinese Academy of Sciences, Beijing 100049, Chin 2 Beihang University, Beijing 100191, China chum Ruhr-University, D-44780 Bochum, Germany 4 Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA 5 Central China Normal University, Wuhan 430079, China titute of Information Technology, Lahore, Defence Road, OﬀRaiwind Road, 54000 Lahore, Pakistan 7 COMSATS Institute of Information Technology, Lahore, Defence Road, OﬀRaiwind Road, 54000 La 7 COMSATS Institute of Information Technology, Lahore, Defence Road, OﬀRaiwind Road, 54000 Lahore, Pakistan 8 8 G.I. Budker Institute of Nuclear Physics SB RAS (BINP), Novosibirsk 630090, Russia 9 9 GSI Helmholtzcentre for Heavy Ion Research GmbH, D-64291 Darmstadt, Germany 10 Guangxi Normal University, Guilin 541004, China 10 Guangxi Normal University, Guilin 541004, China g y, , 11 Guangxi University, Nanning 530004, China g y g 12 Hangzhou Normal University, Hangzhou 310036, China 13 Helmholtz Institute Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, Germany 14 Henan Normal University, Xinxiang 453007, China 15 Henan University of Science and Technology, Luoyang 471003, China 16 University of Science and Technology, Luoyang 4710 16 Huangshan College, Huangshan 245000, China 17 Hunan University, Changsha 410082, China y g 18 Indiana University, Bloomington, Indiana 47405, USA y, g , , 19 (A)INFN Laboratori Nazionali di Frascati, I-00044, Frascati, Italy; (B)INFN and University o 20 (A) S d l ( ) f nali di Frascati, I-00044, Frascati, Italy; (B)INFN and University of Perugia, I-06100, Perugia, Italy FN Laboratori Nazionali di Frascati, I-00044, Frascati, Italy; (B)INFN and University of Perugia, I-06100, 20 ohannes Gutenberg University of Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, G 22 Joint Institute for Nuclear Research, 141980 Dubna, Moscow region, Russia us Liebig University Giessen, II. J. Zhuang()1 B. S. Zou()1 J. H. Zou()1 (BES collaboration) Physikalisches Institut, Heinrich-Buﬀ-Ring 16, D-35392 Giessen, g y , y , g , , 24 KVI-CART, University of Groningen, NL-9747 AA Groningen, The Netherlands 24 KVI-CART, University of Groningen, NL-9747 AA Groningen, The Netherlands 25 University of Groningen, NL-9747 AA Groningen, T 25 Lanzhou University, Lanzhou 730000, China 26 Liaoning University, Shenyang 110036, China Nanjing Normal University, Nanjing 210023, China 28 Nanjing University, Nanjing 210093, China 29 Nankai University, Tianjin 300071, China 30 30 Peking University, Beijing 100871, China 31 Seoul National University, Seoul, 151-747 Korea 32 Shandong University, Jinan 250100, China 33 Shanghai Jiao Tong University, Shanghai 200240, China 34 34 Shanxi University, Taiyuan 030006, China 35 Sichuan University, Chengdu 610064, China 36 Soochow University, Suzhou 215006, China 37 Sun Yat-Sen University, Guangzhou 510275, China 38 Tsinghua University, Beijing 100084, China 38 Tsinghua University, Beijing 100084, China ( ) g y, j g , 39 (A)Istanbul Aydin University, 34295 Sefakoy, Istanbul, Turkey; (B)Dogus University, 34722 Istanbu Turkey; (C)Uludag University, 16059 Bursa, Turkey University of Chinese Academy of Sciences, Beijing 100049, China 41 University of Hawaii, Honolulu, Hawaii 96822, USA 42 University of Minnesota, Minneapolis, Minnesota 55455, USA 43 43 University of Rochester, Rochester, New York 14627, USA 45 University of South China, Hengyang 421001, China 46 University of the Punjab, Lahore-54590, Pakistan 47 (A)University of Turin, I-10125, Turin, Italy; (B)University of Eastern Piedmont, I- University of Turin, I-10125, Turin, Italy; (B)University of Eastern Piedmont, I-15121, Alessandria, Italy; (C)INFN, I-10125, Turin, Italy Italy; (C)INFN, I-10125, Turin, Italy ( ) 48 Uppsala University, Box 516, SE-75120 Uppsala, Sweden 49 49 Wuhan University, Wuhan 430072, China 49 Wuhan University, Wuhan 430072, China 50 Zhejiang University, Hangzhou 310027, China 50 Zhejiang University, Hangzhou 310027, China j g y g 51 Zhengzhou University, Zhengzhou 450001, China a Also at the Novosibirsk State University, Novosibirsk, 630090, Russia b a Also at the Novosibirsk State University, Novosibirsk, 630090, Russia b b Also at Ankara University, 06100 Tandogan, Ankara, Turkey b Also at Ankara University, 06100 Tandogan, Ankara, Turkey Electronics Laboratory, Tomsk State University, Tomsk, 634050, Russia Electronics Laboratory, Tomsk State University, Tomsk, 634050, Russia d Currently at Istanbul Arel University, Kucukcekmece, Istanbul, Turkey d Currently at Istanbul Arel University, Kucukcekmece, Istanbul, Turkey at University of Texas at Dallas, Richardson, Texas 7 f Also at the PNPI, Gatchina 188300, Russia g Also at Bogazici University, 34342 Istanbul, Turkey h Also at the Moscow Institute of Physics and Technology, Moscow 141700, Russia Abstract: Using data samples collected at center-of-mass energies of √s=4.009, 4.230, 4.260, and 4.360 GeV with the BES detector operating at the BEPC collider, we perform a search for the process e+e−→γχcJ (J =0, 1, 2) 041001-3 2 BES detector and Monte Carlo The BES detector at the BEPC collider [17] is a large solid-angle magnetic spectrometer with a geomet- rical acceptance of 93% of 4π solid angle consisting of four main components. The innermost is a small-cell, helium-based (40% He, 60% C3H8) main drift chamber (MDC) with 43 layers providing an average single-hit resolution of 135 μm. The resulting charged-particle momentum resolution for a 1 T magnetic ﬁeld setting is 0.5% at 1.0 GeV/c, and the resolution on the ioniza- tion energy loss information (dE/dx) is better than 6%. The next detector, moving radially outwards, is a time- of-ﬂight (TOF) system constructed of 5 cm thick plastic scintillators, with 176 detectors of 2.4 m length in two layers in the barrel and 96 fan-shaped detectors in the end-caps. The barrel (end-cap) time resolution of 80 ps (110 ps) provides a 2σ K/π separation for momenta up to 1.0 GeV/c. Continuing outward, we have an electro- magnetic calorimeter (EMC) consisting of 6240 CsI(Tl) crystals in a cylindrical barrel structure and two end- caps. The energy resolution at 1.0 GeV is 2.5% (5%) and the position resolution is 6 mm (9 mm) in the barrel (end-caps). Finally, the muon counter consists of 1000 m2 of Resistive Plate Chambers in nine barrel and eight end-cap layers, which provides a 2 cm position resolution. Most of the previous studies of the Y(4260) have utilized hadronic transitions. Except for the clear sig- nal observed in the π+π−J/ψ decay mode, the Belle experiment failed to ﬁnd evidence of the Y(4260) via the e+e−→γISRηJ/ψ process [12]. Based on 13.2 pb−1 of e+e−data collected at √s = 4.260 GeV, the CLEO experiment investigated fourteen hadronic decay chan- nels, but the only charmonium channels with more than 3σ statistical signiﬁcance are π+π−J/ψ, π0π0J/ψ and K+K−J/ψ [13]. The BES Collaboration ﬁrst observed the process e+e−→γX(3872) using data samples taken between √s=4.009 and 4.420 GeV [14], which strongly supports the existence of the radiative transition decays of the Y(4260). To further understand the nature of the Y(4260) state, an investigation into the radiative tran- sitions between the Y(4260) and other lower mass char- monium states, like the χcJ (J =0, 1, 2), is important [15, 16]. The cross sections of e+e−→γχcJ have been evaluated theoretically within the framework of non- relativistic quantum chromodynamics (NRQCD) [16]. Experimentally, the only existing investigation comes from the CLEO experiment [13], which did not observe a signal. 041001-3 Chinese Physics C Vol. 39, No. 4 (2015) 041001 and ﬁnd evidence for e+e−→γχc1 and e+e−→γχc2 with statistical signiﬁcances of 3.0σ and 3.4σ, respectively. The Born cross sections σB(e+e−→γχcJ ), as well as their upper limits at the 90% conﬁdence level (C.L.) are determined at each center-of-mass energy. Key words: heavy quarkonia, decays of hadronic, electron-positron collisions, hadron production by PACS: 14.40.Pq, 13.25.Gv, 13.66.Bc DOI: 10.1088/1674-1137/39/4/041001 ds: heavy quarkonia, decays of hadronic, electron-positron collisions, hadron production by Key words: heavy quarkonia, decays of hadronic, electron-positron collisions, hadron produc 1 Introduction 4.360 GeV, where the χcJ is reconstructed by its γJ/ψ decay mode, and the J/ψ is by its decay to μ+μ−. The decay J/ψ→e+e−is not considered in this analysis due to the high background of Bhabha events. The corre- sponding luminosities of the data samples at diﬀerent CME used in this analysis are listed in Table 1. The charmonium-like state Y(4260) was ﬁrst ob- served in the initial state radiation (ISR) process e+e−→ γISRπ+π−J/ψ by BaBar [1], and later conﬁrmed by the CLEO [2] and Belle [3] experiments. Recently, both BaBar and Belle updated their results with full data sets, and further conﬁrmed the existence of the Y(4260) [4, 5]. Since it is produced through ISR in e+e−annihilation, the Y(4260) has the quantum numbers J P C=1−−. How- ever, there seems to be no c¯c slot available for the Y(4260) in the conventional charmonium family [6]. In addition, a number of unusual features, such as a strong coupling to hidden-charm ﬁnal states, suggest that the Y(4260) is a non-conventional c¯c meson. Possible inter- pretations of this state can be found in Refs. [7–11], but all need further experimental input. Table 1. The center-of-mass energy and Luminos- ity of each data sample. √s/GeV luminosity/pb−1 4.009 482 4.230 1047 4.260 826 4.360 540 2 BES detector and Monte Carlo Table 1. The center-of-mass energy and Luminos- ity of each data sample. 2 BES detector and Monte Carlo The large data sample collected with the BES detector provides a good opportunity to deeply investi- gate these decay modes, which may shed more light on the properties of the Y(4260). A GEANT4 [18] based Monte Carlo (MC) simula- tion software, which includes the geometric description of the detector and the detector response, is used to op- timize the event selection criteria, determine the detec- tion eﬃciency, and estimate the potential backgrounds. Signal MC samples of e+e−→γχcJ are generated for A GEANT4 [18] based Monte Carlo (MC) simula- tion software, which includes the geometric description of the detector and the detector response, is used to op- timize the event selection criteria, determine the detec- tion eﬃciency, and estimate the potential backgrounds. Signal MC samples of e+e−→γχcJ are generated for In this paper, we report on a search for e+e−→γχcJ (J =0, 1, 2) based on the large e+e−annihilation data samples collected with the BES detector at center- of-mass energies (CME) √s =4.009, 4.230, 4.260, and 041001-4 Chinese Physics C Vol. 39, No. 4 (2015) 041001 each CME according to the electric-diplole (E1) tran- sition assumption [19]. Eﬀects of ISR are simulated with KKMC [20] by assuming that γχcJ is produced via Y(4260) decays, where the Y(4260) is described by a Breit-Wigner function with resonance parameters from the world average [21]. For the background studies, an ‘inclusive’ Y(4260) MC sample equivalent to 500 pb−1 integrated luminosity is generated which includes the Y(4260) resonance, ISR production of the known vector charmonium states, and events driven by QED processes. The known decay modes are generated with EvtGen [19] with branching fractions set to their world average val- ues in the Particle Data Group (PDG) [22], and the re- maining events are generated with Lundcharm [23] or PYTHIA [24]. the low energy photon and J/ψ, MγJ/ψ, is used to search for χcJ signals. However, for the data sample taken at √s =4.009 GeV, there is an overlap of the energy dis- tributions of the photons from e+e−→γχc1,2 and from χc1,2 decays, as shown in Fig. 1. To separate the over- lapping photon spectra, the energy of photons from χc1,2 decays is further required to be less than 0.403 GeV at √s=4.009 GeV. Fig. 1. 3 Event selection Charged tracks are reconstructed in the MDC. For each good charged track, the polar angle must satisfy \|cosθ\| < 0.93, and the point of closest approach to the interaction point must be within ±10 cm in the beam di- rection and within ±1 cm in the plane perpendicular to the beam direction. The number of good charged tracks is required to be two with a zero net charge. Charged tracks are identiﬁed as muons if they have E/p < 0.35 c and p > 1.0 GeV/c, where E is the energy deposited in the EMC and p is the momentum measured by the MDC. Photons are reconstructed from isolated showers in the EMC that are at least 20 degrees away from any of the charged tracks. To improve the reconstruction eﬃ- ciency and the energy resolution, the energy deposited in the nearby TOF counters is included. Photon candi- dates are required to have energy greater than 25 MeV in the EMC barrel region (\|cosθ\|<0.8), and 50 MeV in the end-cap region (0.86<\|cosθ\|<0.92). In order to suppress electronic noise and energy deposits that are unrelated to the event, the EMC time t of the photon candidates must be in coincidence with collision events within the range 0⩽t⩽700 ns. At least two photon candidates in the ﬁnal state are required. Fig. 1. The distributions of photon energies in the laboratory frame from e+e−→γχc1,2 and from χc1,2 decays in the exclusive MC samples of e+e−→γχc1, χc1→γJ/ψ (a) and e+e−→γχc2, χc2 →γJ/ψ (b) at √s=4.009 GeV. Dashed lines stand for the ﬁrst radiative photons from e+e−→ γχc1,2 and solid lines for the second radiative pho- tons from χc1,2 decays. 2 BES detector and Monte Carlo The distributions of photon energies in the laboratory frame from e+e−→γχc1,2 and from χc1,2 decays in the exclusive MC samples of e+e−→γχc1, χc1→γJ/ψ (a) and e+e−→γχc2, χc2 →γJ/ψ (b) at √s=4.009 GeV. Dashed lines stand for the ﬁrst radiative photons from e+e−→ γχc1,2 and solid lines for the second radiative pho- tons from χc1,2 decays. 4 Background study To improve the momentum resolution and to reduce backgrounds, a kinematic ﬁt with ﬁve constraints (5C- ﬁt) is performed under the e+e−→γγμ+μ−hypothe- sis, imposing overall energy and momentum conservation and constraining the invariant mass of μ+μ−to the nom- inal J/ψ mass. Candidates with a χ2 5C <40 are selected for further analysis. If more than one candidate occurs in an event, the one with the smallest χ2 5C is selected. Due to the kinematics of the reaction, the ﬁrst radiative pho- ton from e+e−→γχcJ has a high energy while the second radiative photon from χcJ →γJ/ψ has a lower energy at √s=4.230, 4.260, and 4.360 GeV. The invariant mass of The potential backgrounds from e+e−→P+J/ψ, P →γγ (P = π0,η, or η′) can be rejected by requiring \|Mγγ−Mπ0\| > 0.025 GeV/c2, \|Mγγ−Mη\| > 0.03 GeV/c2 and \|Mγγ−Mη′\|>0.02 GeV/c2, where Mγγ is the invari- ant mass of two selected photons. The background from e+e−→γISRψ(2S), ψ(2S)→γχcJ is rejected by applying the 5C kinematic ﬁt. After imposing all the selection criteria above, the remaining dominant background is from radiative dimuon events, which is not expected to peak in the MγJ/ψ distribution. This has been validated by a dedicated simulation study. For other remaining 041001-5 Chinese Physics C Vol. 39, No. 4 (2015) 041001 backgrounds, such as e+e−→π0π0J/ψ, only 3.8 events (normalized to data luminosity) survive and can be ne- glected. extract the numbers of χcJ signal events. In the ﬁt, the shapes of the χcJ signals are described by double Gaus- sian functions, where the means and the standard devi- ations of the double Gaussian functions are determined from a ﬁt to the corresponding signal MC samples at √s=4.260 GeV. These shapes are also used for the other three CME points, as the resolution varies only mildly between √s=4.009–4.360 GeV. This has been validated by MC simulation. Since the dominant background 5 Fit to the mass spectrum The resulting MγJ/ψ distributions, after applying the above selection criteria, at √s=4.009, 4.230, 4.260 and 4.360 GeV are shown in Fig. 2. An unbinned maximum likelihood ﬁt of the MγJ/ψ distribution is performed to Fig. 2. The distribution of γJ/ψ invariant mass, MγJ/ψ, and ﬁt results for data at √s=4.009 (a), 4.230 (b), 4.260 (c) and 4.360 GeV (d). The solid lines show the total ﬁt results. The χcJ signals are shown as dashed lines, dotted lines, and dash-dotted lines, for J = 0, 1, and 2, respectively. The backgrounds are indicated by red dashed lines. Fig. 2. The distribution of γJ/ψ invariant mass, MγJ/ψ, and ﬁt results for data at √s=4.009 (a), 4.230 (b), 4.260 (c) and 4.360 GeV (d). The solid lines show the total ﬁt results. The χcJ signals are shown as dashed lines, dotted lines, and dash-dotted lines, for J = 0, 1, and 2, respectively. The backgrounds are indicated by red dashed lines. Table 2. The results on e+e−→γχcJ Born cross section measurement. Shown in the table are the signiﬁcance σ, detection eﬃciency ϵ, number of signal events from the ﬁts N obs, radiative correction factor (1+δr), vacuum polarization factor (1+δv), upper limit (at the 90% conﬁdence level (C.L.)) on the number of signal events N UP, Born cross section σB and upper limit (at the 90% C.L.) on the Born cross section σUP at diﬀerent CME points. The ﬁrst uncertainty of the Born cross section is statistical, and the second systematic. Table 2. The results on e+e−→γχcJ Born cross section measurement. Shown in the table are the signiﬁcance σ, detection eﬃciency ϵ, number of signal events from the ﬁts N obs, radiative correction factor (1+δr), vacuum polarization factor (1+δv), upper limit (at the 90% conﬁdence level (C.L.)) on the number of signal events N UP, Born cross section σB and upper limit (at the 90% C.L.) on the Born cross section σUP at diﬀerent CME points. The ﬁrst uncertainty of the Born cross section is statistical, and the second systematic. Table 2. The results on e+e−→γχcJ Born cross section measurement. 5 Fit to the mass spectrum As a test, we perform similar analyses to control samples from the J/ψ sideband regions, 2.917 < Mμ+μ−< 3.057 GeV/c2 and 3.137 < Mμ+μ−< 3.277 GeV/c2, by constraining the invariant mass of μ+μ−to 2.987 or 3.207 GeV/c2 in 5C-ﬁt, and ﬁnd no obvious χcJ signals. eﬃciency. The detection eﬃciencies, radiative correction factors as well as the calculated Born cross sections at diﬀerent CME are shown in Table 2. The much lower eﬃciencies for χc1,2 at √s=4.009 GeV are due to the requirement on the photon energy used to separate the overlapping photon spectra as described in Section 3. comes from radiative dimuon events, the correspond- ing MC simulation is used to represent the background shape. To reduce the eﬀect of statistical ﬂuctuations, the dimuon MC shape is smoothed before it is taken as the background function. Fig. 2 also shows the ﬁtted results for the MγJ/ψ distribution at diﬀerent CME. The number of ﬁtted χcJ signal events, as well as the corresponding statistical signiﬁcances (calculated by comparing the ﬁt log likelihood values with and without the χcJ signal) at the four CME points are listed in Table 2. The same ﬁt is applied to the sum of MγJ/ψ distributions of the four CME points. The statistical signiﬁcances for χc0, χc1 and χc2 are found to be 1.2σ, 3.0σ and 3.4σ, respec- tively. The goodness of the ﬁt is estimated by using a χ2 test method with the data distributions regrouped to en- sure that each bin contains more than 7 events. The test gives χ2/d.o.f=39.7/32, where d.o.f is the number of de- grees of freedom. As a test, we perform similar analyses to control samples from the J/ψ sideband regions, 2.917 < Mμ+μ−< 3.057 GeV/c2 and 3.137 < Mμ+μ−< 3.277 GeV/c2, by constraining the invariant mass of μ+μ−to 2.987 or 3.207 GeV/c2 in 5C-ﬁt, and ﬁnd no obvious χcJ signals. Since the χcJ signals are not statistically signiﬁcant at the individual CME points, we also give in Table 2 the upper limits on the Born cross sections at the 90% conﬁdence level (C.L.) under the assumption that no sig- nals are present. The upper limits are derived using a Bayesian method [21], where the eﬃciencies are lowered by a factor of (1−σsys) to take systematic uncertainties into account. 5 Fit to the mass spectrum We also perform a simultaneous ﬁt to the MγJ/ψ dis- tribution at √s=4.009, 4.230, 4.260, and 4.360 GeV, as- suming the production cross section of e+e−→γχcJ at a diﬀerent CME point follows the line shape of the Y(4260) state. In the ﬁt, the line shapes of the χcJ signals and the background are the same as those in the previous ﬁts, and the number of χcJ events at each CME point is expressed as a function of ϵc.m.Lc.m.Rc.m.(1+δr), where ϵc.m. and Lc.m. are the detection eﬃciency and luminos- ity, respectively, and Rc.m. is the ratio of the cross section calculated with the Y(4260) line shape (a Breit-Wigner function with parameters ﬁxed to the PDG values) at diﬀerent CME points to that at √s=4.260 GeV. The corresponding ﬁt result is shown in Fig. 3. The goodness of the ﬁt is χ2/d.o.f =53.3/40 and the statistical signiﬁ- cances for χc0, χc1 and χc2 signals are 0σ, 2.4σ and 4.0σ, respectively. We also found that Γe+e−×B(Y(4260) → γχc1) =(0.11±0.06) eV and Γe+e−×B(Y(4260) →γχc2) =(0.33±0.11) eV. 6 Results The Born cross section at diﬀerent CME is calculated with σB(e+e−→γχcJ)= N obs L·(1+δr)·(1+δv)·B·ϵ, (1) (1) where N obs is the number of observed events obtained from the ﬁt, L is the integrated luminosity, 1+δr is the radiative correction factor for χcJ with the assumption that the e+e−→γχcJ cross section follows the Y(4260) Breit-Wigner line shape [25], 1+δv is the vacuum po- larization factor [26], B is the combined branching ratio of χcJ →γJ/ψ and J/ψ→μ+μ−, and ϵ is the detection 5 Fit to the mass spectrum As a test, we perform similar analyses to control samples from the J/ψ sideband regions, 2.917 < Mμ+μ−< 3.057 GeV/c2 and 3.137 < Mμ+μ−< 3.277 GeV/c2, by constraining the invariant mass of μ+μ−to 2.987 or 3.207 GeV/c2 in 5C-ﬁt, and ﬁnd no obvious χcJ signals. 6 Results The Born cross section at diﬀerent CME is calculated with σB(e+e−→γχcJ)= N obs L·(1+δr)·(1+δv)·B·ϵ, (1) eﬃciency. The detection eﬃciencies, radiative correction factors as well as the calculated Born cross sections at diﬀerent CME are shown in Table 2. The much lower eﬃciencies for χc1,2 at √s=4.009 GeV are due to the requirement on the photon energy used to separate the overlapping photon spectra as described in Section 3. Since the χcJ signals are not statistically signiﬁcant at the individual CME points, we also give in Table 2 the upper limits on the Born cross sections at the 90% conﬁdence level (C.L.) under the assumption that no sig- nals are present. The upper limits are derived using a Bayesian method [21], where the eﬃciencies are lowered by a factor of (1−σsys) to take systematic uncertainties into account. We also perform a simultaneous ﬁt to the MγJ/ψ dis- tribution at √s=4.009, 4.230, 4.260, and 4.360 GeV, as- suming the production cross section of e+e−→γχcJ at a diﬀerent CME point follows the line shape of the Y(4260) state. In the ﬁt, the line shapes of the χcJ signals and the background are the same as those in the previous ﬁts, and the number of χcJ events at each CME point is expressed as a function of ϵc.m.Lc.m.Rc.m.(1+δr), where ϵc.m. and Lc.m. are the detection eﬃciency and luminos- ity, respectively, and Rc.m. is the ratio of the cross section calculated with the Y(4260) line shape (a Breit-Wigner function with parameters ﬁxed to the PDG values) at diﬀerent CME points to that at √s=4.260 GeV. The corresponding ﬁt result is shown in Fig. 3. The goodness of the ﬁt is χ2/d.o.f =53.3/40 and the statistical signiﬁ- cances for χc0, χc1 and χc2 signals are 0σ, 2.4σ and 4.0σ, respectively. We also found that Γe+e−×B(Y(4260) → Table 3. Summary of systematic uncertainties at √s=4.009, 4.230, 4.260, and 4.360 GeV(%). 5 Fit to the mass spectrum √s/GeV 4.009 4.230 4.260 4.360 sources χc0 χc1 χc2 χc0 χc1 χc2 χc0 χc1 χc2 χc0 χc1 χc2 luminosity 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 tracking eﬃciency 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 photon detection 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 kinematic ﬁt 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 branching ratio 4.8 3.6 3.7 4.8 3.6 3.7 4.8 3.6 3.7 4.8 3.6 3.7 vacuum polarization factor 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 χcJ mass resolution 0.3 2.0 7.4 0.0 7.7 7.8 0.0 4.3 6.5 0.0 1.1 2.0 χcJ mass 0.0 0.9 1.4 0.0 0.3 0.2 0.0 0.1 0.1 0.0 0.3 0.4 MC model 0.0 2.2 3.9 0.0 1.2 3.3 0.0 1.9 2.9 0.0 1.5 2.1 ﬁt range 0.1 2.2 2.6 0.0 1.5 2.3 0.0 3.1 2.5 0.0 3.0 3.7 background shape 0.0 3.1 5.6 0.0 0.7 0.3 0.0 1.1 0.4 0.0 0.9 0.1 radiative correction factor 3.0 2.7 3.6 2.6 3.1 2.1 3.5 2.1 2.5 1.8 3.4 3.8 total 6.5 7.3 12.1 6.3 9.8 10.2 6.7 7.7 9.3 6.0 6.9 7.7 ble 3. Summary of systematic uncertainties at √s=4.009, 4.230, 4.260, and 4.360 GeV(%). comes from radiative dimuon events, the correspond- ing MC simulation is used to represent the background shape. To reduce the eﬀect of statistical ﬂuctuations, the dimuon MC shape is smoothed before it is taken as the background function. Fig. 2 also shows the ﬁtted results for the MγJ/ψ distribution at diﬀerent CME. The number of ﬁtted χcJ signal events, as well as the corresponding statistical signiﬁcances (calculated by comparing the ﬁt log likelihood values with and without the χcJ signal) at the four CME points are listed in Table 2. The same ﬁt is applied to the sum of MγJ/ψ distributions of the four CME points. The statistical signiﬁcances for χc0, χc1 and χc2 are found to be 1.2σ, 3.0σ and 3.4σ, respec- tively. The goodness of the ﬁt is estimated by using a χ2 test method with the data distributions regrouped to en- sure that each bin contains more than 7 events. The test gives χ2/d.o.f=39.7/32, where d.o.f is the number of de- grees of freedom. 5 Fit to the mass spectrum Shown in the table are the signiﬁcance σ, detection eﬃciency ϵ, number of signal events from the ﬁts N obs, radiative correction factor (1+δr), vacuum polarization factor (1+δv), upper limit (at the 90% conﬁdence level (C.L.)) on the number of signal events N UP, Born cross section σB and upper limit (at the 90% C.L.) on the Born cross section σUP at diﬀerent CME points. The ﬁrst uncertainty of the Born cross section is statistical, and the second systematic. √s/GeV Nobs signiﬁcance (σ) NUP ϵ (%) 1+δr 1+δv σUP/pb σB/pb χc0 7.0±6.6 1.6 18 36.4±0.2 179 65.0±61.3±4.2 4.009 χc1 4.4±2.6 2.2 9 23.4±0.1 0.738 1.04 5.3 2.4±1.4±0.2 χc2 1.8±1.7 1.5 6 8.7±0.1 18 4.7±4.4±0.6 χc0 0.2±2.3 0.0 7 37.2±0.2 26 0.7±8.0±0.1 4.230 χc1 6.7±4.3 1.9 14 44.4±0.2 0.840 1.06 1.7 0.7±0.5±0.1 χc2 13.3±5.2 2.9 22 42.0±0.2 5.0 2.7±1.1±0.3 χc0 0.1±1.9 0.0 5 36.7±0.2 25 0.5±8.8±0.1 4.260 χc1 3.0±3.0 1.1 7 42.7±0.2 0.842 1.06 1.1 0.4±0.4±0.1 χc2 7.5±3.9 2.3 14 41.7±0.2 4.2 2.0±1.1±0.2 χc0 0.1±0.7 0.0 3 32.4±0.2 23 0.7±5.0±0.1 4.360 χc1 5.2±4.9 2.4 10 31.7±0.2 0.943 1.05 2.9 1.4±1.3±0.1 χc2 4.4±4.5 2.0 9 30.3±0.2 5.0 2.3±2.3±0.2 Chinese Physics C Vol. 39, No. 4 (2015) 041001 Table 3. Summary of systematic uncertainties at √s=4.009, 4.230, 4.260, and 4.360 GeV(%). 5 Fit to the mass spectrum √s/GeV 4.009 4.230 4.260 4.360 sources χc0 χc1 χc2 χc0 χc1 χc2 χc0 χc1 χc2 χc0 χc1 χc2 luminosity 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 tracking eﬃciency 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 photon detection 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 kinematic ﬁt 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 branching ratio 4.8 3.6 3.7 4.8 3.6 3.7 4.8 3.6 3.7 4.8 3.6 3.7 vacuum polarization factor 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 χcJ mass resolution 0.3 2.0 7.4 0.0 7.7 7.8 0.0 4.3 6.5 0.0 1.1 2.0 χcJ mass 0.0 0.9 1.4 0.0 0.3 0.2 0.0 0.1 0.1 0.0 0.3 0.4 MC model 0.0 2.2 3.9 0.0 1.2 3.3 0.0 1.9 2.9 0.0 1.5 2.1 ﬁt range 0.1 2.2 2.6 0.0 1.5 2.3 0.0 3.1 2.5 0.0 3.0 3.7 background shape 0.0 3.1 5.6 0.0 0.7 0.3 0.0 1.1 0.4 0.0 0.9 0.1 radiative correction factor 3.0 2.7 3.6 2.6 3.1 2.1 3.5 2.1 2.5 1.8 3.4 3.8 total 6.5 7.3 12.1 6.3 9.8 10.2 6.7 7.7 9.3 6.0 6.9 7.7 comes from radiative dimuon events, the correspond- ing MC simulation is used to represent the background shape. To reduce the eﬀect of statistical ﬂuctuations, the dimuon MC shape is smoothed before it is taken as the background function. Fig. 2 also shows the ﬁtted results for the MγJ/ψ distribution at diﬀerent CME. The number of ﬁtted χcJ signal events, as well as the corresponding statistical signiﬁcances (calculated by comparing the ﬁt log likelihood values with and without the χcJ signal) at the four CME points are listed in Table 2. The same ﬁt is applied to the sum of MγJ/ψ distributions of the four CME points. The statistical signiﬁcances for χc0, χc1 and χc2 are found to be 1.2σ, 3.0σ and 3.4σ, respec- tively. The goodness of the ﬁt is estimated by using a χ2 test method with the data distributions regrouped to en- sure that each bin contains more than 7 events. The test gives χ2/d.o.f=39.7/32, where d.o.f is the number of de- grees of freedom. 7 Systematic uncertainties The systematic uncertainties in the cross section mea- surements of e+e−→γχcJ are caused by various sources 041001-7 Chinese Physics C Vol. 39, No. 4 (2015) 041001 Fig. 3. Result of the simultaneous ﬁt to MγJ/ψ dis- tributions for all CME data sets assuming that the signals are from decays of the Y(4260). The blue solid line is the total ﬁt result. The χcJ signals are shown as dotted line and dash-dotted line, re- spectively, and the background is shown as the red dashed line. values, and the deviations of the signal yields to the nom- inal values are taken as the systematic uncertainties due to the uncertainties of the signal line shapes. values, and the deviations of the signal yields to the nom- inal values are taken as the systematic uncertainties due to the uncertainties of the signal line shapes. The detection eﬃciency is evaluated using MC sam- ples based on the E1 transition assumption [19] for Y(4260)→γχcJ. Another set of MC samples is gener- ated where the Y(4260)→γχcJ decay is modeled using a phase space distribution, and the diﬀerences of the detec- tor eﬃciencies between the two sets of MC samples are treated as systematic uncertainties from the MC model. To estimate the systematic uncertainty related to the background shape, a control sample is selected from the data by requiring a μ+μ−pair and at least one photon. An alternative background shape is then extracted by re-weighting the γμ+μ−invariant mass spectrum of the control sample, where the weights are the eﬃciency ra- tio of e+e−→(nγ)μ+μ−MC simulated events surviving the signal selection criteria to the same selection criteria for the control sample. A ﬁt with the alternative back- ground shape is performed, and the diﬀerences between the yields of χcJ signal to the nominal ones are taken as the systematic uncertainties due to the shape of the background. Fig. 3. Result of the simultaneous ﬁt to MγJ/ψ dis- tributions for all CME data sets assuming that the signals are from decays of the Y(4260). The blue solid line is the total ﬁt result. The χcJ signals are shown as dotted line and dash-dotted line, re- spectively, and the background is shown as the red dashed line. which all the channels have partially in common. 7 Systematic uncertainties The common sources of systematics include the luminos- ity measurement, reconstruction eﬃciencies for charged tracks and photons, the vacuum polarization factor, kine- matic ﬁt and branching fractions of the decay of the in- termediate states. The systematic uncertainty due to the luminosity measurement is estimated to be 1.0% us- ing Bhabha events [14]. The uncertainty related to the track reconstruction eﬃciency of high-momentum muons is 1.0% per track [27]. The systematic uncertainty re- lated to the photon detection is estimated to be 1.0% per photon [14]. The systematic uncertainty due to 5C- ﬁt is 0.6%, obtained by studying a control sample of ψ(2S) →ηJ/ψ decays. The uncertainty related to the branching fractions of χcJ and J/ψ decays are taken from the PDG [21]. The uncertainty for the vacuum polariza- tion factor is 0.5% [26]. The possible distortions of the Y(4260) line shape due to interference eﬀects with nearby resonances could introduce uncertainties in the radiative correction factor ϵ×(1+δr). To estimate the related systematic uncer- tainties, we instead assume that e+e−→γχcJ are pro- duced via ψ(4040) decays at √s=4.009 GeV, ψ(4160) decays at √s=4.229 and 4.260 GeV, and ψ(4415) decays at √s=4.360 GeV. The variations in the factor ϵ×(1+δr) are taken as the systematic uncertainties due to the ra- diative correction factor. A series of similar ﬁts are performed in diﬀerent ranges of the MγJ/ψ distribution, and the largest dif- ferences on the signal yields to the nominal values are taken as systematic uncertainties. All the systematic uncertainties from the diﬀerent sources are summarized in Table 3. The total system- atic uncertainties are calculated as the quadratic sum of all individual terms. The other systematic uncertainties arising from the χcJ mass resolution, the shift of the χcJ reconstructed mass, the MC model, the shape of the background, the radiative correction factor and the ﬁt range at diﬀerent CME points are discussed below. lation [16]. limits on the Born cross sections at the 90% C.L. are calculated and listed in Table 2, too. These upper limits on the Born cross section of e+e−→γχcJ are compatible with the theoretical prediction from an NRQCD calcu- The BES collaboration thanks the staﬀof BEPC and the IHEP computing center for their strong support. References 96: 162003 14 Ablikim M et al. (BES collaboration). Phys. Rev. Lett., 2014, 112: 092001 1 Aubert B et al. (BABAR collaboration). Phys. Rev. Lett., 2005, 95: 142001 15 MA L, SUN Z F, LIU X H, DENG W Z, LIU X, ZHU S L. Phys. Rev. D, 2014, 90: 034020 2 HE Q et al. (CLEO collaboration). Phys. Rev. D, 2006, 74: 091104(R) 16 CHAO K T, HE Z G, LI D, MENG C. hep-ex/arXiv:1310.8597 ( ) 3 YUAN C Z et al. (Belle collaboration). Phys. Rev. Lett., 2007, 99: 182004 17 Ablikim M et al. (BES collaboration). Nucl. Instrum. Meth- ods A, 2010, 614: 345 18 Agostinelli S et al. (GEANT4 collaboration). Nucl. Instrum. Methods A, 2003, 506: 250 4 Lees J P et al. (BABAR collaboration). Phys. Rev. D, 2012, 86: 051102 19 Lange D J. Nucl. Instrum. Methods A, 2001, 462: 152; PING R G. HEP & NP, 2008, 32: 599602 5 LIU Z Q et al. (Belle collaboration). Phys. Rev. Lett., 2013, 110: 252002 20 Jadach S, Ward B F L, Was Z. Comput. Phys. Commum., 2000, 130: 260; Jadach S, Ward B F L, Was Z. Phys. Rev. D, 2001, 63: 113009 6 Eichten E, Gottfried K, Kinoshita T, Lane K, YAN T. Phys. Rev. D, 1978, 17: 3090; 1980, 21: 203; Barnes T, Godfrey S, Swanson E S. Phys. Rev. D, 2005, 72: 054026 Swanson E S. Phys. Rev. D, 2005, 72: 054026 21 Olive K A et al. (Particle Data Group). Chin. Phys. C, 2014, 38: 090001 7 LIU L et al. (Hadron Spectrum collaboration). JHEP, 2012, 1207: 126 22 Beringer J et al. (Particle Data Group). Phys. Rev. D, 2012, 86: 010001 8 Ebert D, Faustov R N, Galkin V O. Phys. Lett. B, 2006, 634: 214 23 PING R G. Chinese Phys. C, 2008, 32: 599 9 Maiani L, Riquer V, Piccinini F, Polosa A D. Phys. Rev. D, 2005, 72: 031502(R) 24 Sjostrand T, Lonnblad L, Mrenna S. hep-ph/0108264 10 LIU X, ZENG X Q, LI X Q. Phys. Rev. D, 2005, 72: 054023(R) 25 Kuraev E A, Fadin V S. Sov. J. Nucl. Phys., 1985, 41: 466 [Yad. Fiz. 1985, 41: 733] 11 YUAN C Z, WANG P, MO X H. Phys. Lett. B, 2006, 634: 399 26 Actis S et al. Eur. Phys. J. C, 2010, 66: 585 12 WANG X L et al. (Belle collaboration). Phys. 8 Summary The ψ(2S)→γχcJ channel is employed as a control sample to extract the diﬀerences on the mass resolution of the χcJ signal by ﬁtting the MγJ/ψ spectrum. The dif- ferences in the mass resolutions between data and MC are found to be 0.02%, 0.01%, 0.2% for χcJ (J =0, 1, 2). A similar ﬁt is performed, in which the signal shapes are smeared to compensate for the mass resolution diﬀer- ence, and the diﬀerences on the yields of χcJ signal are taken as the systematic uncertainties due to the mass resolution. Using data samples collected at CME of √s=4.009, 4.230, 4.260, and 4.360 GeV with the BES detector, we perform a search for e+e−→γχcJ (J =0, 1, 2) with the subsequent decay χcJ →γJ/ψ and J/ψ→μ+μ−. We ﬁnd evidence for the processes e+e−→γχc1 and e+e−→γχc2 with statistical signiﬁcances of 3.0σ and 3.4σ, respec- tively. No evidence of e+e−→γχc0 is observed. The corresponding Born cross sections of e+e−→γχcJ at dif- ferent CME are calculated and listed in Table 2. Under the assumption of the absence of χcJ signals, the upper An alternative ﬁt is performed shifting the mean of χcJ signal shapes by one standard deviation of the PDG 041001-8 Chinese Physics C Vol. 39, No. 4 (2015) 041001 References Rev. D, 2013, 87: 051101(R) 27 Ablikim M et al. (BES collaboration). Phys. Rev. D, 2012, 86: 071101 13 Coan T E et al. (CLEO collaboration). Phys. Rev. Lett., 2006, E et al. (CLEO collaboration). Phys. Rev. Lett., 2006 041001-9
https://openalex.org/W2769350678	https://link.springer.com/content/pdf/10.1007%2Fs10494-017-9877-z.pdf	English	null	Generation Mechanisms of Rotating Stall and Surge in Centrifugal Compressors	Flow, turbulence and combustion	2,017	cc-by	7,059	Flow Turbulence Combust (2018) 100:705–719 https://doi.org/10.1007/s10494-017-9877-z Generation Mechanisms of Rotating Stall and Surge in Centrifugal Compressors Elias Sundstr¨om1 · Bernhard Semlitsch2 · Mihai Mih˘aescu1 Received: 24 May 2017 / Accepted: 7 November 2017 / Published online: 23 November 2017 © The Author(s) 2017. This article is an open access publication Abstract Flow instabilities such as Rotating Stall and Surge limit the operating range of centrifugal compressors at low mass-flow rates. Employing compressible Large Eddy Simulations (LES), their generation mechanisms are exposed. Toward low mass-flow rate operating conditions, flow reversal over the blade tips (generated by the back pressure) causes an inflection point of the inlet flow profile. There, a shear-layer induces vortical structures circulating at the compressor inlet. Traces of these flow structures are observed until far downstream in the radial diffuser. The tip leakage flow exhibits angular momentum imparted by the impeller, which deteriorates the incidence angles at the blade tips through an over imposed swirling component to the incoming flow. We show that the impeller is incapable to maintain constant efficiency at surge operating conditions due to the extreme alteration of the incidence angle. This induces unsteady flow momentum transfer down- stream, which is reflected as compression wave at the compressor outlet traveling toward the impeller. There, the pressure oscillations govern the tip leakage flow and hence, the inci- dence angles at the impeller. When these individual self-exited processes occurs in-phase, a surge limit-cycle establishes. Keywords Rotating stall · Surge · Centrifugal compressor · LES 1 KTH - Mechanics, Competence Center for Gas Exchange (CCGEx), Osquars Backe 18. Royal Institute of Technology (KTH), Department of Mechanics, Stockholm, 10044, Sweden Elias Sundstr¨om elias@mech.kth.se Nomenclature Latin symbols ˙m mass-flow rate [kg/s] Ma Mach number [-] mass-flow rate [kg/s] Mach number [-] Elias Sundstr¨om elias@mech.kth.se Elias Sundstr¨om elias@mech.kth.se 1 KTH - Mechanics, Competence Center for Gas Exchange (CCGEx), Osquars Backe 18. Royal Institute of Technology (KTH), Department of Mechanics, Stockholm, 10044, Sweden 2 Whittle Laboratory - Department of Engineering, University of Cambridge, 1 JJ Thomson Avenue., Cambridge CB3 0DY, UK 2 Whittle Laboratory - Department of Engineering, University of Cambridge, 1 JJ Thomson Avenue., Cambridge CB3 0DY, UK Flow Turbulence Combust (2018) 100:705–719 706 p pressure [Pa] T temperature [K] U mean velocity [m/s] D2 exducer diameter [m] Greek symbols β angle [o] ρ density [kg/m3] γ ratio of specific heats [-] η isentropic efficiency ( p02 p01 γ −1 γ −1)/( T02 T01 −1) [- phase angle [radian] Abbreviations BPF Blade Passing Frequency PSD Power Spectral Density LES Large Eddy Simulation PIV Particle Image Velocimetry RO Rotating Order. Normalized angular velocity Subscript 0 total quantity 1 upstream of the impeller 2 downstream of the impeller ref variable is referred to reference value b blade r, θ, z radial, tangential and axial coordinates 1 Introduction The operating range of compressors is bounded by flow instabilities at low mass-flow rates; e.g. rotating stall and surge [1]. Centrifugal compressors are commonly employed in auto- motive turbochargers to increase the specific efficiency of internal combustion engines [2]. A wide compressor functionality range is essential to supply boost pressure at all engine operating conditions [3]. The understanding of the generation mechanisms leading to these flow instabilities would allow a more efficient flow control design, which yet puzzles researchers and engineers. Rotating stall is a localized phenomenon restricted to the vicinity of the impeller, where stall cells propagate circumferentially across the blade passages. Different rotating stall appearances have been documented [1, 4] and control models have been developed [5]. Bousquet et al. [6] described the generation process in centrifugal compressors as a momen- tum balance between the incoming flow stream and the leakage flow at the impeller blade tip, inducing reversed flow. Rotating stall has been reported to be a precursor of surge [7, 8], while other researches [9] observed surge without traces of rotating stall. Fink et al. [10] highlighted by investigating different machinery setups that surge is an insta- bility affecting and being characterized by the entire system. They described surge as a cyclic emptying and filling process of the volume between the downstream plenum and the impeller. Based on this description, they developed a low-order model, which could reproduce the Flow Turbulence Combust (2018) 100:705–719 707 system behavior. Although the general behavior of surge has been characterized, the flow dynamics around the impeller remained yet undisclosed. system behavior. Although the general behavior of surge has been characterized, the flow dynamics around the impeller remained yet undisclosed. The visualization of flow structures via experimental methods is challenged by the confinement of the geometry and the violent flow fluctuations at off-design operating conditions [11–13]. Nonetheless, Particle Image Velocimetry (PIV) measurements of the ingested flow upstream of the impeller have been performed by Guillou et al. [14], who reported strong swirling back flow at the outer perimeter at off design operating conditions. Semlitsch et al. [15, 16] could confirm by studying the same ported shroud compressor numerically that this reversed flow originates from the tip leakage and back flow jets gen- erated via the ported shroud. 1 Introduction Semlitsch and Mih˘aescu [17] suggested that the increased tip leakage swirling the inflow toward off design operating conditions degenerates the inci- dence flow angles such that the rotor blades might be incapable to maintain a constant back pressure and cause surging. This emphasizes the practicality of unsteady computational methods such as LES for the investigation of flow structures in centrifugal compressors. Further, the potential of stability analysis via numerical methods to predict the onset of stall was shown by Sun et al. [18], Stein et al. [19] and Sundstr¨om et al. [20]. Vortical flow structures circulating at the impeller inlet have been visualized by Sundstr¨om et al. [21] and Broatch et al. [22]. These, could be associated to rotating stall. The aim of the present work is to understand the link of rotating stall and surge in centrifugal compressors and the generation of these phenomena. Therefore, we perform compressible LES calculations to investigate the flow structures at different operating points. We show that the vortical flow structures roll off the inflection point radius of the inlet profile and are therefore, linked to the tip leakage back-flow. The modes associated with these flow structures are analyzed using the modal decomposition techniques and Power Spectral Density (PSD). Further, we present the flow field at surge with a statistical analysis of the incidence flow angles, the flow field around the blades and the transferred flow momentum in order to reveal the generation mechanism of surge. 2 Geometry and Numerical Methodology The investigated centrifugal compressor is presented in Fig. 1, where the impeller contains ten main blades with an exducer diameter D2 = 88 mm, and the vaneless diffuser has an area ratio of 0.57. The compressor is equipped with a ported shroud, which is supported by four asymmetrically arranged ribs. The main objective of the ported shroud is to extend the stable operating range of the compressor map (see e.g. Yang et al. [23]). Four different operating conditions are considered at a constant speedline of 64000 rpm to investigate the evolution of flow instabilities developing toward the low mass-flow rate limit. Details to the operating conditions are provided in Table 1. Case D corresponds to a stable operating condition, whereas the other cases are chosen in the operating regimes of rotating stall and surge. An idealized installation is considered to mimic the experimental setup at the University of Cincinnati [14]. Air is entrained from quiescent, standard ambient conditions via a bell mouth inlet. The hemispherical shaped inlet is located three diffuser diameters upstream from the bell mouth entrance to the compressor, where a non-reflective boundary condition treatment is applied. The region close to the compressor can be considered as a compact Flow Turbulence Combust (2018) 100:705–719 708 Fig. 1 Side and front views of the centrifugal compressor CAD geometry. Locations of the pressure sensors D0 and IS1 are indicated as well as the location of planes used during data post-processing. The IS1 point is located in the middle of the ported shroud cavity, oriented 127◦clockwise from the vertical and 0.22D2 from the diffuser back wall. The impeller section intersects the impeller region at 50% blade span. The P1 and P2 planes intersect the inlet duct upstream of the impeller eye. These planes are located 0.85D2 and 0.76D2, respectively, upstream from the diffuser back wall. The bottom subfigure depicts inlet and outlet boundaries of the computational domain. The inset at the bottom right shows a zoomed in view of the blade passage grid resolution at 50% blade span Fig. 1 Side and front views of the centrifugal compressor CAD geometry. Locations of the pressure sensors D0 and IS1 are indicated as well as the location of planes used during data post-processing. The IS1 point is located in the middle of the ported shroud cavity, oriented 127◦clockwise from the vertical and 0.22D2 from the diffuser back wall. 1The back pressure and thus the mass flow is regulated via a valve in the experiments [14], which has been modeled by a mass flow rate boundary condition in the simulations due to the lack of the valve geometry information. The valve exhibits different impedance characteristics delaying the pressure reflection as com- pared to the numerical boundary condition. Therefore, surge occurs at a lower frequency in the experiments as in the simulations. Nonetheless, it can be observed that the employed numerical method captures all other tonalities. 2This represents another difference to the experimental setup, where the compressor is driven by the turbine. 2This represents another difference to the experimental setup, where the compressor is driven by the turbine. 3The disagreement can be explained by the differences in the experimental and numerical setup, as mentioned in the section “Geometry and Numerical Methodology”. 4The limit cycle is obtained from the raw monitored system variables for Case A, i.e. p02/p01(t), η(t), ˙m(t). These signals are first low-pass filtered and then approximated with a Fourier curve fit of 1st order. This yields the surge frequency, the phase angle, and their respective amplitudes. Since the phase angle is 90o, an ellipse shape in the phase space diagram is obtained with a semi-major axis (p02/p01 amplitude) and semi-minor axis ( ˙m amplitude). 2 Geometry and Numerical Methodology The impeller section intersects the impeller region at 50% blade span. The P1 and P2 planes intersect the inlet duct upstream of the impeller eye. These planes are located 0.85D2 and 0.76D2, respectively, upstream from the diffuser back wall. The bottom subfigure depicts inlet and outlet boundaries of the computational domain. The inset at the bottom right shows a zoomed in view of the blade passage grid resolution at 50% blade span acoustic source field, which radiates acoustical waves in all directions toward the acous- tic farfield at the speed of sound. To prevent such waves from begin reflected at the inlet boundary, i.e. back toward the compressor, the freestream velocity on the inlet cell face is corrected. The boundary normal velocity component on the cell face is obtained from char- acteristics, which involves extrapolation from the neighboring cell center velocity. A more detailed explanation of the non-reflecting boundary treatment can be found in the work by Giles [24]. Correspondingly to the experimental setup, the compressor outlet duct is ten diffuser diameters long. At the outlet, the mass-flow is fixed and so is the total temperature.1 Thus, the outlet pressure is allowed to float about the specific target pressure according to the experimental conditions. The walls are considered as adiabatic2 with non-slip boundary conditions. The finite-volume compressible solver within STAR-CCM+ is utilized for the simula- tion of the flow. It is characterized by the integral form of the conservation equations of Flow Turbulence Combust (2018) 100:705–719 709 Table 1 Imposed inlet and outlet boundary conditions Case ˙m [kg/s] pin [kPa] Tin [K] T0,exit [K] A 0.050 101.325 300 365 B 0.085 101.325 300 365 C 0.105 101.325 300 365 D 0.280 101.325 300 344 mass, momentum, and energy. The air flow is assumed to behave as an ideal gas, where the dynamic viscosity and thermal conductivity are assumed to be functions of the local tem- perature only. An implicit LES approach is adopted to reproduce the unsteady flow in the compressor. An implicit second-order upwind scheme is used for time marching. For the calculation of the convective and diffusion gradients, an implicit, bounded central third-order scheme is employed. The governing system of equations is solved as a coupled system with Gauss- Seidel relaxation in an algebraic multigrid approach. 2 Geometry and Numerical Methodology To handle the impeller rotation, the domain is divided into stationary and rotating regions, which are interfaced by the sliding mesh technique requiring information interpolation at each time step. A solid body rotation at an angular velocity of the impeller is applied to revolve the grid about the compressor axis. The computational domain is discretized by a fine unstructured polyhedral grid consisting of approximately nine million finite volumes. A low-Reynolds number wall-resolved grid with y+ = 1 distribution on average is used. This is obtained with 10 prismatic cell layers, a total wall prism layer height of 0.5 mm and using a geometric stretching factor of 1.5. The simulations have been computed for 0.2 s to converge the spectral averages corre- sponding to 200 rotor revolutions. More specifications of the numerical methodology have been documented in previous publications, see i.e. Sundstr¨om et al. [20, 21], where also a grid sensitivity study and validation with experimental data were performed. Therefore, the presently employed approach can be considered as a reliable technique to capture the flow dynamics in the compressor. 3 Results The operating conditions under investigation are characterized by the fluctuations of the global performance parameters, as shown in Fig. 2.3 Small variations to the mean value can be observed for stable operating condition, while the perturbations are enhanced toward low mass-flow rate operating conditions. These are the fundamental properties of flow instabilities manifesting on the positive slope of the characteristics. With surge, the perfor- mance parameters oscillate and form a limit cycle,4 where the amplitude and frequency are governed by the entire system [25]. Flow Turbulence Combust (2018) 100:705–719 710 Fig. 2 The performance map illustrates the location of the chosen operating conditions. The variation of the total pressure ratio and isentropic efficiency during operation is illustrated with horizontal and vertical bars, respectively. A limit cycle is established for Case A, which is depicted with a circle and two arrows in order to show the phase direction. Experimental data [26] is added to guide the reader Fig. 2 The performance map illustrates the location of the chosen operating conditions. The variation of the total pressure ratio and isentropic efficiency during operation is illustrated with horizontal and vertical bars, respectively. A limit cycle is established for Case A, which is depicted with a circle and two arrows in order to show the phase direction. Experimental data [26] is added to guide the reader The observed attributes at off-design operating conditions express themselves also in the flow-field, which is shown in Fig. 3. The flow velocities are lower at low mass-flow rates when the static back pressure is higher. This causes a higher residence time of the flow, manifested in the form of circulating the flow in the radial diffuser with intensified turbulent fluctuations. The turbulent kinetic energy levels increase significantly the further the mass-flow rate is reduced. The level is specifically increased at the interface between the diffuser and the volute, subject to flow unsteadiness at the trailing edge, which is similar with observations made by Lennemann and Howard [27]. The higher back pressure pushes the flow through the blade tip gap toward the shroud inlet. This back flow, which increases the further the mass-flow rate is reduced, is quantified in Fig. 4. Due to the rotation of the rotor, the back flow exhibits inherently a swirling motion in the same rotational direction as the wheel. 3 Results With the mixing process of the back flow and the freshly entrained air, the entire flow upstream of the impeller spins around the impeller axis. The high turbulent kinetic energy levels (Fig. 3) indicate the mixing of the back flow with the entrained stream upstream of the impeller, in the shroud entrance. Toward surge operating conditions, the formation of an annular separated, radial out- wards and inwards swirling flow can be clearly observed in the P1 plane shown in Fig. 3 (in terms of streamlines) and on the P2 plane in Fig. 4. The flow profiles on the P2 plane (shown in Fig. 4) reveal a significant amount of back flow, which acts as a blockage for the freshly entrained air. Therefore, the inflow is funneled causing the radial flow profile, while the back flow spreads outwards due to the centrifugal forces. Both the radial and axial velocity profiles yield an inflection point, a possible onset mechanism for shear-layer insta- bility. Coinciding with this shear-layer interface, an annular increased region of turbulence kinetic energy can be observed in the P1 plane shown in Fig. 3. The spectra shown in Fig. 5 exhibit tonalities on top of a broadband distributed fluc- tuation character. These can be related to the angular velocity of the impeller shaft (RO), blade passing frequency (BPF), rotating stall (RS) and surge (S). There are also frequencies related to duct resonance, where multiples (2 and 5) of the rotating order are amplified. For the stable, near design operating condition (Case D) only the RO=1, RO=5, and the blade passing frequency RO=10 with its higher harmonic RO=20 can be observed, respectively. Flow Turbulence Combust (2018) 100:705–719 711 Fig. 3 Flow-field characterization at the four investigated operating points in terms of time-averaged Mach number and turbulent kinetic energy. Streamlines of the velocity field are overlapped to indicate the flow direction. For the location of the post-processing planes see Fig. 1. Here, the P1 plane is offset to the right relative the front plane Additionally, a tonality in the range of approximately 30 −50% of the rotating order unity Fig. 3 Flow-field characterization at the four investigated operating points in terms of time-averaged Mach number and turbulent kinetic energy. Streamlines of the velocity field are overlapped to indicate the flow direction. For the location of the post-processing planes see Fig. 1. 5Rotating stall was observed at approximately 480 Hz, 425 Hz and 320 Hz for the Cases A-C, respectively. 3 Results Here, the P1 plane is offset to the right relative the front plane Additionally, a tonality in the range of approximately 30 −50% of the rotating order unity for all low mass-flow rate operating conditions is observed.5 Due to the influence of the dominant surge oscillation at a lower frequency, the rotating stall establishes as a narrow- band amplification over a finite frequency range for Case A. Further, another clearly distinct peak at RO=0.04 (43 Hz) can be observed, including one harmonic at RO=0.08. Similarly, Additionally, a tonality in the range of approximately 30 −50% of the rotating order unity for all low mass-flow rate operating conditions is observed.5 Due to the influence of the dominant surge oscillation at a lower frequency, the rotating stall establishes as a narrow- band amplification over a finite frequency range for Case A. Further, another clearly distinct peak at RO=0.04 (43 Hz) can be observed, including one harmonic at RO=0.08. Similarly, 5Rotating stall was observed at approximately 480 Hz, 425 Hz and 320 Hz for the Cases A-C, respectively. Flow Turbulence Combust (2018) 100:705–719 712 Fig. 4 Time averaged velocity profile components in radial, tangential and axial directions. All profiles are evaluated along a vertical line at the plane P2. The blade tip speed, Uref , is used for normalization. The gray horizontal lines indicates the circumferential radius at which the rotating-stall vortical structures are circulating in the P1 plane. This location is close to the inflection point Fig. 4 Time averaged velocity profile components in radial, tangential and axial directions. All profiles are evaluated along a vertical line at the plane P2. The blade tip speed, Uref , is used for normalization. The gray horizontal lines indicates the circumferential radius at which the rotating-stall vortical structures are circulating in the P1 plane. This location is close to the inflection point this peak also spans a range of frequencies, which is due to the period variation over time. It suggests a complex situation with multiple period doubling until the signal may eventually come back to the same initial starting point. In between the tonalities, the spectrum is broad- band. Thus the signal in those frequency ranges is stochastic with no apparent coherent flow structure. 3.1 Flow Field Decomposition Revealing Rotating Stall and Surge Modal decomposition of the sampled simulation data was performed to analyze the insta- bilities associated with the flow phenomena. The flow field is therefore reconstructed in terms of mean and solely the modal oscillation at the particular frequency corresponding to rotating stall (Case B) and surge (Case A). Hence, the modal decomposition is utilized to filter out the frequency of interest from the unsteady flow. The reconstructed sequence as function of its phase is shown in Fig. 6, while quantitative analysis of the impeller work is presented in Fig. 7. p g At the rotating stall frequency of approximately 30 −50% of the rotating order unity (Case B), the most prominent feature is a co-rotating vortex pair being rolled off at the inner side of the shear layer formed by the back flow in the compressor inlet. Its evolution is shown in Fig. 6 in the P1 plane. Following streamlines of the phase angle sequence, it is noteworthy that the vortex pair propagates only a half revolution per rotating stall cycle. Inspecting the streamlines close to the volute tongue over a rotating stall cycle in Fig. 6, it can be observed that the flow intensely recirculates into the volute (between = 3π/2 and 0) while the flow discharges into the compressor exit pipe at other times of the cycle (between = π/2 and π). Hence, the forming shear layer under the volute tongue [15] is excited by the perturbations generated via the stall cells. Further, the strength of the vortex changes, which depends on its circumferential location. This is noted by the coiling up of the streamlines around the vortex cores. It is influenced by the non-axisymmetric pressure distribution in the radial diffuser at low mass flow rate operating conditions [28]. It is also influenced by the back flow distribution [29]. At = 3π/2, the two streamlines on the Flow Turbulence Combust (2018) 100:705–719 713 Fig. 5 The Power Spectral Density (PSD) at probe location IS1 (see Fig. 1 for location orientation) is shown for all investigated operating conditions, respectively. Experimental data [26] is added to guide the reader. Comparison for Case B and Case C was omitted because the experimental data exhibits non-investigated fea- tures, who’s origin is undetermined and is not visible in the numerical data. The frequency axis is normalized with the angular velocity of the impeller shaft, represented as the Rotating Order (RO). 3.1 Flow Field Decomposition Revealing Rotating Stall and Surge A RO=1 corresponds to one full rotation Fig. 5 The Power Spectral Density (PSD) at probe location IS1 (see Fig. 1 for location orientation) is shown for all investigated operating conditions, respectively. Experimental data [26] is added to guide the reader. Comparison for Case B and Case C was omitted because the experimental data exhibits non-investigated fea- tures, who’s origin is undetermined and is not visible in the numerical data. The frequency axis is normalized with the angular velocity of the impeller shaft, represented as the Rotating Order (RO). A RO=1 corresponds to one full rotation P1 plane, seeded outside of the shear-layer, diverge radially outwards due to the centrifu- gal force. Streamlines seeded inside of the shear-layer eventually end up in the co-rotating vortex pair. The manifestation of this flow instability, i.e. rotating stall, evolves similarly as described by Alekseenko et al. [30] who analyzed a simplified scenario where helical vortices where found in a swirling flow. The radial gradient of the radial velocity component (shown in Fig. 4) generates the instability causing the vortices. The rotation of the co-rotating vor- tices around the impeller axis occurs due to radial gradient of the circumferential velocity component. The essential flow feature of surge is the global response of the compression system, which is characterized by flow recirculation causing the emptying and filling process. By a phase angle of 3π/2 shown in Fig. 6 for Case A, the compressor undergoes the filling phase of surge. The flow incidence angles at the leading edge of the impeller reach a minimum (as shown in Fig. 7), which coincide with similar inflow angles as observed for Case B. There- fore, the flow momentum transferred downstream reaches high values and the streamlines shown in Fig. 6, especially in the blade passages, indicate an efficient mass flow transport downstream. The high flow momentum transported downstream causes the pressure to build up in the compressor discharge pipe, which reaches a maximum at a phase angle of 2π (phase shift of π/2 compared to the momentum). The phase lag of the pressure behind the moment is evidently shown in Fig. 7. By the phase angle 0 in the surge cycle, more flow is Flow Turbulence Combust (2018) 100:705–719 714 Fig. 6 Velocity streamlines and Mach number evolution of the surge and rotating stall modes are shown. 3.1 Flow Field Decomposition Revealing Rotating Stall and Surge Case A (left column) is phase averaged at the surge frequency tonality and Case B (right column) is phase averaged at the rotating stall frequency tonality. For location of post-processing planes see Fig. 1. Here, the P1 plane is offset to the right of the front plane pushed by the high pressure in the discharge pipe and recirculated in the volute underneath the tongue By = π/2 the compressor undergoes the emptying phase where the pressure Fig. 6 Velocity streamlines and Mach number evolution of the surge and rotating stall modes are shown. Case A (left column) is phase averaged at the surge frequency tonality and Case B (right column) is phase averaged at the rotating stall frequency tonality. For location of post-processing planes see Fig. 1. Here, the P1 plane is offset to the right of the front plane pushed by the high pressure in the discharge pipe and recirculated in the volute underneath the tongue. By = π/2 the compressor undergoes the emptying phase, where the pressure at the discharge pipe decays rapidly. The streamlines at the inducer reverse mid-way through the blade passages with large flow separation zones, resulting in a high flow discharge angle and low flow momentum transport, as shown in Fig. 7. The two seeded streamlines on the P1 plane are subsequently being trapped with an extremely long residence time and forms Flow Turbulence Combust (2018) 100:705–719 715 Fig. 7 Quantities characterizing the impeller performance are presented as function of the cycle phase at the dominant frequency for the particular case, i.e. Case A, surge; Case B & C, rotating stall; Case D, time-averaged. The quantities, i.e. flow incidence and discharge angles, upstream and downstream flow momentum and total pressure ratio, are circumferentially averaged at 50% blade span. The inducer blade angle is β1b = 60◦and the blade back sweep angle is β2b = 40◦, defined relative the meridional, see sketch in the upper right corner Fig. 7 Quantities characterizing the impeller performance are presented as function of the cycle phase at the dominant frequency for the particular case, i.e. Case A, surge; Case B & C, rotating stall; Case D, time-averaged. The quantities, i.e. flow incidence and discharge angles, upstream and downstream flow momentum and total pressure ratio, are circumferentially averaged at 50% blade span. 3.1 Flow Field Decomposition Revealing Rotating Stall and Surge The inducer blade angle is β1b = 60◦and the blade back sweep angle is β2b = 40◦, defined relative the meridional, see sketch in the upper right corner multiple turns around the center axis. The impeller is not capable to retain its efficiency. Again, the pressure lags π/2 behind the low flow momentum indicating a resonance phenomenon. As explained in the previous paragraph, the crucial factor leading to surge is the flow incidence angle at the impeller blade tip, with impact on the rotor efficiency to push fluid downstream. Figure 8 presents the modal flow perturbation at the surge frequency (case A), in terms of flow streamlines overlapped on the turbulence kinetic energy data. The data, cir- cumferentially averaged, reveals the variations of the incoming flow incidence angle, see e.g. cycle phase = 3π/2 as compared with cycle phase = 0. Moreover, the stream- lines indicate large regions of flow separation off the blade surfaces toward the impeller discharge. This complements Fig. 7, which shows at surge (Case A) that only very little flow momentum is transferred downstream of the impeller. Thus, only a proportion of the impeller cord imparts flow momentum. By the phase angle = 0 the streamlines close to the blade pressure side and in the mid- dle of the blade passage depict downstream directed flow and the turbulence kinetic energy level is at a minimum, as shown in Fig. 8. Close to the blade suction side, the streamlines describe a circular motion (located toward the rear of the blade). They are spiraling outwards and finally are discharged downstream, into the diffuser. Qualitatively, this feature is typical for an emerging boundary layer separation. By = π/2 all seeded streamlines are now going in the upstream direction depicting reversed flow of the surge mode and the turbulence Flow Turbulence Combust (2018) 100:705–719 716 Fig. 8 Phase evolution of the modal flow perturbation within the blade’s passages at the surge frequency (Case A). Flow streamlines on top of normalized turbulence kinetic energy. The data is circumferentially averaged. Between = 3π/2 and = 0 the incoming flow streamlines are directed downstream. From = π/2 to = π the streamlines depict complete flow reversal for an averaged blade passage Fig. 8 Phase evolution of the modal flow perturbation within the blade’s passages at the surge frequency (Case A). 3.1 Flow Field Decomposition Revealing Rotating Stall and Surge Flow streamlines on top of normalized turbulence kinetic energy. The data is circumferentially averaged. Between = 3π/2 and = 0 the incoming flow streamlines are directed downstream. From = π/2 to = π the streamlines depict complete flow reversal for an averaged blade passage kinetic energy distribution shows a strong gradient between the pressure and suction sides of the blade. The streamline seeded close to the blade suction side initially describes an out- ward spiraling motion before its being discharged upstream. Since the seeded streamlines are completely reversed, the impeller is not producing any notable downstream momentum (see also Fig. 7 at = π/2). Toward phase angle = π in Fig. 8, the turbulence levels have now dropped. The seeded streamlines still depict flow reversal, but the angle at which the flow moves upstream from the impeller is almost parallel with the impeller’s inlet plane. The compressor is gradually recovering from a minimum pressure level of the mode cycle (see also Fig. 7). By cycle phase of = 3π/2 all streamlines show downstream oriented flow, apart from the streamline seeded close to the blade suction side. This correlates with flow incidence and discharge angles reaching their minimum levels, but also to that the upstream and downstream flow momentum are at their maximum levels. From = 3π/2 to = 2π the flow mode perturbation will gradually go back to the phase evolution stated at = 0, and the surge limit cycle will repeat. 4 Conclusions The unsteady compressible flow approaching surge operating conditions in a centrifugal compressor has been analyzed using LES. The numerical setup and the boundary condi- tions were chosen to replicate the cold gas-stand experiment at the University of Cincinnati. Therefore, validation of the numerical predictions with experimental data was possible and documented by Sundstr¨om et al. [20, 21]. Focusing on flow instabilities, the flow field evolution from design to off-design con- ditions was assessed. Tonalities in the pressure spectra corresponding to rotating stall and surge have been shown for particular low mass-flow rate operating conditions. Flow modal decomposition analysis was employed to investigate the phenomena causing rotating stall and surge. The observation of back flow emergence toward off-design operating conditions has been documented by many investigations, both numerically [31] and experimentally [26]. 717 Flow Turbulence Combust (2018) 100:705–719 This back flow, streaming over the blade tips, is generated by the increased back pressures at low mass-flow rate conditions. We showed that flow incidence angles were altered by the back flow swirl, which lead to a reduction of entrained flow momentum and hence rotor effi- ciency. Surge was shown to be an extreme case, where the flow incidence angle and hence the downstream flow momentum transfer oscillate in a feedback loop with the build-up of back pressure in the discharge pipe causing a limit cycle. In a surge limit cycle it was shown that the pressure ratio and the entrained flow momen- tum are phase shifted 90◦. Thus, at peak pressure ratio, the flow momentum is 90◦from reaching its minimum. The incidence angle is seen to be in phase with the momentum. When the incidence angle and the momentum reach their maximum and minimum, respectively, the pressure ratio has reduced significantly and is well on its way toward the minimum value. But since the back pressure drops it allows the flow momentum to increase and the impeller gradually recovers, and starts to produce gradually more downstream radial momentum. However, it only increases until some critical pressure ratio. Beyond this limit the momentum starts to drop again, and the surge limit cycle repeats. At all low mass-flow rate operating conditions, the back flow over the blade tips interacts with the freshly entrained stream in the shroud entrance region, generating a strong shear- layer. 4 Conclusions In the presence and energized by the swirling back-flow the shear-layer can give rise to helical vortex formation circulating in the impeller inlet. The tonal signature of this flow instability can be traced far downstream into the radial diffuser and the volute. The asymmetric pressure distribution in the volute at off-design operating conditions results in an amplification of vortex strength toward the volute tongue. Therefore, the induced pressure oscillations are especially notable as modulation of the shear-layer under the volute tongue. Further, the perturbations induced at the impeller were shown to affect the achieved boost pressure ratio. p It was shown that the back flow at the impeller eye, manifesting due to tip-leakage, is essential for the occurrence of both phenomena, i.e. rotating stall and surge. However, the strength of the generated shear-layer in the shroud entrance governs rotating stall, whereas the induced swirl is decisive for surge. The shear-layer strength was also shown to be affected by the swirling motion of the back-flow, as quantified with the Reynolds stresses upstream of the impeller eye. It was found that the flow separates near the shroud wall with flow reversal going in the upstream direction, i.e. for operating conditions close to the surge line. This flow reversal induces a strong shear-layer interface. Seeded streamlines on the inner side of the interface were shown to curl up into two vortex pairs. Moreover, since a strong swirl component is present under surge condition, these vortices are seen to co-rotate in the same direction as the impeller rotation. For Case B, i.e. not in the deep surge condition, this feature is seen to be relatively distinct in the point spectrum, which means that one rotating stall period is similar with the next following one. For Case A, the rotating stall feature is seen to be more spread out over a larger frequency range. Thus, in presence of strong swirl the strength and frequency of co-rotating vortex pair are modulated. By means of seeded streamlines, it was qualitatively shown that the co-rotating vortex pair gains strength, when one of the vortices is aligned with the volute tongue. Acknowledgements This work was executed within the Competence Center for Gas Exchange (CCGEx). The work received support from the Swedish Energy Agency (STEM), Volvo Cars, Volvo GTT, and SCA- NIA. 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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter- national 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. 4 Conclusions Computational resources were provided via the Swedish National Infrastructure for Computing (SNIC) via HPC2N and PDC, the Parallel Computing Center at KTH. Prof. Ephraim J. Gutmark and his team at University of Cincinnati provided CAD geometry and available experimental data for validation purposes. 718 Flow Turbulence Combust (2018) 100:705–719 References Semlitsch, B., JyothishKumar, V., Mihaescu, M., Fuchs, L., Gutmark, E., Gancedo, M.: Numerical flow analysis of a centrifugal compressor with ported and without ported shroud. 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https://openalex.org/W1976429675	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113194&type=printable	English	null	Metabolism of Androstenone, 17β-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3β-Hydroxysteroid Dehydrogenase in This Process	PloS one	2,015	cc-by	6,266	Metabolism of Androstenone, 17b-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3b-Hydroxysteroid Dehydrogenase in This Process Gang Chen , Ying Bai , Li Ren , Dan Zhu , Yanhua Li , Meiying Fang , Huda Al Kateb , Olena Doran 1 Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China, 2 College of Animal Science and Technology, China Agricultural University, Beijing, China, 3 Department of Pathology, Air Force General Hospital, Beijing, China, 4 Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, United Kingdom Received June 15, 2014; Accepted October 20, 2014; Published January 15, 2015 Copyright: 2015 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. ability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within t Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All rele Funding: This work was supported by Natural Science Foundation of China (31171691, 31371779), International Science & Technology Cooperation Program of China (2012DFA31140), Biotechnology and Biological Sciences Research Council (BBSRC) UK-China Partnering Award (BB/H531435/1), and EU Marie Curie International Research Staff Exchange Scheme (FP7-PEOPLE-2009-IRSES, 246760). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: meiying@cau.edu.cn biosynthesis, or via enhancing the rate of androstenone metabo- lism [8]. It has been reported that enzymes of hydroxysteroid dehydrogenase family (HSDs), namely 3bHSDs and 17bHSDs, play the central role in steroid metabolism [9]. Data of the literature showed that porcine 3bHSD enzyme catalyzes the conversion of androstenone to its hydroxyl form in pig liver [10]. Testosterone is the main active androgen. It can be either irreversibly converted to estrogens by aromatase, or can be transformed to other active form such as dihydrotestosterone [9]. Testosterone metabolism in pig hepatocytes has been studied previously and it was established that 4-androstene-3,17-dione was its main metabolite [13]. 17b-Estradiol and dihydrotestosterone are also known as bioactive steroids of the endocrine systems. 17b- Estradiol directly influences reproduction of sows and indirectly influences off-springs development in terms of body weight and composition [3]. Biological role of dihydrotestosterone in pigs has not been extensively studied and has not been fully understood. To the best of our knowledge, relationship between 3bHSD and 17b- estradiol and dihydrotestosterone metabolism has not been investigated. Metabolism of Androstenone, 17b-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3b-Hydroxysteroid Dehydrogenase in This Process Gang Chen1, Ying Bai2, Li Ren3, Dan Zhu1, Yanhua Li1, Meiying Fang2, Huda Al-Kateb4, Olena Doran4 1 Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China, 2 College of Animal Science and Technology, China Agricultural University, Beijing, China, 3 Department of Pathology, Air Force General Hospital, Beijing, China, 4 Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, United Kingdom Metabolism of Androstenone, 17b-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3b-Hydroxysteroid Dehydrogenase in This Process Gang Chen1, Ying Bai2, Li Ren3, Dan Zhu1, Yanhua Li1, Meiying Fang2, Huda Al-Kateb4, Olena Doran4 1 Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China, 2 College of Animal Science and Technology, China Agricultural University, Beijing, China, 3 Department of Pathology, Air Force General Hospital, Beijing, China, 4 Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, United Kingdom Abstract Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17b-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3b-hydroxysteroid dehydrogenase (3bHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3bHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3bHSD enzyme was essential for metabolism of androstenone to 5a-androst-16-en-3b-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17b- estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3bHSD enzyme was not involved in 17b-estradiol metabolism. 5a- Dihydrotestosterone-17b-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3bHSD enzyme activity as demonstrated by inhibition study. Citation: Chen G, Bai Y, Ren L, Zhu D, Li Y, et al. (2015) Metabolism of Androstenone, 17b-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3b-Hydroxysteroid Dehydrogenase in This Process. PLoS ONE 10(1): e113194. doi:10.1371/journal.pone.0113194 Received June 15, 2014; Accepted October 20, 2014; Published January 15, 2015 Chemicals and reagents Androstenone, 17b-estradiol, dihydrotestosterone, b-estradiol 17-(b-D-glucuronide) sodium salt, b-glucuronidase from bovine liver (type B-1), 3a-androstenol, 3b-androstenol, apigenin, and trilostane were from Sigma-Aldrich (Shanghai, China). Stock solutions of the steroids were prepared in methanol at concentra- tions of 1 g/L. Working solutions of steroids were prepared by diluting the stock solutions in methanol. All other reagents and solvents (HPLC grade) were purchased from Fisher Scientific (service in Beijing, China). Cell culture media and other cell culture reagents were purchased from Hyclone (Beijing, China). Isolation of primary hepatocytes and treatments with steroids Primary pig hepatocytes were isolated from Large White male pigs (3–5 days old) using procedure described by Chen et al. [13]. The experimental protocol for using animals was approved by the Animal Ethics Committee of the China Agricultural University, Beijing, China (permission number: 2011-11-23-1). Cell viability was assessed by 0.2% trypan blue exclusion and was greater than 90% in all the cases. Approximately 56106 cells were plated into 10 cm Petri dishes with 10 ml of Dulbecco modified Eagle’s medium (DMEM) containing 20% fetal bovine serum (FBS) supplemented with insulin (10 mg/L), penicillin (100 U/mL) and streptomycin (100 mg/mL). Hepatocytes were incubated for 24 h in a humidified atmosphere at 37uC with 5% of CO2. Steroid metabolites extraction Aims of this study were to use time of flight mass spectrometry coupled with liquid chromatography (LC-TOF-MS) in order to (i) characterize products of androstenone, 17b-estradiol and dihy- drotestosterone metabolism, and (ii) investigate the role of 3bHSD enzyme in metabolism of androstenone, 17b-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. Extraction procedure for steroid metabolites was as follows: two mL of cell culture medium was taken in duplicate from each hepatocytes incubation and split into two samples, 1 mL per sample. One sample was treated with 0.1 mL of 30% trichlor- oacetic acid (TCA) to precipitate protein. The protein precipita- tion procedure was optimized by analyzing protein residues using HPLC at ultraviolet wavelength (240 nm). Another sample was pre-treated with b-glucuronidase prior to protein precipitation. The pre-treatment involved incubation with 2 mL of PBS buffer (pH = 5.0) in the presence of b-glucuronidase (2.08 mkat) for 16 h at 37uC. The samples were centrifuged at 17226 g for 10 min. All of the supernatants were collected and loaded onto a SPE cartridge (Bond Elut-C18, 500 mg/3 mL, Agilent, Lake Forest, CA, USA) preconditioned with 3 mL of methanol and water. The cartridges were washed with 3 mL of water and 3 mL 5% of methanol and then vacuum dried. The analytes were eluted with 9 mL of acetonitrile (ACN). The elutants were evaporated under nitrogen and reconstituted with 400 mL of 62.5% methanol. Solvents were passed through 0.22 mm filter paper (Jinteng company, Tianjin, China), and then transferred to inserts for analysis. The recoveries were determined by analyzing spiked samples in a basal medium. The recoveries for androstenone, 17b- estradiol and dihydrotestosterone were in a range of 60–95%. 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism One of the issues with investigation of steroids metabolism is a lack of appropriate analytical methods which would allow to differentiate steroids from their metabolites. Physiological concen- trations of steroid metabolites are very low and their structure is very similar to the structures of corresponding steroids [10,11]. During the last decade, liquid chromatography mass spectrometry with electrospray ionization (ESI) has been receiving increasing attention in relation to its application to steroids profile analysis [12]. Mass spectrometry with the time of flight mass analyzer (TOF) is another promising technology for identification of steroid metabolites because of its high resolution, high accuracy in mass detection, and a wide range of m/z scan. incubated with different concentrations of inhibitors (1, 10, 25 and 50 mM) for 48 h. The cell viability was analyzed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madi- son, WI) reagent following manufacture’s protocol. The cell culture medium was collected at the following time points: 0, 6, 12, 18, 24, 30, 36, 42 and 48 h after adding steroids and/or inhibitors, and was stored at 280uC until metabolites analysis. Cells treated with basal growth medium were used as control. The experiments were repeated in three different days. Experiments on each of the three days were conducted in triplicate. Mass spectrometry analysis Steroid metabolites were analyzed by LC-TOF-MS (QSTAR Elite, AB Sciex, ON, Canada) equipped with an electrospray ion source (TurboIonSpray) and controlled by Analyst QS 2.0 software. ESI source was run on both, a positive and negative mode. Spectrometry parameters were as follows: ion source GS1 and GS2 were 0.41 and 0.34 MPa, respectively. Curtain gas was 0.14 MPa. Collision gas was 0.04 MPa. All the gases were supplied with nitrogen (purity$99.995%). IonSpray voltage was 5500 V (ESI+) and 24200 V (ESI–). Ion source temperature was 500uC. Declustering Potential (DP) was 60 V (ESI+) and 260 V (ESI–). The Q1 mass monitoring was conducted using a scan mode in the range from 200 to 600 m/z. Mass intensity above 100 counts was set for acquiring product ions on TOF analyzer. An information dependent acquisition (IDA) method was set to optimize data acquisition. p After the incubation, the primary pig hepatocytes were washed three times with phosphate buffer saline solution (PBS) before they were treated with steroids and/or inhibitors. In steroid metabolism studies, hepatocytes were incubated in 10 ml of basal growth medium containing phenol red-free DMEM with 10% of charcoal treated FBS fortified with 10 mM (final concentration) of androstenone, 17b-estradiol or dihydrotestosterone. In the inhibi- tion experiments, 10 mM (final concentration) of trilostane or apigenin, the specific inhibitors of 3bHSD and 17bHSD respectively, were added to hepatocytes simultaneously with androstenone, 17b-estradiol or dihydrotestosterone. Solutions of both, the inhibitors and steroids, were prepared in methanol. Concentration of methanol in the cell culture medium was always less than 1%, and it did not have any effect on hepatocyte metabolism. The concentration choice was based on results of cytotoxicity test. In the cytotoxicity text, the steroids were Chromatographic separation was conducted using HPLC coupled to mass spectrometry (1200 SL, Agilent Technologies, USA) equipped with a binary pump, vacuum solvent degasser, column oven and autosampler (Agilent Technologies, Waldbronn, Germany). The column used was Zorbax Eclipse Plus C18 (2.16100 mm, 3.5 mm) with a guard column (2.1612.5 mm, 5 mm) (Agilent Technologies, USA). For the ESI+ mode, the mobile phase was 0.1% formic acid in water (A) and methanol (B). For the ESI– mode, the mobile phase was 0.1% ammonium in water (A) and methanol (B). Introduction Steroid hormones are essential for a range of physiological processes in mammals. They act as body chemical messengers, and play critical role in organism development and maturation [1]. Steroids also play an important role in regulation of pig metabolism and pig meat quality. Androgen metabolism results in production of a group of non-hormonal androstanes, which act as pheromones and influence physiology or behavior of mammals [4]. Accumulation of one of the androstanes, 5a-androst-16-ene-3- one (androstenone) in adipose tissue of entire male pigs is associated with a pork quality defect, boar taint, which is unpleasant ‘urine-like’ odour [5]. Other compounds contributing to boar taint are skatole and indole which are formed during tryptophan metabolism [2]. Boar taint can be prevented by surgical castration which reduces the level of steroids, including androstenone [6]. However, due to the EU initiative to ban surgical castration by 2018, boar taint becomes an increasing issue for international pig meat industry and introduces the challenge of reduction of androstenone level by the means other than castrations [7]. Decreasing androstenone level in pork can be achieved either via reduction of the rate of androstenone January 2015 \| Volume 10 \| Issue 1 \| e113194 1 PLOS ONE \| www.plosone.org 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 50% solvent B at 9.1 and 15 min. The flow rate was 300 mL/min, and the sample injection volume was 20 mL. glucuronidation. To check this hypothesis, b-glucuronidase was added to the cell culture medium. Presence of hydroxylated androstenone with the m/z+ = 257.2257 was determined in the medium after incubation with b-glucuronidase using LC-TOF-MS (Figure 1E). Comparing the samples with authentic standards of 5a-androst-16-en-3a-ol (3a-A) and 5a-androst-16-en-3b-ol (3b-A) allowed to establish the presence of hydroxylated androstenone in the medium after incubation with b-glucuronidase. Therefore, this hydroxylated androstenone was defined as 3b-A (Figure 1F). These experiments confirmed that androstenone was hydroxylated to 3b-A prior to glucuronidation reaction. Statistical analysis Data were analyzed by the Statistical Analysis System, version 9.0 (SAS Institute, Cary, NC, USA). Anova procedure was used to evaluate the group differences, and the means of groups were compared by Duncan’s multiple comparison. P,0.05 was regarded as statistically significant. Results The inhibitory study demonstrated that incubation of the primary isolated hepatocytes in the presence of 10 mM of trilostane, the specific inhibitor of 3bHSD, resulted in 70% decrease in the formation of androstenone metabolite in the cell culture medium. Incubation of hepatocytes with 10 mM of apigenin, the specific inhibitor of 17bHSD, did not affect significantly the level of androstenone metabolite (Table 2). Cytotoxicity study The results of cell viability incubated at different concentration of steroids and HSD inhibitors were given in Table 1. The cell survival rate of 80% was regarded as a threshold for evaluating the chemicals with or without toxicological effect. It can be seen that steroids and inhibitors at 10 mM have cell viability higher than 80%, and therefore was chosen for further experiment. Androstenone metabolism Abundance of 17b-estradiol precursor ion (m/z2 = 271.2106) in the medium before cell culturing is shown in Figure 2A. 17b- Estradiol was completely metabolized by pig hepatocytes after 6 h of incubation in the medium (Figure 2B). Investigation of 17b- estradiol metabolites by LC-TOF-MS established the presence of two compounds with m/z2 445.2190 and 447.2298, which correspond to molecular ions of glucuronide-conjugated estrone (E1-G) (Figure 2C) and glucuronide-conjugated estradiol (E2-G) (Figure 2D) respectively. The ratio of E1-G to E2-G was 5:1. When the cell culture medium was hydrolyzed by b-glucuronidase, both estrone and 17b-estradiol were detected. The ratio estrone: 17b-estradiol was similar to the ratio E1-G: E2-G. Using b- estradiol-39-glucuronide and b-estradiol-179-glucuronide authentic standards, this study established that glucuronidation of E2-G occurred at 39-hydroxyl group (Figure 2E). Figure 1A presents data on abundance of androstenone precursor ion (m/z+ = 273.2198) in the incubation medium before cell culturing. A time-course of androstenone metabolism showed that androstenone was non-detectable in the cell culture medium after 24 h of incubation (Figure 1B). LC-TOF-MS analysis of androstenone metabolites in ESI+ ionization mode identified a metabolite ion (m/z+ = 257.2245), which corresponds to a precursor ion of androstenone without a 3-keto group (Figure 1C). Analysis of androstenone metabolite in ESI– ionization mode demonstrated the presence of another ion with m/z2 = 449.2424 (Figure 1D). A molecular formula of this ion was determined using MetID software. The analysis demonstrated that this ion corresponds to a glucuronide-conjugated androstenone (A–G). Further investigation was conducted to determine association between the results obtained with the two different ionization approaches. The fraction of m/z+ = 257.2245 from post-HPLC column was collected and analyzed in ESI– mode. The results demonstrated a sole mass spectrometric peak of m/z2 = 449.2424, with the retention time corresponding to the peak of androstenone metabolite in the cultured medium samples analyzed in ESI– mode. These experiments confirmed that the ion m/z+ = 257.2245 is the glucuronide-conjugated androstenone which lost glucuronide structure (C6H8O6, MW = 176) in ESI+ ionization. There was no significant inhibition of E2-G and E1-G formation in the presence of trilostane and apigenin (Table 2). Mass spectrometry analysis The linear gradient used was as follows: 50% to 95% solvent B at 0–5 min; 95% solvent at 9 min, PLOS ONE \| www.plosone.org January 2015 \| Volume 10 \| Issue 1 \| e113194 2 Dihydrotestosterone metabolism Abundance of dihydrotestosterone precursor ion (m/z+ = 291.2352) in the medium before cell culturing is presented in Figure 3A. Similarly to 17b-estradiol, dihydrotestosterone was also rapidly metabolized by pig hepatocytes after 6 h of incubation (Figure 3B). LC-TOF-MS analysis identified a metabolite with m/ z2 = 465.2554 (RT = 4.82 min) in ESI– mode (Figure 3C). Molecular formula calculation allowed to establish that this compound matches glucuronide-conjugated dihydrotestosterone (DHT-G). Since dihydrotestosterone contains only one 17b- It is known that glucuronidation occurs at the hydroxyl group of compounds. Since androstenone does not have a hydroxyl group, we suggested that androstenone was hydroxylated by a specific enzyme at the 3-keto group first, and then was subjected to Table 1. Cytotoxicity of steroids and HSD inhibitors in isolated pig hepatocytes. Concentration (mM) Androstenone 17b-Estradiol Dihydrotestosterone Trilostane Apigenin 1 90.663.8 109.264.8 96.760.1 91.860.1 97.763.2 10 89.067.2 91.660.4 84.363.9 82.462.1 83.565.9 25 77.260.6 89.868.0 79.162.2 79.162.9 64.261.7 50 78.766.8 83.661.6 76.164.7 69.261.8 69.860.9 Cells (<56106 cells/plate) were incubated in the presence of different concentrations of steroids and inhibitors (0, 1, 10, 25 and 50 mM) for 48 h. Cell viability was evaluated by analyzing the luminescence intensity (RLU) normalized to mean value of cells without treatment. Data presented as Mean 6 SD. Effects of steroids and inhibitors at different concentrations vs. cell viability were evaluated by one-way anova. Cell viability did not differ significantly (p.0.05) in the presence of steroids and inhibitors at any of the concentrations studied. The experiments were conducted in three independent batches, with triplicate repeats for each experiment. doi:10.1371/journal.pone.0113194.t001 Table 1. Cytotoxicity of steroids and HSD inhibitors in isolated pig hepatocytes. Table 1. Cytotoxicity of steroids and HSD inhibitors in isolated pig hepatocytes. Cells (<56106 cells/plate) were incubated in the presence of different concentrations of steroids and inhibitors (0, 1, 10, 25 and 50 mM) for 48 h. Cell viability was evaluated by analyzing the luminescence intensity (RLU) normalized to mean value of cells without treatment. Data presented as Mean 6 SD. Effects of steroids and inhibitors at different concentrations vs. cell viability were evaluated by one-way anova. Cell viability did not differ significantly (p.0.05) in the presence of steroids and inhibitors at any of the concentrations studied. The experiments were conducted in three independent batches, with triplicate repeats for each experiment. Dihydrotestosterone metabolism doi:10.1371/journal.pone.0113194.t001 January 2015 \| Volume 10 \| Issue 1 \| e113194 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism Figure 1. Ion chromatograms and mass spectra (inset) of androstenone and its metabolites. (A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5a-androst-16-en-3b-glucuronide (m/z2 = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5a-androst-16-en- 3b-ol after enzyme hydrolysis by b-glucuronidase; (F) a mixture of authentic standards 5a-androst-16-en-3a-ol (3a-A) and 5a-androst-16-en-3b-ol (3b- A). doi:10.1371/journal.pone.0113194.g001 Figure 1. Ion chromatograms and mass spectra (inset) of androstenone and its metabolites. (A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5a-androst-16-en-3b-glucuronide (m/z2 = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5a-androst-16-en- 3b-ol after enzyme hydrolysis by b-glucuronidase; (F) a mixture of authentic standards 5a-androst-16-en-3a-ol (3a-A) and 5a-androst-16-en-3b-ol (3b- A). doi:10 1371/journal pone 0113194 g001 doi:10.1371/journal.pone.0113194.g001 hydroxyl group, the metabolite was defined as 5a-dihydrotestos- terone-17b-glucuronide. Experiments with hydrolysis of the cell culture medium by b-glucuronidase provided further confirmation of presence of glucuronide structure. production [14]. Moreover, an excessive accumulation of androstenone in pig adipose tissue contributes to an unpleasant odour of meat from some entire male pigs which makes the meat unsuitable for human consumption [5]. Therefore, developing strategies for controlling androstenone level in pork is one of the key challenges of pig industry. Accumulation of androstenone in pig adipose tissue can be due to a high rate of androstenone biosynthesis and/or low rate of androstenone metabolism. Therefore, understanding the pathways controlling metabolism of androstenone in pigs is important for improving meat quality. In addition to androstenone, the other two steroids, 17b-estradiol Enzyme inhibition study showed that trilostane reduces DHT-G formation in the hepatocytes by almost 86% (Table 2). Incubation with apigenin did not have any effect on DHT-G level. Discussion The level of steroid hormones in pigs influences animal growth rate and fat accumulation, which has impact on economics of pig Table 2. Peak heights (cps) of the ion chromatograms of steroids metabolites in cell culture medium. Metabolites C Apigenin Trilostane Androstenone glucuronide 3.460.5 3.760.9 1.060.6 Estrone glucuronide 16.562.2 15.361.1 13.561.7 Estradiol 3b-glucuronide 3.160.8 2.660.1 2.760.4 Dihydrotestosterone 17b-glucuronide 11.861.1 11.560.5 1.660.2 Data are presented as Mean 6 SD. () presents statistical significance of differences between groups at p,0.01. The peak height values are presented after division by 1000 (cps/1000). C = steroid metabolites analyzed after 48 h incubation of cell culture in absence of enzyme inhibitors. Apigenin = steroid metabolites analyzed after 48 h of cell culture in the presence of apigenin, the specific inhibitor of 17bHSD. Trilostane = steroid metabolites analyzed after 48 h of incubation of cell culture in the presence of trilostane, the specific inhibitor of 3bHSD. The cell density was approx. 56106 cells/plate. The experiments were conducted in three independent batches with triplicate repeats for each experiment. doi:10.1371/journal.pone.0113194.t002 Table 2. Peak heights (cps) of the ion chromatograms of steroids metabolites in cell culture medium. Metabolites C Apigenin Trilostane Androstenone glucuronide 3.460.5 3.760.9 1.060.6 Estrone glucuronide 16.562.2 15.361.1 13.561.7 Estradiol 3b-glucuronide 3.160.8 2.660.1 2.760.4 Dihydrotestosterone 17b-glucuronide 11.861.1 11.560.5 1.660.2 Data are presented as Mean 6 SD. () presents statistical significance of differences between groups at p,0.01. The peak height values are presented after division by 1000 (cps/1000). C = steroid metabolites analyzed after 48 h incubation of cell culture in absence of enzyme inhibitors. Apigenin = steroid metabolites analyzed after 48 h of cell culture in the presence of apigenin, the specific inhibitor of 17bHSD. Trilostane = steroid metabolites analyzed after 48 h of incubation of cell culture in the presence of trilostane, the specific inhibitor of 3bHSD. The cell density was approx. 56106 cells/plate. The experiments were conducted in three independent batches with triplicate repeats for each experiment. doi:10.1371/journal.pone.0113194.t002 ble 2. Peak heights (cps) of the ion chromatograms of steroids metabolites in cell culture medium. Table 2. Peak heights (cps) of the ion chromatograms of steroids metabolites in cell culture mediu ( p ) C = steroid metabolites analyzed after 48 h incubation of cell culture in absence of enzyme inhibitors. Apigenin = steroid metabolites analyzed after 48 h of cell culture in the presence of apigenin, the specific inhibitor of 17bHSD. Discussion Trilostane = steroid metabolites analyzed after 48 h of incubation of cell culture in the presence of trilostane, the specific inhibitor of 3bHSD. The cell density was approx. 56106 cells/plate. The experiments were conducted in three independent batches with triplicate repeats for each experiment. doi:10 1371/journal pone 0113194 t002 January 2015 \| Volume 10 \| Issue 1 \| e113194 PLOS ONE \| www.plosone.org 4 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism PLOS ONE \| www plosone org Figure 2. Ion chromatograms and mass spectra (inset) of 17b- estradiol and its metabolites. (A) 17b-estradiol in the medium in absence of isolated hepatocytes; (B) 17b-estradiol in the medium in the presence of isolated hepatocytes. No 17b-estradiol was found after 6 h of cell culture; (C) identified 17b-estradiol metabolite estrone-3- glucuronide (m/z2 = 445.2190). The samples were analyzed in ESI– ionization mode; (D) identified 17b-estradiol metabolite b-estradiol-3- glucuronide (m/z2 = 447.2298) in ESI– ionization mode; (E) a mixture of authentic standards b-estradiol-17-glucuronide and b-estradiol-3-glu- curonide. doi:10.1371/journal.pone.0113194.g002 and dihydrotestosterone can have an impact on pig physiology [15,16]. It has been recently reported that 17b-estradiol and dihydrotestosterone can alter porcine nuclear receptor expression and thus modulate activity of enzymes involved in steroid metabolism including enzymes controlling androstenone metabo- lism [17]. The present study used LC-TOF-MS to investigate metabolism of androstenone, 17b-estradiol, and dihydrotestosterone in isolated pig hepatocytes as an in vitro model. The results showed that androstenone formed a 3b-androstenol structure during the phase I metabolism (reduction). The inhibition study with trilostane and apigenin as specific inhibitors of 3bHSD and 17bHSD respec- tively, showed that 3bHSD but not 17bHSD is the key enzyme catalyzing the first stage of the hepatic androstenone metabolism. This finding is consistent with previous reports that b-androstenol is the main product of pig hepatic androstenone metabolisms, and that the process of conversion of androstenone to b-androstenol is catalyzed by 3bHSD [8,18]. Our study also investigated the second stage of androstenone metabolism (conjugation) and established the formation of glucuronidated androstenone struc- ture. Previous reports suggested the existence of glucuronidated androstenone form on the basis of observation that 3a- and 3b- androstenol glucuronide are present in pig urinary system [1], and the formation of conjugates may facilitate the metabolic clearance of the steroids [19]. The present study provided direct evidence of existence of androstenone glucuronide structure in experiments on isolated pig hepatocytes. Discussion Data of the literature on the other main steroid hormone, testosterone report 4-androstene-3,17-dione as the main product of the stage I of the hepatic testosterone metabolism [13]. However, the phase II of the porcine hepatic testosterone metabolism has not been fully investigated, and the formation of conjugated testosterone metabolite cannot be excluded. Some research group observed the formation of androstenone sulfocon- jugate in primary cultured porcine hepatocytes. These data were obtained using an analytical approach for sample hydroxylation and GC-MS derivatization [20]. In the present study, formation of sulfoconjugated androstenone was not observed. One explanation for this discrepancy might be tissue-specific formation of different androstenone conjugates. Sulfoconjugated androstenone (but not 16-androstene glucuronide conjugates) was reported to be present in pig plasma and testis [19]. Another reason for discrepancy between results of this paper and data of the literature might be the fact that in vitro cell culture systems have a lower concentra- tion of sulfate when compared to in vivo conditions, which does not facilitate sulfo-conjugation. Sinclair et al. (2005) suggested that glucuronidation of 16-androstene steroids is the main route of their metabolic clearance [19]. This hypothesis is supported by finding of this paper that androstenone glucuronide is a predominant hepatic conjugate which carry out the main clearance functions in pigs. Our finding is in a line with other papers which reported the presence of androstenone glucuronide in urine [1]. To summarize, the present paper suggests that androstenone metabolic pathway includes (i) transformation of PLOS ONE \| www.plosone.org January 2015 \| Volume 10 \| Issue 1 \| e113194 PLOS ONE \| www.plosone.org 5 January 2015 \| Volume 10 \| Issue 1 \| e113194 References 1. Gower DB (1972) 16-Unsaturated C 19 steroids. A review of their chemistry, biochemistry and possible physiological role. J Steroid Biochem 3: 45–103. 15. Gray MA, Peacock JN, Squires EJ (2009) Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants. Xenobiotica 39: 915–930. 1. Gower DB (1972) 16-Unsaturated C 19 steroids. A review of their chemistry, biochemistry and possible physiological role. J Steroid Biochem 3: 45–103. y p p y g J 2. Yokoyama MT, Carlson JR (1979) Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole. Am J Clin Nutr 32: 173–178. 2. Yokoyama MT, Carlson JR (1979) Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole. Am J Clin Nutr 32: 173–178. 16. Gray MA, Pollock CB, Schook LB, Squires EJ (2010) Characterization of porcine pregnane X receptor, farnesoid X receptor and their splice variants. Exp Biol Med 235: 718–736. 3. Robic A, Faraut T, Prunier A (2014) Pathways and genes involved in steroid hormone metabolism in male pigs: A review and update. J Steroid Biochem 140: 44–55. 17. Gray MA, Squires EJ (2013) Effects of nuclear receptor transactivation on boar taint metabolism and gene expression in porcine hepatocytes. J Steroid Biochem 133: 110–119. 4. Brooks RI, Pearson AM (1986) Steroid hormone pathways in the pig, with special emphasis on boar odor: a review. J Anim Sci 62: 632–645. 18. Nicolau-Solano SI, McGivan JD, Whittington FM, Nieuwhof GJ, Wood JD, et al. (2006) Relationship between the expression of hepatic but not testicular 3beta-hydroxysteroid dehydrogenase with androstenone deposition in pig adipose tissue. J Anim Sci 84: 2809–2817. 5. Patterson RLS (1968) 5a-androst-16-ene-3-one:–Compound responsible for taint in boar fat. J Sci Food Agric 19: 31–38. 6. Zamaratskaia G, Squires EJ (2009) Biochemical, nutritional and genetic effects on boar taint in entire male pigs. Animal 3: 1508–1521. 19. Sinclair PA, Squires EJ, Raeside JI, Renaud R (2005) Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar. J Steroid Biochem 96: 217–228. 7. Hansen LL, Mejer H, Thamsborg SM, Byrne DV, Roepstorff A, et al. (2007) Influence of chicory roots (Cichorium intybus L) on boar taint in entire male and female pigs. Anim Sci 82: 359–368. 20. Sinclair PA, Hancock S, Gilmore WJ, Squires EJ (2005) Metabolism of the 16- androstene steroids in primary cultured porcine hepatocytes. J Steroid Biochem 96: 79–87. 8. Conclusions This study employed an effective time of flight mass spectrom- etry approach to investigate metabolism of steroids androstenone, 17b-estradiol and dihydrotestosterone in isolated pig hepatocytes as a model system. The study provided novel data on pig hepatic steroid metabolism. In particular, this is the first report which directly confirms the formation of glucuronide-conjugated andros- tenone and involvement of 3bHSD in this process. The study also reported involvement of dehydrogenase enzymes in regulation of 17b-estradiol and dihydrotestosterone metabolism in pig hepato- cytes. Dihydrotestosterone is one of the most active androgens in the endocrine system of various species [24]. This study reported the formation of glucuronide conjugated dihydrotestosterone in primary cultured pig hepatocytes. Formation of DHT-G was greatly inhibited by trilostane, the specific 3bHSD inhibitor, whereas apigenin, the inhibitor of 17bHSD, did not have any effect on the DHT-G formation. It was previously reported that dihydrotestosterone can be metabolized to 5a-androstane-3a,17b- diol (3a-Diol) and 5a-androstane-3b,17b-diol (3b-Diol) in a purified enzyme system [22]. These metabolites were not found in the present study. One explanation for discrepancy between data of the literature and this study might be the use of different experimental systems: the processes taking place in purified 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism metabolite in 17b-estradiol metabolism is somewhat surprising, since the reduction of keto-steroid is the main reaction in the hepatic tissue [22]. The enzyme inhibition experiments undertak- en in the present study showed that 3bHSD and 17bHSD are not involved in 17b-estradiol metabolism. Regarding the second stage of 17b-estradiol metabolism, the present study demonstrated that glucuronidation of 17b-estradiol occurred at 3-hydroxyl but not at 17-hydroxyl group. A mechanism of this stereo selection remains unknown. It has been reported that glucuronidation of steroids is catalyzed by UDP-glucuronosyltransferase (UGT) [23]. The stereo selection depends on UGT enzyme sub-type and it is tissue- specific. It was previously reported that sulfoconjugated estrone (E1S) was the main metabolite and a storage form of estrogens in pigs [19]. The present study did not detect E1S in the in vitro cell culture system. This suggest that sulfation of estradiol may occur in other organs, such as testis, which is known as the main organ of steroids genesis [19]. enzyme systems might be different to the processes taking place in primary cultured hepatocytes. Another possible explanation for this discrepancy might be a sequential metabolic pathway of dihydrotestosterone. For example, 3a-Diol and 3b-Diol metabo- lites may undergo further glucuronidation and sulfation as reported by Stephan et al. (2004) [22]. It has also been reported that DHT-G can be further metabolized at 3 keto-group to form 3a-androstanediol-17-glucuronide [25]. Considering that DHT-G only accounts for 1/6 of total dihydrotestosterone, formation of other dihydrotestosterone metabolites cannot be excluded. Lack of clarify regarding dihydrotestosterone metabolism suggests its multiple and complex metabolic pathway. Author Contributions Conceived and designed the experiments: GC LR MF. Performed the experiments: GC YB DZ YL. Contributed to the writing of the manuscript: GC MF HA OD. 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism chromatograms and mass spectra (inset) of dihydrotestosterone and its metabolite. (A) dihydrotestosterone in the ence of isolated hepatocytes; (B) dihydrotestosterone in the medium in the presence of isolated hepatocytes. No dihydrotestosterone er 6 h of cell culture; (C) identified dihydrotestosterone metabolite dihydrotestosterone-17-glucuronide (m/z2 = 465.2554). The nalyzed in ESI– ionization mode. rnal.pone.0113194.g003 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism 3ßHSD Catalyzation in Porcine Hepatic Steroids Metabolism Figure 3. Ion chromatograms and mass spectra (inset) of dihydrotestosterone and its metabolite. (A) dihydrotestosterone in the medium in absence of isolated hepatocytes; (B) dihydrotestosterone in the medium in the presence of isolated hepatocytes. No dihydrotestosterone was found after 6 h of cell culture; (C) identified dihydrotestosterone metabolite dihydrotestosterone-17-glucuronide (m/z2 = 465.2554). The samples were analyzed in ESI– ionization mode. doi:10.1371/journal.pone.0113194.g003 fully understood. The present study investigated the role of the two HSD enzymes, 3bHSD and 17bHSD, in metabolism of two steroids, 17b-estradiol and dihydrotestosterone in isolated pig hepatocytes. It was found that over 80% of 17b-estradiol was oxidized at C-17 position to form estrone, with follow-on glucuronidation at 39-hydroxyl group and formation E1-G. Less than 20% of 17b-estradiol underwent direct phase II metabolism and formed E2-G. The observation that E1-G is the main androstenone to 3-hydroxyl structure by 3bHSD enzyme; (ii) sulfoconjugation of androstenone, which might be important for storage of androstenone in testis, and (iii) metabolism of androstenone 3-hydroxyl structure to glucuronide conjugate in the liver. The glucuronide conjugated androstenone can be excreted with urine. Although enzymes of HSD family are highly expressed in pig liver [8,21], the physiological role of pig hepatic HSD has not been January 2015 \| Volume 10 \| Issue 1 \| e113194 January 2015 \| Volume 10 \| Issue 1 \| e113194 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 References Doran E, Whittington FM, Wood JD, McGivan JD (2004) Characterisation of androstenone metabolism in pig liver microsomes. Chem-Biol Interact 147: 141–149. 21. Chen G, Bourneuf E, Marklund S, Zamaratskaia G, Madej A, et al. (2007) Gene expression of 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase in relation to androstenone, testosterone, and estrone sulphate in gonadally intact male and castrated pigs. J Anim Sci 85: 2457–2463. 9. Payne AH, Hales DB (2004) Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 25: 947–970. 10. Penning TM, Lee SH, Jin Y, Gutierrez A, Blair IA (2010) Liquid chromatography-mass spectrometry (LC-MS) of steroid hormone metabolites and its applications. J Steroid Biochem 121: 546–555. gonadally intact male and castrated pigs. J Anim Sci 85: 2457–246 22. Steckelbroeck S, Jin Y, Gopishetty S, Oyesanmi B, Penning TM (2004) Human cytosolic 3alpha-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3beta-hydroxysteroid dehydrogenase activity: implications for steroid hormone metabolism and action. J Biol Chem 279: 10784–10795. 11. Mareck U, Geyer H, Opfermann G, Thevis M, Schanzer W (2008) Factors influencing the steroid profile in doping control analysis. J Mass Spectrom 43: 877–891. 12. De Brabander HF, Le Bizec B, Pinel G, Antignac JP, Verheyden K, et al. (2007) Past, present and future of mass spectrometry in the analysis of residues of banned substances in meat-producing animals. J Mass Spectrom 42: 983–998. 23. Zhou J, Tracy TS, Remmel RP (2010) Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites. Drug Metab Dispos 38: 431–440. 13. Chen G, Li S, Dong X, Bai Y, Chen A, et al. (2012) Investigation of testosterone, androstenone, and estradiol metabolism in HepG2 cells and primary culture pig hepatocytes and their effects on 17betaHSD7 gene expression. PloS One 7; e52255. 24. Luu-The V (2013) Assessment of steroidogenesis and steroidogenic enzyme functions. J Steroid Biochem 137: 176–182. 25. Jin Y, Duan L, Lee SH, Kloosterboer HJ, Blair IA, et al. (2009) Human cytosolic hydroxysteroid dehydrogenases of the aldo-ketoreductase superfamily catalyze reduction of conjugated steroids: implications for phase I and phase II steroid hormone metabolism. J Biol Chem 284: 10013–10022. 14. Lundstrom K, Matthews KR, Haugen JE (2009) Pig meat quality from entire males. Animal 3: 1497–1507. 7 PLOS ONE \| www.plosone.org 7 January 2015 \| Volume 10 \| Issue 1 \| e113194 January 2015 \| Volume 10 \| Issue 1 \| e113194
https://openalex.org/W3037442684	https://link.springer.com/content/pdf/10.1007/s00296-020-04637-x.pdf	English	null	Adequately dosed aerobic physical activity in people with axial spondyloarthritis: associations with physical therapy	Rheumatology international	2,020	cc-by	7,637	Rheumatology INTERNATIONAL Rheumatology International (2020) 40:1519–1528 https://doi.org/10.1007/s00296-020-04637-x PHYSICAL MEDICINE AND REHABILITATION Adequately dosed aerobic physical activity in people with axial spondyloarthritis: associations with physical therapy Bas Hilberdink1 · Thea Vliet Vlieland1 · Florus van der Giesen2 · Floris van Gaalen2 · R Andreas Peeters4 · Marta Fiocco5,6 · Salima van Weely1 Received: 8 April 2020 / Accepted: 19 June 2020 / Published online: 28 June 2020 © The Author(s) 2020 Abstract Introduction This study aimed to compare the engagement in moderate- and vigorous-intensity PA in axSpA patients with and without current physical therapy (PT). p y py Methods In this cross-sectional study, a survey, including current PT treatment (yes/no) and PA, using the ‘Short QUes- tionnaire to ASsess Health-enhancing PA’ (SQUASH), was sent to 458 axSpA patients from three Dutch hospitals. From the SQUASH, the proportions meeting aerobic PA recommendations (≥ 150 min/week moderate-, ≥ 75 min/week vigorous- intensity PA or equivalent combination; yes/no) were calculated. To investigate the association between PT treatment and meeting the PA recommendations, odds ratios (OR) with 95% confidence intervals (95% CI) were estimated using logistic regression models, adjusting for sex, age, health status and hospital. g j g g p Results The questionnaire was completed by 200 patients, of whom 68%, 50% and 82% met the moderate-, vigorous- or combined-intensity PA recommendations, respectively. Ninety-nine patients (50%) had PT treatment, and those patients were more likely to meet the moderate- (OR 2.09 [95% CI 1.09–3.99]) or combined-intensity (OR 3.35 [95% CI 1.38–8.13]) PA recommendations, but not the vigorous-intensity PA recommendation (OR 1.53 [95% CI 0.80–2.93]). Aerobic exercise was executed in 19% of individual PT programs. Conclusion AxSpA patients with PT were more likely to meet the moderate- and combined-intensity PA recommendations, whereas there was no difference in meeting the vigorous-intensity PA recommendation. Irrespective of having PT treatment, recommendations for vigorous-intensity PA are met by only half of the patients. Implementation should thus focus on aerobic PA in patients without PT and on vigorous-intensity PA in PT programs. Keywords Axial spondyloarthritis · Physical activity · Exercise · Physical therapy words Axial spondyloarthritis · Physical activity · Exercise · Physical therapy * Bas Hilberdink B.Hilberdink@lumc.nl Thea Vliet Vlieland t.p.m.vliet_vlieland@lumc.nl Florus van der Giesen f.j.van_der_giesen@lumc.nl Floris van Gaalen f.a.van_gaalen@lumc.nl Robbert Goekoop r.goekoop@hagaziekenhuis.nl Andreas Peeters peeters@rdgg.nl Marta Fiocco m.fiocco@lumc.nl Salima van Weely s.f.e.van_weelij@lumc.nl Sa a va Wee y s.f.e.van_weelij@lumc.nl 1 Department of Orthopaedics, Rehabilitation and Physical Therapy, Leiden University Medical Center, j11, P.O. Introduction Axial spondyloarthritis (axSpA) is a chronic inflamma- tory rheumatic disease, with back pain and stiffness as main symptoms and encompassing both non-radiographic and radiographic axSpA (ankylosing spondylitis) [1, 2]. The literature shows that axSpA is associated with both decreased cardiorespiratory fitness [3–6] and an increased risk of cardiovascular disease [7, 8], which are interrelated [9–12]. Adequately dosed aerobic physical activity (PA) according to public health recommendations improves cardiorespiratory fitness in people with axSpA [13, 14] and might reduce the cardiovascular risk. For this reason, it is advocated in international recommendations on PA in people with rheumatic and musculoskeletal conditions [15]. Aerobic PA concerns PA executed with moderate or vigorous intensity. Recent studies suggest that vigorous- intensity PA is most effective in improving cardiorespira- tory fitness and reducing cardiovascular risk [10, 16–18] and it shows to be both beneficial and safe for people with axSpA [19, 20]. Therefore, especially vigorous-intensity PA should be pursued by people with axSpA, at least by those without an increased risk of cardiovascular compli- cations during exercise. Another limitation of previous studies on aerobic PA among people with axSpA, besides not distinguishing between moderate- and vigorous-intensity PA and between leisure time and work-related aerobic PA, is that none of the studies so far took the role of physical therapy (PT) into account. This is striking as relatively many axSpA patients have PT treatment [32] and it is generally acknowledged that apart from other health professionals, physical thera- pists play an important role in the promotion of PA [15]. However, it appears that aerobic PA may not be included in PT treatments often [32] and that the aerobic PA employed in exercise programs for people with axSpA is often inad- equately dosed [20, 33–35]. To implement aerobic PA recommendations in peo- ple with axSpA, it is important to know what the focus of implementation activities should be, both in patients with and without PT treatment. Due to the physical limitations for which axSpA patients seek PT treatment, it is not necessarily expected that patients with PT are more inclined to meet the aerobic PA recommendations. Moreover, PT programs may not include (advice on) aerobic PA [32]. Abstract Box 9600, 2300 RC Leiden, the Netherlands 2 Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands 3 Haga Hospital, The Hague, the Netherlands 4 Reinier de Graaf Gasthuis, Delft, the Netherlands 5 Department of Biomedical Data Science, Medical Statistics, Leiden University Medical Center, Leiden, the Netherlands 6 Mathematical Institute, Leiden University, Leiden, the Netherlands B.Hilberdink@lumc.nl Thea Vliet Vlieland t.p.m.vliet_vlieland@lumc.nl Florus van der Giesen f.j.van_der_giesen@lumc.nl Floris van Gaalen f.a.van_gaalen@lumc.nl Robbert Goekoop r.goekoop@hagaziekenhuis.nl Andreas Peeters peeters@rdgg.nl Marta Fiocco m.fiocco@lumc.nl 1 Department of Orthopaedics, Rehabilitation and Physical Therapy, Leiden University Medical Center, j11, P.O. Box 9600, 2300 RC Leiden, the Netherlands 2 Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands 3 Haga Hospital, The Hague, the Netherlands 4 Reinier de Graaf Gasthuis, Delft, the Netherlands 5 Department of Biomedical Data Science, Medical Statistics, Leiden University Medical Center, Leiden, the Netherlands 6 Mathematical Institute, Leiden University, Leiden, the Netherlands :(0123 1 3456789) 3 Rheumatology International (2020) 40:1519–1528 1520 leisure time and work-related aerobic PA either, whereas leisure time PA appears to have greater health benefits [27–31] and is probably more easily modifiable than work- related PA. This superiority of leisure time PA could prob- ably be caused by the difference in the nature of activities or by more opportunities to rest when desired and recover between sessions [27, 29]. Introduction Given the lack of knowledge on the association between having PT treatment and meeting aerobic PA recommendations among people with axSpA, the aim of the present study was to compare the engagement in moderate- and vigorous-intensity PA (during work and leisure time) in axSpA patients with and without PT treatment. This raises the question to what extent people with axSpA are actually engaged in aerobic PA, either or not with vigorous intensity. A previous study reported that evi- dence on PA engagement of people with axSpA is limited and heterogeneous in nature [3]. Nevertheless, it appears that the engagement in adequately dosed aerobic PA is insufficient, in particular in vigorous-intensity aerobic PA [21–24]. Three studies, all using accelerometers, showed that people with axSpA engaged less in vigorous-intensity PA than population controls, while the total amount of PA was comparable [21–23]. Another study found that more people with axSpA comply with the moderate-intensity PA recommendation (57%) than with the vigorous-inten- sity PA recommendation (32%), using a non-validated PA questionnaire [24]. That study used the PA recommen- dation prescribing moderate-intensity PA for ≥ 30 min on ≥ 5 days per week or vigorous-intensity PA for ≥ 20 min on ≥ 3 days. Other studies on aerobic PA in people with axSpA [3, 21, 23, 25] were based on the recommenda- tion by the World Health Organization (WHO) [26], which does not state a required minimum frequency, but pre- scribes ≥ 150 min of moderate-intensity PA, ≥ 75 min of vigorous-intensity PA per week or an equivalent combina- tion of this. It was reported that this recommendation was met by approximately half of patients [21, 23, 25], but no distinction was made between the proportions of people meeting the moderate- or vigorous-intensity PA recom- mendations. None of the studies distinguished between Study design and setting This cross-sectional, multicenter study consisted of a once- only survey among people with axSpA living in the south- western region of the Netherlands. In this survey, partici- pants were asked whether they had either individual or group PT treatment, to compare PA of patients with and without any guidance from a physical therapist. In the Netherlands, PT for people with axSpA can both be offered on an indi- vidual basis or by means of axSpA-specific supervised group exercise. This group exercise usually consists of weekly land- and water based exercises supervised by a physical therapist and is organized by local patient associations for people with a rheumatic disease [34]. The study obtained ethical approval from the Leiden University Hospital Ethical 1 3 3 1521 Rheumatology International (2020) 40:1519–1528 committee (P14.326). The reporting of this study was done in accordance with the checklist for cross-sectional stud- ies from the ‘Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement’. on home exercise; coping; and PA and sports); Exercise (including active joint range of motion exercises; mus- cle strengthening exercises; aerobic exercises; balance exercises; and relaxation exercises); Manual treatment (including passive mobilization; and massage); and Applying physical modalities (including thermotherapy; kinesiotaping; electrotherapy; ultrasound; and dry nee- dling). Assessments The survey was self-developed and first pilot-tested by patient representatives affiliated with the Dutch Arthritis Society. It measured the following variables: a. Demographic and clinical characteristics: sex, age, year of diagnosis and use of medication related to axSpA (painkillers (acetaminophen or opioid painkillers); Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), biological Disease-Modifying Antirheumatic Drugs (DMARDs); synthetic DMARDs; no medication related to axSpA). b. Health status, using the Assessment of Spondyloarthritis International Society Health Index (ASASHI), which is a valid, reliable and responsive questionnaire measuring functioning, health and disease impact in people with axSpA [36, 37]. The ASAS HI includes 17 questions and results in a score between 0 and 17, with a lower score indicating a better health status. c. PT treatment, by asking whether they had PT treatment, either at the time the study was conducted (current PT; yes/no) or ever in the past (yes/no). Moreover, it was asked whether they were or had been treated individually in a practice (yes/no) and/or in a group with axSpA-spe- cific group exercise therapy (yes/no). Furthermore, for individual PT, the duration (> 5 years, > 3 years, > 1 year , > 6 months or < 6 months), frequency (less than weekly, weekly, twice weekly, more than twice weekly) and contents (15 treatment options) of PT treatment were recorded. These 15 treatment options were clustered according to the four groups of treatment modalities as described in the national physical therapists’ profes- sional profile developed by the Royal Dutch Society of Physical Therapy [38]: Counseling (including education Patients In 2015, registers of three hospitals in the southwestern region of the Netherlands (Leiden University Medical Center in Leiden, Haga Hospital in The Hague and Reinier de Graaf Gasthuis in Delft) were screened for patients with a con- firmed diagnosis of axSpA who had ever visited the rheu- matology outpatient clinic. The survey was sent by postal mail to eligible patients, including an invitation letter on behalf of their treating rheumatologist, an information leaf- let, an informed consent form and a pre-stamped envelope. No reminders were sent. d. d. Aerobic physical activity, using the validated Dutch version of the ‘Short QUestionnaire to ASsess Health- enhancing PA’ (SQUASH) [39, 40]. The SQUASH consists of 17 items asking respondents to recall PA as performed during a regular week in the past 12 months, yielding the time duration per PA intensity and the type of aerobic PA. The SQUASH categorizes PA into PA during commuting, (light and heavy) work, (light and heavy) household, walking, cycling, gardening, odd jobs and sports. For the purpose of this study, these categories were dichotomized into leisure time PA, including recreational walking, recreational cycling, exercise and sports, and non-leisure time PA, which includes PA during commuting, work, household, gar- dening and odd jobs. Using the compendium of Ains- worth [41], a research assistant (JP) assigned the cor- rect MET-values to the corresponding activities. The SQUASH uses a syntax to categorize the activities into light-, moderate- and vigorous-intensity PA, by com- bining activities’ MET-values with both participants’ age and a subjective effort-score (slow, average, fast) that participants assigned to each activity. Aerobic PA includes all PA performed with at least moderate-inten- sity. The SQUASH data were used to calculate whether patients met the moderate- (≥ 150 min/week), vigorous- (≥ 75 min/week) and/or combined-intensity (≥ 75 min/ week vigorous- and/or ≥ 150 min/week moderate- or vigorous-intensity PA) aerobic PA recommendations by the WHO [26]. This was examined both for PA during all daily activities and during leisure time specifically. Patients The questionnaire was sent to 458 axSpA patients of whom 206 returned it (response rate 45%). Six of them were excluded because the SQUASH data were either missing (n = 3) or invalid (n = 3). Patient characteristics are presented in Table 1, for the total group and for patients with and without PT separately. The majority of patients was male (69%), the median age 57 years and the median disease duration 23 years. The median ASAS HI score was 5.3, indicating moderate health status [37]. Ninety-nine patients had PT treatment at the time the study was conducted: 77 had individual PT treatment in a private practice only, 11 participated in axSpA-specific group exercise therapy only and 11 had both individual PT treatment in a private practice and group exercise therapy Min minimum value, Max maximum value, NSAID non-steroidal anti-inflammatory drugs, DMARD disease-modifying antirheumatic drugs, ASAS HI assessment of spondyloarthritis international society health index Statistical analyzes The returned questionnaires were scanned and analyzed by Cardiff® Software (California, United States) and manu- ally checked and corrected afterwards. Descriptive statistics were used to describe patient characteristics, the proportions meeting the aerobic PA recommendations and the engaged types of leisure time and non-leisure time aerobic PA. This was done for the total group of participants and for patients with and without PT guidance separately. Results were reported as percentages or medians with minimum (Min) and maximum (Max) values, where appropriate. To investigate the differences in characteristics between patients with and without PT, the median test for 1 3 Rheumatology International (2020) 40:1519–1528 1522 (on two different days). The group exercise therapy con- sisted of a standardized program comprising weekly land- and water based mobility and strengthening exercises and sports (mostly volleyball) in most patients [34]. Table 2 shows the duration, frequency and contents of current indi- vidual PT treatment. Among the 88 participants who were receiving individual PT at the time the study was conducted, the duration of treatment was more than five years in 66 patients (75%) and the treatment took place less than once a week in 44 patients (50%). Furthermore, the individual PT treatment included counseling in 67 (76%), exercise in 47 (53%), manual treatment in 80 (91%) and the application of physical modalities in 24 (27%). Regarding contents with a direct link to aerobic PA recommendations, education on PA and sports was reported by 37 patients (42%) and aerobic exercise during PT treatment by 17 (19%). Among the 101 participants without current PT, 84 had PT treatment ever in the past. No statistically significant differences regarding sex, age, disease duration, medication use, ASAS HI score and being employed were found between patients with and without PT. independent samples was used for continuous data and Pear- son’s chi-square test for categorical data. In addition, six logistic regression models were estimated with meeting the moderate, vigorous or combined-intensity PA recommenda- tions, both during all daily activities and during leisure time, as the dependent variables and current PT treatment (indi- vidual and/or group) as independent variable. To control for confounding, sex, age, health status and hospitals were included in the models as independent variables. All statisti- cal analyzes were performed with IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, N.Y., USA). f a Acetaminophen or opioid painkillers P-value of chi-square test (for nominal variables) or median test (for continuous variables) for difference e, Max maximum value, NSAID non-steroidal anti-inflammatory drugs, DMARD disease-modifying antirheumati t of spondyloarthritis international society health index Aerobic PA recommendations Table 3 presents the proportions of participants meeting the aerobic PA recommendations during all daily activities and during leisure time. This table shows that for all daily PA, the moderate, vigorous- and combined-intensity PA rec- ommendations were met by 68%, 50% and 82% of the par- ticipants, respectively. With respect to meeting the aerobic PA recommendations by taking only leisure time PA into Table 1 Differences in characteristics between axial spondyloarthritis patients with (n = 99) and without (n = 101) current physical therapy (PT), participating in a survey on physical activity and PT P-value of chi-square test (for nominal variables) or median test (for continuous variables) for differences between patients with and without PT a Acetaminophen or opioid painkillers Min minimum value, Max maximum value, NSAID non-steroidal anti-inflammatory drugs, DMARD disease-modifying antirheumatic drugs, ASAS HI assessment of spondyloarthritis international society health index Total group (n = 200) With PT (n = 99) Without PT (n = 101) p-value* Sex, male, n (%) 138 (69) 70 (71) 68 (68) 0.679 Age, years, median (Min–Max) 57 (23–93) 59 (23–85) 54 (23–93) 0.066 Disease duration, years, median (Min–Max) 23 (1–58) 25 (1–58) 17 (2–58) 0.127 Medication use, n (%) Painkillera 78 (39) 42 (42) 36 (36) 0.326 NSAID 123 (62) 64 (65) 59 (58) 0.365 Biological DMARD 77 (39) 39 (39) 38 (38) 0.797 Synthetic DMARD 25 (13) 11 (11) 14 (14) 0.557 None 16 (8) 5 (5) 11 (11) 0.128 ASAS HI score, median (Min–Max) 5.3 (0–14.9) 6.0 (0–13.4) 5.0 (0–14.9) 0.669 Being employed, n % 110 (55) 55 (56) 55 (54) 0.990 ristics between axial spondyloarthritis patients with (n = 99) and without (n = 101) current physical therapy (PT), sical activity and PT ween axial spondyloarthritis patients with (n = 99) and without (n = 101) current physical therapy (PT), ity and PT Table 1 Differences in characteristics between axial spondyloarthritis patients with (n = 99) and without (n = 101) cu participating in a survey on physical activity and PT 1 3 1523 Rheumatology International (2020) 40:1519–1528 patients with PT are significantly more likely to meet the moderate- (OR 2.09 [95% CI 1.09–3.99]) and combined- intensity (OR 3.35 [95% CI 1.38–8.13]) PA recommenda- tions than patients without current PT after adjusting for sex, age, health status and hospital. Discussion This study found that people with axSpA who were having PT treatment were more likely to meet the moderate- and combined-intensity aerobic PA recommendations than those without PT, whereas there were no differences in meeting the vigorous-intensity PA recommendation. Irrespective of current PT treatment, the proportion of participants meeting the vigorous-intensity PA recommendation was relatively low and often not attained with leisure time activities.i The finding that having PT treatment was associated with meeting aerobic PA recommendations was not necessarily expected, because PT programs may not include aerobic PA and those who need PT treatment are expected to have more physical limitations and may thus be less physically active. In our study, PT treatment was related to more aero- bic PA, but this did not pertain to vigorous-intensity PA. Given the cross-sectional design of the study, it remains unclear whether the association between PT and aerobic PA is a result of PT treatment or that axSpA patients who are already relatively active are more inclined to seek PT treatment. Either way, the findings show that specifically axSpA patients without PT should be better educated on the benefits of aerobic PA. It is recently recommended that all health professionals in rheumatology should promote aerobic PA [15], but especially physical therapists could play an important role in such education, in particular since account, the proportions of participants meeting the moder- ate-, vigorous- and combined-intensity PA recommendations were 48%, 42% and 67%, respectively. Moreover, 68% of the participants engaged in any moderate-intensity leisure time activities, whereas 50% of participants engaged in any vigorous-intensity leisure time activities. Aerobic PA recommendations When only including leisure time PA, patients with PT are more likely to meet the moderate-intensity PA recommendation (OR 1.86 [95% CI 1.03–3.36]) than patients without PT, with no differences for the vigorous- or combined-intensity PA recommendations. Table 2 Duration, frequency and contents of individual physical ther- apy (PT) in people with axial spondyloarthritis (axSpA) participating in a survey on physical activity and PT (n = 88) in a survey on physical activity and PT (n 88) AxSpA patients with individual PT (n = 88) PT duration, n (%) > 5 years 66 (75) 3–5 years 8 (9) 1–3 years 5 (6) 6 months-1 year 4 (5) < 6 months 5 (6) PT frequency, n (%) More than twice weekly 0 Twice weekly 13 (15) Weekly 30 (34) Less than weekly 44 (50) PT contents, n (%) Counseling 67 (76) Education on home exercise 54 (61) Education on coping 31 (35) Education on physical activity and sports 37 (42) Exercise 47 (53) Active joint range of motion exercises 28 (32) Muscle strengthening exercises 36 (41) Aerobic exercises 17 (19) Balance exercises 11 (13) Relaxation exercises 3 (3) Manual treatments 80 (91) Passive mobilization 62 (71) Massage 50 (57) Physical modalities 24 (27) Thermotherapy 9 (10) Kinesiotaping 2 (2) Electrotherapy or ultrasound 16 (18) Dry needling 4 (5) Types of aerobic activities Figure 2 presents the proportions of axSpA patients with and without PT engaging weekly in different forms of leisure or non-leisure time aerobic PA. There were no statistically significant differences between the proportions of partici- pants with and without PT engaging in the different types of aerobic activities, besides engagement in group exercise and aqua-aerobics; these types of aerobic PA were executed by significantly more patients with PT. This difference is likely to be due to participants with group PT, which often consists of group exercise and hydrotherapy in the Netherlands [34]. In both groups, it appeared that walking (69%) and cycling (57%) were the most frequently performed aerobic activities. PT and aerobic PA recommendations To study the association between PT treatment and aerobic PA, only current PT treatment was considered, since almost all participants (92%) had ever had PT. The differences between patients with and without current PT regarding the meeting of aerobic PA recommendations are shown in Fig. 1 and Table 3. Table 3 shows that, considering all daily PA, 1 Rheumatology International (2020) 40:1519–1528 1524 Table 3 Differences in meeting combined-, moderate- and vigorous-intensity physical activity (PA) recommendations during all daily activities and during leisure time between axial spondyloarthritis patients with (n = 99) and without (n = 101) current physical therapy (PT), participating in a survey on physical activity and PT Odds ratio adjusted for sex, age, health status and affiliated hospitals using multivariate logistic regression models Total group (n = 200) With PT (n = 99) Without PT (n = 101) OR 95% CI Meeting combined-intensity PA recommendation With all daily PA, n (%) 164 (82) 88 (89) 76 (75) 3.35 1.38–8.13 With leisure time PA, n (%) 133 (67) 72 (73) 61 (60) 1.81 0.94–3.49 Meeting moderate-intensity PA recommendation With all daily PA, n (%) 136 (68) 74 (75) 62 (61) 2.09 1.09–3.99 With leisure time PA, n (%) 96 (48) 55 (56) 41 (41) 1.86 1.03–3.36 Meeting vigorous-intensity PA recommendation With all daily PA, n (%) 100 (50) 54 (55) 46 (46) 1.53 0.80–2.93 With leisure time PA, n (%) 84 (42) 42 (42) 42 (42) 1.01 0.53–1.90 OR odds ratio. CI Confidence Interval. All daily PA = PA during commuting, household, work, garden- ing and odd jobs and leisure PA. Leisure time PA = recreational walking and cycling, exercise and sports. Combined-intensity PA recommendation = 150 min/week at least moderate-intensity PA or 75 min/week vigorous-intensity PA. Moderate-intensity PA recommendation = 150 min/week moderate-intensity PA. Vigorous-intensity PA recommendation = 75 min/week vigorous-intensity PA Fig. 2 Proportions of axSpA patients with and without physical therapy (PT) engaging in different forms of leisure time aerobic PA (with > 2% patients participating) and other aerobic PA types Fig. 1 Proportions of axSpA patients with and without physical ther- apy (PT) meeting the combined-, moderate- and vigorous-intensity PA recommendations, both when including all daily PA and when only including leisure time PA Fig. Table 3 Differences in meeting combined-, moderate- and vigorous-intensity physical activity (PA) recommendations during all daily activities and during leisure time between axial spondyloarthritis patients with (n = 99) and without (n = 101) current physical therapy (PT), participating in a survey on physical activity and PT PT and aerobic PA recommendations This comparison appears to not have been studied before and is important information to account for when implementing the aerobic PA recommendation. This study has a number of limitations. First, because of the cross-sectional study design, no conclusions can be drawn about any causal relationships between having PT and aerobic PA. Second, and as already addressed, using a self- report questionnaire the amount of PA might have been over- estimated [49]. Another limitation of the SQUASH is that it does not measure sedentary time. Moreover, it asks par- ticipants to recall their PA during a regular week in the past twelve months, whereas the groups compared in this study are based on having PT treatment at the time the study was conducted. As 89% of participants with individual PT were treated for more than 12 months, possibly not in all patients with PT, but at least in most of them, the actual influence of PT treatment on PA have been measured. Finally, the gener- alizability of our study is limited because the response rate was moderate (45%) and patients were recruited from only three hospitals in one region of the Netherlands. Although the participants of this study were relatively old [3], their sex ratio [3] and the proportion with PT [50] were comparable to other studies. An important note when implementing vigorous-inten- sity PA is that caution is needed with sedentary individu- als and people with an increased risk of cardiovascular complications during exercise [17, 43, 44]. Still, for most axSpA patients, vigorous-intensity PA should ultimately be aimed for, since this appears to have more health benefits [10, 16–18] and is more time-efficient [45], while time is an important exercise barrier in axSpA [19, 46, 47]. Regarding the types of actual activities, about half of the participants did not engage in any vigorous-intensity PA dur- ing leisure time at all. Studies reporting on the superiority of leisure time PA suggest that possible explanations for the greater benefits of leisure time PA are the difference in nature of activities and the presence of more opportunities to rest and recover when needed [27, 29]. The observation that recreational walking and cycling were the most popu- lar forms of aerobic PA in our study could guide physical therapists in their advice and guidance on specific activities that are likely to be maintained in daily life. PT and aerobic PA recommendations 1 Proportions of axSpA patients with and without physical ther- apy (PT) meeting the combined-, moderate- and vigorous-intensity PA recommendations, both when including all daily PA and when only including leisure time PA Fig. 2 Proportions of axSpA patients with and without physical therapy (PT) engaging in different forms of leisure time aerobic PA (with > 2% patients participating) and other aerobic PA types most individuals with axSpA have PT treatment at some point during their disease course, as confirmed in the pre- sent study. However, there is room for improvement in those with PT as well. Our study showed that education on PA is currently only provided in 42% of axSpA patients with indi- vidual PT in the Netherlands. In addition, aerobic exercise was only executed during PT in 19% of individual PT pro- grams. This is unfavorable, as guided practice is one of the most important intervention components to optimize exer- cise behavior of axSpA patients [42]. Ideally, axSpA patients could experience and practice vigorous-intensity PA under supervision of a physical therapist. Therefore, aerobic PA should be included more often in individual PT programs, in particular with vigorous intensity.i The finding that particularly vigorous-intensity PA was performed insufficiently by relatively many axSpA patients is consistent with previous findings [21–24]. Similar to patients without PT, only half of those with PT met the vigorous-intensity PA recommendation. This finding could be related to results from previous studies, showing that appropriately dosed aerobic PA is often not included in (PT) exercise programs [33, 34]. A recent study on content of PT 1 3 3 Rheumatology International (2020) 40:1519–1528 1525 in axSpA patients found that in the Netherlands, aerobic exercises are only performed during individual PT in 22% of patients [32]. Hence, when implementing vigorous-inten- sity PA among people with axSpA, barriers and facilitators of both patients and therapists should be accounted for. A cross-sectional study examining these barriers and facili- tators [19] found that motivation, disease symptoms and group heterogeneity could act as both barriers and facilita- tors according to patients and physical therapists. An imple- mentation strategy could include education for therapists on how to motivate patients for vigorous-intensity PA and how to tailor and adjust it to varying symptoms, individual preferences and other potential variances among individual patients, such as the presence of comorbidity. axSpA patients with and without PT. PT and aerobic PA recommendations It is neverthe- less conceivable that preferences for recreational activities may vary not only among individuals but among countries as well. In conclusion, axSpA patients with PT were more likely to meet the moderate- and combined-intensity but not the vigorous-intensity aerobic PA recommendations than those without PT. These findings imply first of all that in axSpA patients without PT, aerobic PA must be promoted. Sec- ond, as vigorous-intensity PA appears insufficiently imple- mented among those with PT, additional education of physi- cal therapists regarding the importance of and requirements for vigorous-intensity exercise as an essential element of PT programs for axSpA patients seems warranted. With the education of physical therapists, it should be noted that only 19% of patients with PT reported executing aerobic exercise as part of their PT treatment. This may indicate that there is a window of opportunity for physical therapists to increase patients’ engagement with vigorous-intensity PA. Future research should thus focus on interventions to optimize aerobic PA in axSpA patients without PT and on the imple- mentation of vigorous-intensity exercise in PT programs. Overall, the proportion of patients meeting the WHO PA recommendation in the current study was much higher than in previous studies, namely 82% as opposed to around 50% in previous studies [21, 23, 25]. It is conceivable that the discrepancy might be due to the use of the SQUASH questionnaire. Another recent Dutch study using the SQUASH questionnaire among the general population and people with osteoarthritis found even slightly higher proportions of participants meeting the combined-inten- sity PA recommendation [48]. Nevertheless, despite the probable overestimation of the amount of aerobic PA, the current results are useful to compare subgroups within a population; the SQUASH has indeed shown to be fairly valid and reliable for within group comparisons [39, 40, 49]. Therefore, the SQUASH can be regarded as a valid measure to investigate the main objective of this study; to compare moderate- and vigorous-intensity PA between Acknowledgements The authors wish to acknowledge and thank all participants from the three participating hospitals. The authors also thank the Dutch Arthritis Society for funding this study (grant number: BP 14-1-161) and their patient representatives for pilot-testing the sur- vey. Finally, the authors wish to thank Joséphine Peeters for her assis- tance in processing the data from the SQUASH questionnaire in SPSS. Funding Dutch Arthritis Society (ReumaNederland), grant number: BP 14-1-161. Conflict of interest Authors Bas Hilberdink, Thea Vliet Vlieland, Florus van der Giesen, Floris van Gaalen, Robbert Goekoop, Andreas PT and aerobic PA recommendations Funding Dutch Arthritis Society (ReumaNederland), grant number: BP 14-1-161. Conflict of interest Authors Bas Hilberdink, Thea Vliet Vlieland, Florus van der Giesen, Floris van Gaalen, Robbert Goekoop, Andreas 1 3 1526 Rheumatology International (2020) 40:1519–1528 for cardiovascular disease risk management in patients with rheu- matoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. Ann Rheum Dis 76(1):17–28. https://doi. org/10.1136/annrheumdis-2016-209775 Peeters, Marta Fiocco and Salima van Weely declare that they have no conflict of interest. Peeters, Marta Fiocco and Salima van Weely declare that they have no conflict of interest. Peeters, Marta Fiocco and Salima van Weely declare that they have no conflict of interest. Related congress abstract publication: We presented the data as a poster at the Annual European Congress of Rheumatology (EULAR 2019) and an abstract has therefore been published: Hilberdink, B., Van der Giesen, F., Vliet Vlieland, T., Van Gaalen, F., Ronday, K., Peeters, A., Van Weely, S. (2019). FRI0376 Differences in physical activity between axial spondyloarthritis patients with and without physical therapy. Annals of the Rheumatic Diseases, 78, 870–871. 9. 9. Al-Mallah MH, Sakr S, Al-Qunaibet A (2018) Cardiorespira- tory fitness and cardiovascular disease prevention: an update. Curr Atheroscler Rep 20(1):1. https://doi.org/10.1007/s1188 3-018-0711-4 10. Sassen B, Cornelissen VA, Kiers H, Wittink H, Kok G, Vanhees L (2009) Physical fitness matters more than physical activity in controlling cardiovascular disease risk factors. Eur J Cardiovasc Prev Rehabil 16(6):677–683. https://doi.org/10.1097/HJR.0b013 e3283312e94 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 11. 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https://openalex.org/W2577801124	https://europepmc.org/articles/pmc5398984?pdf=render	English	null	Modified Criteria for Radiographic Response Assessment in Glioblastoma Clinical Trials	Neurotherapeutics	2,017	cc-by	11,219	Neurotherapeutics (2017) 14:307–320 DOI 10.1007/s13311-016-0507-6 Neurotherapeutics (2017) 14:307–320 DOI 10.1007/s13311-016-0507-6 REVIEW REVIEW Modified Criteria for Radiographic Response Assessment in Glioblastoma Clinical Trials Benjamin M. Ellingson1,2,3,5 & Patrick Y. Wen4 & Timothy F. Cloughesy5,6 Published online: 20 January 2017 Published online: 20 January 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com pseudoresponse, and a confirmed durable response in newly diagnosed and recurrent glioblastoma trials. Abstract Radiographic endpoints including response and progression are important for the evaluation of new glioblas- toma therapies. The current RANO criteria was developed to overcome many of the challenges identified with previous guidelines for response assessment, however, significant chal- lenges and limitations remain. The current recommendations build on the strengths of the current RANO criteria, while addressing many of these limitations. Modifications to the current RANO criteria include suggestions for volumetric re- sponse evaluation, use contrast enhanced T1 subtraction maps to increase lesion conspicuity, removal of qualitative non- enhancing tumor assessment requirements, use of the post- radiation time point as the baseline for newly diagnosed glio- blastoma response assessment, and Btreatment-agnostic^ re- sponse assessment rubrics for identifying pseudoprogression, Keywords Glioblastoma .GBM .ResponseAssessment .T1 Subtraction . RANO * Benjamin M. Ellingson bellingson@mednet.ucla.edu Introduction Approximately 89,000 new primary brain tumors are diag- nosed in the United States each year, for which 27% are glio- mas and 32.8% are malignant [1]. Glioblastoma (GBM) oc- curs in approximately 46% of gliomas [1] and has a poor prognosis of around 14 months median survival [2] and less than 10% of patients live longer than 5 years from diagnosis [3]. The current standard of care for newly diagnosed GBM patients consists of maximum safe surgical resection followed by external beam radiation therapy plus concomitant and ad- juvant temozolomide [2], particularly in patients that demon- strate O6-methylguanine-methyltransferase (MGMT) pro- moter methylation. At recurrence there is no consensus as to the standard of care as no therapeutic options have produced substantial survival benefit [4]. * Benjamin M. Ellingson bellingson@mednet.ucla.edu 1 UCLA Brain Tumor Imaging Laboratory, Center for Computer Vision and Imaging Biomarkers, University of California Los Angeles, 924 Westwood Blvd., Suite 615, Los Angeles, CA 90024, USA Although overall survival (OS) is the standard for deter- mining GBM treatment efficacy, using OS as an endpoint when studying new therapeutic strategies can be problematic because of potential influence of therapies prior to or subse- quently following the therapy being studied. For example, it is difficult to definitively conclude that bevacizumab has no ef- ficacy in GBM when a large percentage of patients in the placebo arms in both III trials studying efficacy of bevacizumab (i.e. AVAglio and RTOG-0825) eventually crossed over and received bevacizumab (31% in AVAglio [5] and 48% in RTOG-0825 [6]). If bevacizumab increased OS when given at any time during treatment, we may expect 2 Department of Radiological Sciences, University of California Los Angeles, Los Angeles, CA, USA 3 Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA 4 Center for Neuro-Oncology, Dana-Farber/Brigham and Women’s Cancer Center, Harvard Medical School, Boston, MA, USA 5 UCLA Neuro-Oncology Program, University of California Los Angeles, Los Angeles, CA, USA 6 Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA 308 Ellingson et al. significantly improving upon the Levin criteria [19] and the WHO oncology response criteria [20]. Known Limitations for Current Response Criteria Although contrast enhancement has been used to assess brain tumor response for more than 60 years and contrast enhancement is generally a strong surrogate of brain tumor disease, there are caveats and exceptions that have been discovered as a result of different treatment mechanisms that affect vascular permeability. For example, increased vascular permeability from cytotoxic therapies including radiotherapy and anti-neoplastic treatments have been shown to result in increased contrast enhancement in the context of therapeutic benefit, a phenomena known as Bpseudoprogression.^ Additionally, clinical studies exam- ining the efficacy of new anti-angiogenic agents have noticed a substantial decrease in contrast enhancement [22–31] resulting in high response rates, ranging from 28 to 63% in bevacizumab [32–34] and 50% in cediranib [31] compared with < 10% using other chemotherapies [35–38], which translated into prolonged PFS but no difference in OS [31, 32]. It was assumed this high response rate was due to the use of contrast enhancement as the primary tool for evaluation in the Macdonald criteria, which resulted in a Bpseudoresponse^[39], where contrast enhancement is falsely reduced due to changes in vascular permeability independent of anti-tumor effect. The goal of this modified response criteria is to mean- ingfully evaluate radiographic response and progression while simultaneously allowing therapies that may have transient effects on contrast enhancement but therapeutic benefit to be treated equally. This is particularly important in the context of platform trials, where many different therapies may be compared against a common control and there is a significant risk of over or under estimating tumor burden with a single evaluation time point. By allowing patients to stay on therapy longer, a more com- prehensive and accurate assessment of therapeutic benefit can be performed on retrospective examination. A univer- sal set of principles and guidelines, rather than treatment- specific response criteria, may allow us to fully under- stand the possible therapeutic benefits and potential limi- tations of promising new therapies for patients with GBM. Introduction By standardizing the definition of radiographic response using quantitative bidirec- tional measurements and accounting for corticosteroid use in neurological status, similar to the response evaluation criteria in solid tumors (RECIST) [21], the new BMacdonald criteria^ utilized measurements of contrast enhancing tumor size com- bined with other clinical metrics to determine treatment re- sponse and tumor progression by stratifying response into four categories: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The origi- nal Macdonald criteria continues to be the fundamental frame- work for response assessment and radiographic interpretation of treatment changes in neuro-oncology, having been used for more than 20 years. both treatment arms to have similar median OS since most patients eventually were treated with bevacizumab, disguising any therapeutic effects of the drug. Together, these results suggest OS may not be a suitable endpoint when studying new therapeutics or when there is a high chance of cross over in the control arm. To overcome the limitations associated with using OS as the primary endpoint in studies involving new therapeutics, progression-free survival (PFS) and objective response rate (ORR) should be considered important end points [7]. However, PFS and ORR also have challenges, as determina- tion of response and progression using anatomic imaging techniques may suffer from issues associated with measure- ment variability and discordance in interpretation between ra- diologists [8]. Therefore, it is important to develop both new response guidelines for identifying these issues as well as new imaging tools for better differentiating treatment-related changes from changes associated with non-responsive, grow- ing tumor. Brief History of Radiologic Response Assessment in GBM The formation of new blood vessels, or angiogenesis, is critical for the growth of malignant brain tumors [9–11]. Malignant gliomas with high neovascularity or vascular permeability [12–14] are often associated with higher proliferation rates [15] and higher degree of aggressivity. Because of this associ- ation, imaging techniques aimed at identifying abnormal vas- cularity or vascular permeability, including contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used for diagnosis and clinical manage- ment of brain tumors, as they have been shown to contain the most aggressive portions of the tumor [16, 17]. In addition to increased response rates, studies examining tumor relapse/progression while on anti-angiogenic agents note a tendency for growth of nonenhancing, infiltrative tumor prior to emergence of contrast enhancement [25]. Approximately 30-40% of patients are estimated to experi- ence non-enhancing tumor progression prior to changes in contrast enhancement [40, 41]. Malignant gliomas are known to contain proportions of both neovascularized and infiltrative tumor [42, 43] and the relative proportions are thought to reflect different biological phenotypes [44–48]. In 2010, ex- pert opinion and examination of these limitations resulted in In 1990, Macdonald et al. [18] introduced the first radio- graphic response assessment specific to brain tumors by Modified RANO Criteria for Glioblastoma 309 the creation of a formal Response Assessment in Neuro- Oncology (RANO) criteria [49] to comprehensively reform the Macdonald criteria using previously documented perspec- tives and approaches [50–52]. integrated as an early radiographic endpoint. Further, new immunotherapy agents can also cause inflammation lead- ing to changes in T2 signal intensity that is ambiguous with regard to interpretation of changes in tumor biology. Although the RANO criteria corrects for a number of insufficiencies identified in the Macdonald criteria includ- ing inclusion of the evaluation of nonenhancing tumor pro- gression and issues associated with pseudoresponse and pseudoprogression, there remain significant limitations to the current standard RANO criteria given recent data. For example, the current RANO criteria requires use of bidi- rectional measurements of contrast enhancing tumor size, which have been shown to overestimate tumor volume [53] and result in higher reader discordance [8, 54–59], presum- ably due to differences in head tilt and accurate identifica- tion of longest and perpendicular diameter in relatively irregular tumors. Image Acquisition Requirements In response to a need for better standardization of image acquisition in GBM clinical trials [64], a recent consensus paper was published outlining an Binternational brain tu- mor imaging protocol (BTIP)^ (Table 1) with recom- mended sequences and parameters [65]. At the core of this recommended protocol is parameter matched, pre- and post-contrast 3D (volumetric) inversion recovery gra- dient recalled echo (IR-GRE) images with less than 1.5- mm isotropic resolution, which allows for both bidimensional and volumetric measurements of enhancing tumor. When possible, this protocol should be employed for prospective clinical trials. If volumetric acquisition is not employed, or if retrospec- tive evaluations of existing trial data are performed, then slice thickness plus interslice gap should be less than 5 mm. If the sum of the slice thickness and gap exceeds 5 mm, then slightly modified definitions of measurable disease should be used (e.g. measurable disease = largest perpendicular diame- ters > 2× slice thickness + gap). Brief History of Radiologic Response Assessment in GBM Other studies have shown reasonable agreement between bidimensional and volumetric mea- surements [60, 61], suggesting quick bidimensional assess- ment of contrast enhancing tumor size may be a practical alternative to more sophisticated volumetric segmentation. Additionally, the thresholds used to define response and progression is relatively arbitrary and not optimized based on scientific data showing the best correlation with surviv- al benefit or time to treatment failure. (Note: The efficacy of these thresholds remains to be sufficiently challenged). Also, the use of thresholds based on Bpercentage change^ with respect to baseline tumor size are significantly biased toward small tumors where relatively low absolute changes in tumor size are interpreted as a large percentage change [61]. This is particularly an issue in newly diagnosed GBM studies, where patients with tiny tumors often progress ear- ly due to triggering of progression (PD) when Bnon-mea- surable disease^, defined as having the two largest perpen- dicular diameters of a contrast enhancing target lesion less than 10mm, reaches the subtle threshold of Bmeasurable disease^. Lastly, although changes in non-enhancing disease were added to the RANO criteria in an attempt to identify non-enhancing tumor progression, particularly in the presence of anti-angiogenic therapy, retrospective eval- uations in clinical trials have shown it results in PD approximately a month prior to contrast enhancing disease progression [62], does not result in significant differences in prediction of OS [62, 63], and is one of the most controversial aspects of RANO evaluation due to the subjective nature of the interpretation and high adjudica- tion rates. Further, studies have shown that specific aspects of non-enhancing tumor progression (e.g. circumscribed vs. infiltrative T2 changes) result in dramatically different post-progression survival in GBM patients [41], suggest- ing evaluation of non-enhancing tumor progression using T2 and/or FLAIR may be more complex than once thought Updated Strategies for Response Assessment in Neuro-Oncology: Modified RANO Criteria Based on these various challenges, an update to the current response criteria is necessary in an attempt to establish a gen- eral framework for response assessment in neuro-oncology that is agnostic to the mechanism of action of the particular therapy (e.g. anti-angiogenic, anti-neoplastic, immunotherapy, etc.), each of which has its own challenges associated with interpretation of radiographic changes, and is updated based on recent scientific evidence and current clinical convention. In order to advance the RANO criteria and address these chal- lenges we propose the following Bmodified^ RANO criteria for use in evaluating therapeutic efficacy in patients with GBM. Contrast Enhanced T1-Weighted Digital Subtraction Maps for Increased Lesion Conspicuity Table 1 International Standardized Brain Tumor Imaging Protocol (BTIP) minimum image acquisition requirements for 1.5T and 3T MR systems Variable 3D T1w Preb Ax 2D FLAIRj Ax 2D DWI Contrast Injectiona Ax 2D T2wh,i 3D T1w Postb Sequence IR-GREe,f TSEc SS-EPIg TSEc IR-GREe,f Plane Sagittal/Axial Axial Axial Axial Sagittal/Axial Mode 3D 2D 2D 2D 3D TR [ms] 2100m >6000 >5000 >2500 2100m TE [ms] Min 100-140 Min 80-120 Min TI [ms] 1100n 2000-2500k 1100n Flip angle 10°-15° 90°/≥160° 90°/180° 90°/≥160° 10°-15° Frequency ≥172 ≥256 ≥128 ≥256 ≥172 Phase ≥172 ≥256 ≥128 ≥256 ≥172 NEX ≥1 ≥1 ≥1 ≥1 ≥1 FOV 256mm 240mm 240mm 240mm 256mm Slice thickness ≤1.5mm ≤4mml ≤4mml ≤4mml ≤1.5mm Gap/Spacing 0 0 0 0 0 Diffusion optionsp b = 0, 500, 1000 s/mm2 ≥3 directions Parallel imaging Up to 2x Up to 2x Up to 2x Up to 2x Up to 2x Approximate scan time 5-10 min 4-8 min 2-4 min 4-8 min 5-10 min Ax = Axial; ADC = apparent diffusion coefficient; FLAIR = fluid attenuated inversion recovery; DWI = diffusion-weighted imaging; 3D = three dimen- sional; TSE = turbo spin echo; EPI = echo planar imaging; SS-EPI = single-shot echo planar imaging; GE-EPI = gradient echo echo planar imaging; dardized Brain Tumor Imaging Protocol (BTIP) minimum image acquisition requirements for 1.5T and 3T MR systems Ax = Axial; ADC = apparent diffusion coefficient; FLAIR = fluid attenuated inversion recovery; DWI = diffusion-weighted imaging; 3D = three dimen- sional; TSE = turbo spin echo; EPI = echo planar imaging; SS-EPI = single-shot echo planar imaging; GE-EPI = gradient echo echo planar imaging; 2DFL = two-dimensional FLASH (fast low angle shot) gradient recalled echo; MPRAGE = magnetization prepared rapid gradient-echo; A/P = anterior to posterior; R/L = right to left; NEX = number of excitations or averages; FOV = field of view; TE = echo time; TR = repetition time; TI = inversion time; PD = proton density; DSC = dynamic susceptibility contrast; IR-GRE = inversion-recovery gradient-recalled echo b Post-contrast 3D T1-weighted images should be collected with equivalent parameters to pre-contrast 3D T1-weighted images d FL2D = two-dimensional fast low angle shot (FLASH; Siemens) is equivalent to the spoil gradient recalled echo (SPGR; GE) or T1- fast field echo (FFE; Philips), fast field echo (FastFE; Toshiba), or the radiofrequency spoiled steady state acquisition rewound gradient echo (RSSG; Hitachi). Contrast Enhanced T1-Weighted Digital Subtraction Maps for Increased Lesion Conspicuity A fast gradient echo sequence without inversion preparation is desired e IR-GRE = inversion-recovery gradient-recalled echo sequence is equivalent to MPRAGE = magnetization prepared rapid gradient-echo (Siemens & Hitachi) and the inversion recovery spoiled gradient-echo (IR-SPGR or Fast SPGR with inversion activated or BRAVO; GE), 3D turbo field echo (TFE; Philips), or 3D fast field echo (3D Fast FE; Toshiba) e IR-GRE = inversion-recovery gradient-recalled echo sequence is equivalent to MPRAGE = magnetization prepared rapid gradient-echo (Siemens & Hitachi) and the inversion recovery spoiled gradient-echo (IR-SPGR or Fast SPGR with inversion activated or BRAVO; GE), 3D turbo field echo (TFE; Philips), or 3D fast field echo (3D Fast FE; Toshiba) f A 3D acquisition without inversion preparation will result in different contrast compared with MPRAGE or another IR-prepped 3D T1-weighted sequences and therefore should be avoided f A 3D acquisition without inversion preparation will result in different contrast compared with MPRAGE or another IR-prepped 3D T1-weighted sequences and therefore should be avoided g In the event of significant patient motion, a radial acquisition scheme may be used (e.g. BLADE [Siemens], PROPELLER [GE], MultiVane [Philips], RADAR [Hitachi], or JET [Toshiba]); however, this acquisition scheme is can cause significant differences in ADC quantification and therefore should be used only if EPI is not an option. Further, this type of acquisition takes considerably more time g In the event of significant patient motion, a radial acquisition scheme may be used (e.g. BLADE [Siemens], PROPELLER [GE], MultiVane [Philips], RADAR [Hitachi], or JET [Toshiba]); however, this acquisition scheme is can cause significant differences in ADC quantification and therefore should be used only if EPI is not an option. Further, this type of acquisition takes considerably more time Dual echo PD/T2 TSE is optional for possible quantification of tissue T2. Contrast Enhanced T1-Weighted Digital Subtraction Maps for Increased Lesion Conspicuity Quantification of contrast enhancing tumor size or volume should be performed on contrast-enhanced T1-weighted digi- tal subtraction maps (Fig. 1) in order to increase lesion con- spicuity and better predict tumor burden in the presence of reduced vascular permeability as occurs during anti- angiogenic therapy [66] and/or T1 shortening from blood 310 Ellingson et al. Contrast Enhanced T1-Weighted Digital Subtraction Maps for Increased Lesion Conspicuity For this sequence, the PD echo is recommended to have a TE < 25ms i Advanced sequences can be substituted into this time slot, so long as 3D post-contrast T1-weighted images are collected between 4 and 8 min after contrast injection i Advanced sequences can be substituted into this time slot, so long as 3D post-contrast T1-weighted images are collected between 4 and 8 min after contrast injection j 3D FLAIR is an optional alternative to 2D FLAIR, with sequence parameters as follows per EORTC guidelines: 3D TSE/FSE acquisition; TE = 90- 140ms; TR = 6000-10000ms; TI = 2000-2500ms (chosen based on vendor recommendations for optimized protocol and field strength); GRAPPA ≤2; Fat Saturation; Slice thickness ≤1.5mm; Orientation Sagittal or Axial; FOV ≤250 mm x 250 mm; Matrix ≥244x244 j 3D FLAIR is an optional alternative to 2D FLAIR, with sequence parameters as follows per EORTC guidelines: 3D TSE/FSE acquisition; TE = 90- 140ms; TR = 6000-10000ms; TI = 2000-2500ms (chosen based on vendor recommendations for optimized protocol and field strength); GRAPPA ≤2; Fat Saturation; Slice thickness ≤1.5mm; Orientation Sagittal or Axial; FOV ≤250 mm x 250 mm; Matrix ≥244x244 hould be chosen based on the magnetic field strength of the system (e.g. TI ≈2000ms for 1.5T and TI ≈2500ms for 3T) k Choice of TI should be chosen based on the magnetic field strength of the system (e.g. TI ≈2000ms for 1.5T and TI ≈2500ms for 3T) l k Choice of TI should be chosen based on the magnetic field strength of the system (e.g. TI ≈2000ms for 1.5T and TI ≈2500ms for 3T) l In order to ensure comparable SNR older 1.5T MR systems can use contiguous (no interslice gap) images with 5mm slice thickness or increase NEX for k Choice of TI should be chosen based on the magnetic field strength of the system (e.g. TI ≈2000ms for 1.5T and TI ≈2500ms for 3T) l In order to ensure comparable SNR older 1.5T MR systems can use contiguous (no interslice gap) images with 5mm slice thickness or increase NEX for slice thickness ≤4mm k Choice of TI should be chosen based on the magnetic field strength of the system (e.g. Bidimensional and/or Volumetric Measurements Similar to the current RANO criteria, two-dimensional, per- pendicular measurements of contrast enhancing tumor size, excluding the resection cavity along with any cysts or areas of central macroscopic necrosis, should be used for response assessment if volumetric tools are not available. Table 2 out- lines suggested volumetric conversions from two- to three- dimensional measurements for consistency in response defi- nitions, as outlined by Chappell et al. [70]. It is important to note that the field remains conflicted on whether or not enhancing disease should be included in tumor size measurements, or whether it is more appropriate to mon- itor total enhancing lesion volume, which may include central macroscopic necrosis and any cystic components (but exclud- ing surgically resected tissue). Scientific studies have shown that both approaches for quantifying change in tumor size as a surrogate of treatment response are valuable. Multiple studies utilizing the Macdonald and RANO criteria have shown that change in enhancing disease size using bidimensional mea- surements, excluding necrosis and cystic components, can be used to predict survival in a variety of therapies. A recent study from the BRAIN trial, a phase II trial of bevacizumab with or without irinotecan in recurrent GBM, confirmed that change in the volume of enhancing disease can be used to predict survival benefit [66]. However, a recent study exam- ining growth rates in treatment naïve presurgical GBMs showed that changes in enhancing disease only may not be Contrast Enhanced T1-Weighted Digital Subtraction Maps for Increased Lesion Conspicuity TI ≈2000ms for 1.5T and TI ≈2500ms for 3T) l In order to ensure comparable SNR older 1.5T MR systems can use contiguous (no interslice gap) images with 5mm slice thickness or increase NEX for slice thickness ≤4mm l In order to ensure comparable SNR older 1.5T MR systems can use contiguous (no interslice gap) images with 5mm slice thickness or increase NEX for slice thickness ≤4mm For Siemens and Hitachi scanners. GE, Philips, and Toshiba scanners should use a TI = 400-450ms for similar contrast m i d i hi hili d hib h ld f i il For Siemens and Hitachi scanners. GE, Philips, and Toshiba scanners should use a TI = 400-450ms for similar contrast m For Siemens and Hitachi scanners. GE, Philips, and Toshiba scanners should use a TR = 5-15ms for similar contrast m For Siemens and Hitachi scanners. GE, Philips, and Toshiba scanners should use a TR = 5-15ms for similar contrast identification and delineation of subtly enhancing bone and soft tissue lesions [69]. products or calcifications [67, 68]. Further, the American College of Radiology (ACR) recommends this approach for products or calcifications [67, 68]. Further, the American College of Radiology (ACR) recommends this approach for identification and delineation of subtly enhancing bone and soft tissue lesions [69]. 311 Modified RANO Criteria for Glioblastoma Fig. 1 Construction of contrast enhanced T1-weighted subtraction maps in a recurrent glioblastoma patient treated with bevacizumab. A) Pre-contrast T1- weighted MR image. B) Post- contrast T1-weighted MR image. C) T1 subtraction map calculated by voxel-wise subtraction of pre- contrast from post-contrast T1- weighted images highlighting areas of increased contrast enhancement. Red arrows show two subtly enhancing lesions that are easily identified on T1 subtraction maps Post-Radiation MRI Examination as the Reference for Evaluating Radiographic Response in Newly Diagnosed GBM Post-Radiation MRI Examination as the Reference for Evaluating Radiographic Response in Newly Diagnosed GBM The current RANO criterion defines the post-surgical MRI scan as the baseline for treatment response evaluation; however, we propose using the post-radiation examination (i.e. the first scan following completion of concurrent radiation therapy and che- motherapies such as temozolomide and/or experimental thera- peutics) as the baseline for response assessment because reli- ability of tumor assessment on the post-surgical MR scans can be problematic for a number of reasons. First, this scan is typ- ically acquired prior to a final pathological diagnosis, thus pa- tients are not yet enrolled in a clinical trial and therefore the imaging protocol may not be consistent with trial recommen- dations, leading to a mismatch between the baseline and sub- sequent follow-up time points. Secondly, post-operative MR scans are often contaminated with post-surgical changes in- cluding blood products and increased vascular permeability from surgical trauma. Thirdly, steroid dose can be highly var- iable during this time and may be poorly annotated, as patients are typically not yet enrolled in clinical trials at this point. Additionally, the timing of the post-operative MR scans can be highly variable from patient to patient, depending on the complexity of the surgery and potential intraoperative compli- cations, and institution by institution, as many factors including availability of inpatient MR scanners can lead to different timing of the post-surgical MRI evaluation. This variability inevitably leads to differing degrees of post-surgical artifacts and fluid levels on the resulting images. Together, these factors appear to indicate the post-surgical MRI examination may not be a reliable reference scan for accurately determining radio- graphic changes, despite post-surgical residual enhancing vol- ume being a significant prognostic factor as outlined above. Fig. 2 Algorithm for identifying measurable and target lesions Non-measurable disease should be defined as lesions that are too small to be measured (less than 1 cm in both perpen- dicular dimensions), lesions that lack contrast enhancement (non-enhancing disease), or lesions that contain a poorly de- fined margin that cannot be measured or segmented with confidence. Definition of Measurable Disease, Non-Measurable Disease, and Target Lesions Measurable disease should be defined as contrast enhancing lesions with a minimum size of both perpendicular measure- ments greater than or equal to 10mm (Fig. 2). For example, if the largest diameter is 15 mm but the perpendicular diameter is 8 mm, this would constitute non-measurable disease. Additionally, in the event that the BTIP protocol is not used, if the slice thickness plus interslice gap is greater than 5mm, then the minimum size for both perpendicular measurements should be twice the sum of the slice thickness and interslice gap (e.g. if the slice thickness is 5mm with 1.5mm interslice gap, the minimum tumor size on both perpendicular dimen- sions should be 13 mm). Up to a total of five target measurable lesions should be defined and ranked from largest to smallest (Fig. 2). State of disease Change in bidimensional product Estimated volumetric change Complete response (CR) 100% Decrease 100% Decrease Partial response (PR) ≥50% Decrease ≥65% Decrease Progressive disease (PD) ≥25% Increase ≥40% Increase Stable disease (SD) <50% Decrease to <25% Increase <65% Decrease to <40% Increase 312 Ellingson et al. Fig. 2 Algorithm for identifying measurable and target lesions Correction for BBaseline Tumor Volume^ in Newly Diagnosed and Recurrent GBM Perhaps the most compelling argument for using the post- radiation scan as the baseline for determining response assess- ment is the highly unpredictable, transient radiographic changes that often accompany the initial chemoradiation phase (i.e. ex- ternal beam radiation therapy plus concurrent temozolomide) with or without experimental therapeutics. Within 1 month after completion of standard chemoradiation therapy, approximately 50% of patients will experience radiographic changes sugges- tive of early tumor progression in reference to the post-surgical MRI exam, of which 50% are likely to have pseudoprogression (i.e. 25% of all patients at 1 month post-chemoradiation are estimated to have pseudoprogression) [94]. This proportion of patients with both early progression and pseudoprogression de- creases steadily during the subsequent standard adjuvant che- motherapy phase, which forms the basis for current RANO recommendations of excluding patients in recurrent GBM trials who progressed within 3 months after completion of chemora- diation. Many clinicians are reluctant to change therapy based on this examination due to the relatively high incidence of An abundance of single center, multicenter, and phase I-III trials have confirmed that baseline contrast enhancing tumor size (volume or bidirectional measurements) is a significant prognostic factor contributing to overall survival (OS) in GBM. In newly diagnosed GBM, both extent of resection [3, 71–87] and post-surgical residual volume [83–85, 88–92] have been shown to be prognostic. Similarly, baseline pre- treatment contrast enhanced tumor size has also been shown to be prognostic for OS in recurrent GBM [53, 66, 93]. However, from a clinical trial perspective, post-surgical resid- ual enhancing tumor volume may be a more practical mea- surement to obtain, as pre-surgical MRI scans are often not available or collected as part of clinical trials because patients are not enrolled until after surgery and diagnosis. Thus, care should be made to make sure baseline tumor size is a stratifi- cation factor during randomization (i.e. prospectively bal- anced across treatment arms) and used as a covariate in statis- tical models evaluating treatment efficacy. Modified RANO Criteria for Glioblastoma 313 and the patient should continue on therapy until confirmed PD is observed. treatment-related radiographic changes directly after comple- tion of concurrent chemotherapy and radiation, and instead use this scan as a new baseline in which to interpret subsequent changes in tumor size. Detailed Definitions Used for Modified Radiographic Response Assessment Criteria Radiographic response should be determined in comparison to the tumor measurements obtained at baseline (post-radiation scan will be baseline for newly diagnosed GBM and pre- treatment scans will be the baseline for recurrent GBM) for determination of response, and the smallest tumor measure- ment at either pre-treatment baseline or following initiation of therapy for determining progression. Because novel treatments are likely to result in a higher than normal incidence of treatment-related increase in contrast enhancement (Bpseudoprogression^, PsP) or decrease in con- trast enhancement (Bpseudoresponse^, PsR), patients should continue therapy with close observation (e.g. 4-8 week inter- vals) if there is a suspicion of PsP or PsR. If subsequent im- aging studies and/or clinical observations demonstrate that progression in fact has occurred, the date of confirmed pro- gression should be noted as the scan at which the potential progression was first identified. Definitions for complete re- sponse, partial response, progressive disease, and stable dis- ease should be defined as follows for all target lesions. 2. Steroid dose should be the same or lower compared with baseline scan. 3. Stable or improved clinical assessments. Note: Patients with non-measurable disease only at base- line cannot have PR; the best response possible is stable dis- ease (SD). Progressive Disease (PD): Defined by any of the following: 1. At least two sequential scans separated by at ≥4 weeks both exhibiting ≥25% increase in sum of products of per- pendicular diameters or ≥40% increase in total volume [54, 70, 96] of enhancing lesions. The first scan exhibiting ≥25% increase in sum of products of perpendicular diam- eters or ≥40% increase in total volume [54, 70, 96] of enhancing lesions should be compared to the smallest tumor measurement obtained either at baseline (if no de- crease) or best response (on stable or increasing steroid dose) and is noted as Bpreliminary PD.^ If the second scan at least 4 weeks later exhibits a subsequent ≥25% increase in sum of products of perpendicular diameters or ≥40% increase in total volume of enhancing lesions relative to the Bpreliminary PD^ scan, it is considered Bconfirmed PD^ and the patient should discontinue therapy. If the second scan at least 4 weeks later exhibits SD or PR/ CR, this scan showing Bpreliminary PD^ is noted as 1. Correction for BBaseline Tumor Volume^ in Newly Diagnosed and Recurrent GBM Additionally, experimental therapeutics that significantly alter vascular permeability, including anti- angiogenic and immunotherapies, when used concurrently with radiation therapy and temozolomide often demonstrate dramat- ic and transient changes in contrast enhancement that quickly stabilize following completion of radiation [95]. Despite the improved lesion conspicuity on T1 subtraction maps in the settings of these therapies, these early changes between the post-surgical, pre-radiation exam and the post-radiation exam may not accurately reflect true changes in tumor burden nor predict long-term survival benefit [95]. 2. Patients must be off corticosteroids (or on physiologic replacement doses only). 3. Stable or improved clinical assessments (i.e. neurological examinations). Note: Patients with non-measurable disease only at base- line cannot have CR; the best response possible is stable dis- ease (SD). Partial Response (PR): Requires all of the following: 1. ≥50% decrease in sum of products of perpendicular diam- eters or ≥65% decrease in total volume [54, 70, 96] of all measurable enhancing lesions compared with baseline, sustained for at least 4 weeks. The first scan exhibiting ≥50% decrease in sum of products of perpendicular diam- eters or ≥65% decrease in total volume [54, 70, 96] of all measurable enhancing lesions compared with baseline is considered Bpreliminary PR^. If the second scan exhibits PD with respect to the Bpreliminary PR^ scan, then the response is not sustained, noted as pseudoresponse, PsR, and is now considered Bpreliminary PD^ (note confirmed PD requires at least two sequential increases in tumor volume). If the second scan exhibits SD, PR, or CR, it is considered a durable PR and the patient should continue on therapy until confirmed PD is observed. Symptomatic Deterioration & Reporting Clinical Status Bpseudoprogression^, PsP, and the patient should contin- ue on therapy until a second increase in tumor size relative to the PsP scan is observed. Note that any new measurable (>10mm x 10mm) enhancing lesions should not be immediately considered PD, but instead should be added to the sum of bidimensional products or total vol- ume representing the entire enhancing tumor burden. Patients with global deterioration of health status requiring dis- continuation of treatment without objective evidence of disease progression at that time, and not either related to study treat- ment or other medical conditions, should be reported as PD due to Bsymptomatic deterioration.^ Every effort should be made to document the objective progression even after discontinuation of treatment due to symptomatic deterioration. Neurological exam data should be provided to the independent radiologic facility as Bstable, better, worse^ in case report forms or from study sponsor. Clinical status should be recorded as Bworse^ if the neurological exam is worse, otherwise the clinical status should be set to Bnot worse.^ In the event that necessary clinical data is not available, clinical status should be recorded as Bnot available^ and that particular time point can only be reviewed for PD (otherwise Bnon-evaluable^). Neurological data must be within ±7 days of the time-point response date, otherwise the data is considered Bnot available^. 2. p g g In the case where the baseline or best response demon- strates no measurable enhancing disease (visible or not visible), then any new measurable (>10mm x 10mm) en- hancing lesions are considered PD after confirmed by a subsequent scan ≥4 weeks exhibiting ≥25% increase in sum of products of perpendicular diameters or ≥40% in- crease in total volume of enhancing lesions [54, 70, 96] relative to the scan first illustrating new measurable dis- ease. The first scan exhibiting new measurable disease is noted as Bpreliminary PD.^ If the second scan at least 4 weeks later exhibits a subsequent ≥25% increase in sum of products of perpendicular diameters or ≥40% increase in total volume [54, 70, 96] of enhancing lesions relative to the Bpreliminary PD^ scan it is considered Bconfirmed PD^ and the patient should discontinue therapy. Stable Disease (SD): Requires all of the following: 1. Does not qualify for CR, PR, or PD as defined above. Note this also applies to patients that demonstrate PsR when the confirmation scan does not show PD or PsP when the confirmation scan does not show PR/CR. 1. Does not qualify for CR, PR, or PD as defined above. Note this also applies to patients that demonstrate PsR when the confirmation scan does not show PD or PsP when the confirmation scan does not show PR/CR. 2. In the event that corticosteroid dose was increased (for new symptoms/signs) without confirmation of disease progression on neuroimaging, and subsequent follow-up imaging shows that the steroid increase was required be- cause of disease progression, the last scan considered to show stable disease will be the scan obtained when the corticosteroid dose was equivalent to the baseline dose. Overall Objective Status The overall objective status for an evaluation should be deter- mined by combining the patient’s radiographic response on tar- get lesions, new disease, neurological status, and steroid dose/ usage as defined in Table 3 for patients with measurable (>10mm x 10mm) disease. Note that patients with possible PsP or pseudoresponse should be given the Objective Status of BPreliminary Progression^ or BPreliminary Response^, respec- tively. Once PsP, pseudoresponse, or true progression/response are confirmed, the Objective Status can be changed accordingly. 4. Failure to return for evaluation as a result of death or deteriorating condition. Symptomatic Deterioration & Reporting Clinical Status If the second scan at least 4 weeks later exhibits SD, CR, PR, or becomes non-measurable, this scan showing Bpreliminary PD^ is noted as Bpseudoprogression^, PsP, and the patient should continue on therapy until a second increase in tumor size relative to the Bpreliminary PD^, or PsP, scan is observed. Note that any new measurable (>10mm x 10mm) enhancing lesions on the subsequent scan following the preliminary PD scan should not be immediately considered confirmed PD, but instead should be added to the sum of bidimensional products or total volume representing the entire enhancing tumor burden. Steroid Use and Dose Steroid use should be derived from the concomitant medications on the case report forms and recorded as BYes^, BNo^, or Bnot available^. A value of BNo^ should be assigned if, at the time- point, the subject is not on steroids or on physiologic replacement doses only (<1.5 mg dexamethasone or equivalent per day). Steroid dose should be derived from the concomitant med- ications on the case report forms. Average steroid dose no greater than 2 mg change from baseline should be abstracted to Bstable^. If outside this range the steroid dose should be abstracted to Bincreased^ or Bdecreased^ accordingly. Steroid data should be within ±5 days of the time-point response date, otherwise the data is considered Bnot available^. 3. Clear clinical deterioration not attributable to other causes apart from tumor (e.g. seizures, medication adverse ef- fects, therapy complications, stroke, infection) or attribut- able to changes in steroid dose. Detailed Definitions Used for Modified Radiographic Response Assessment Criteria At least two sequential scans separated by at ≥4 weeks both exhibiting ≥25% increase in sum of products of per- pendicular diameters or ≥40% increase in total volume [54, 70, 96] of enhancing lesions. The first scan exhibiting ≥25% increase in sum of products of perpendicular diam- eters or ≥40% increase in total volume [54, 70, 96] of enhancing lesions should be compared to the smallest tumor measurement obtained either at baseline (if no de- crease) or best response (on stable or increasing steroid dose) and is noted as Bpreliminary PD.^ If the second scan at least 4 weeks later exhibits a subsequent ≥25% increase in sum of products of perpendicular diameters or ≥40% increase in total volume of enhancing lesions relative to the Bpreliminary PD^ scan, it is considered Bconfirmed PD^ and the patient should discontinue therapy. If the second scan at least 4 weeks later exhibits SD or PR/ CR, this scan showing Bpreliminary PD^ is noted as Complete Response (CR): Requires all of the following: 1. Disappearance of all enhancing measurable and non- measurable disease sustained for at least 4 weeks. The first scan exhibiting disappearance of all enhancing mea- surable and non-measurable disease is considered Bpreliminary CR^. If the second scan exhibits measurable enhancing disease with respect to the Bpreliminary CR^ scan, then the response is not sustained, noted as pseudoresponse, PsR, and is now considered Bpreliminary PD^ (note confirmed PD requires at least two sequential increases in tumor volume). If the second scan continues to exhibit disappearance of enhancing dis- ease or emergence of non-measurable disease (less than 10mm bidimensional product), it is considered a durable CR 1. Disappearance of all enhancing measurable and non- measurable disease sustained for at least 4 weeks. The first scan exhibiting disappearance of all enhancing mea- surable and non-measurable disease is considered Bpreliminary CR^. If the second scan exhibits measurable enhancing disease with respect to the Bpreliminary CR^ scan, then the response is not sustained, noted as pseudoresponse, PsR, and is now considered Bpreliminary PD^ (note confirmed PD requires at least two sequential increases in tumor volume). If the second scan continues to exhibit disappearance of enhancing dis- ease or emergence of non-measurable disease (less than 10mm bidimensional product), it is considered a durable CR 314 Ellingson et al. Symptomatic Deterioration & Reporting Clinical Status Detailed Modified Radiographic Response Assessment Rubric In order to provide both clinical guidelines for continuing therapy beyond suspected radiographic progression if the 315 Modified RANO Criteria for Glioblastoma Table 3 Guidelines for determining comprehensive objective status Target lesions (current scan) Target lesions (previous scan) New sites of measurable diseasea Neurological status Steroid usage Steroid dose Overall objective status CR Not Evaluated No Stable/Better No N/A Preliminary CR PR Not Evaluated No Stable/Better Any Stable/Decreasing Preliminary PR PD Not Evaluated Yes or No Stable/Better Any Stable/Increasing Preliminary PD PD Preliminary or Confirmed PR/CR No Stable/Better Any Stable/Increasing Preliminary PD SD Preliminary or Confirmed CR/PR or SD/NE No Stable/Better Any N/A SD PR Preliminary PR Yes or No Stable/Better Any Stable/Decreasing Confirmed PR SD Preliminary PR Yes or No Stable/Better Any Stable/Decreasing SD (Preliminary PR →Confirmed PR) SD Preliminary CR Yes or No Stable/Better Any Stable/Decreasing SD (Preliminary CR →Confirmed CR) CR Preliminary CR No Stable/Better No N/A Confirmed CR SD Preliminary PD No Stable/Better Any Stable/Decreasing SD (Confirmed PsP) CR/PR/SD PD/NE CR/PR/SD/PD/NE Yes or No Worse Any Stable/Increasing Confirmed PD PD Preliminary PD Yes or No Any Yes Stable/Increasing Confirmed PD a Note that new sites of measurable disease are added to the sum of bidimensional products or total lesion volume, or constitutes preliminary PD in the case of no measurable disease at baseline or best response Table 3 Guidelines for determining comprehensive objective status a Note that new sites of measurable disease are added to the sum of bidimensional products or total lesion volume, or constitutes preliminary PD in the case of no measurable disease at baseline or best response baseline for response evaluation in newly diagnosed GBM and (2) considering only objectively defined, measurable enhancing disease in the definition of response and pro- gression (i.e. exclusion of qualitatively assessed T2/FLAIR changes). treating physician believes there may be a therapeutic benefit and to provide criteria for defining progression and early drug failure while also allowing for the possi- bility of PsP and PsR, a modified response rubric similar to those described recently [97] should be employed. Two different rubrics should be used depending on whether the patient is newly diagnosed or enrolled in a trial for recur- rent disease. 1 Note the post-surgical, pre-RT scan is often corrupted with post-surgical blood products and other surgical changes. Further, it is difficult to standardize in terms of post-surgical timing, where delays in scanning often lead to in- creased T1 shortening on pre-contrast T1-weighted images due to blood prod- ucts, etc., and therefore may not be an adequate representation of baseline tumor burden. Newly Diagnosed GBM (Fig. 3) Newly diagnosed GBM patients will initially undergo a pre- entry MRI scan for initial diagnosis prior to entry in the study and prior to therapy. The post-operative scan [MRI(0)] is It is important to note that the primary differences between conventional RANO and the proposed modified criteria are: (1) use of the post-radiation time point as the Fig. 3 Modified radiographic response assessment rubric for management of both pseudoprogression and pseudoresponse in newly diagnosed glioblastoma Fig. 3 Modified radiographic response assessment rubric for management of both pseudoprogression and pseudoresponse in newly diagnosed glioblastoma Fig. 3 Modified radiographic response assessment rubric for management of both pseudoprogression and pseudoresponse in newly diagnosed glioblastoma 316 Ellingson et al. Fig. 4 Modified radiographic response assessment rubric for recurrent glioblastoma Fig. 4 Modified radiographic response assessment rubric for recurrent glioblastoma Details Common to Both Newly Diagnosed and Recurrent GBM desired in order to assess residual enhancing disease volume for use as a covariate in survival analyses, as described previ- ously. Patients will then start on standard or experimental therapy with concurrent radiation therapy (RT). The Post-RT scan [MRI(1)] will be required and used as the baseline scan for which response will be determined.1 Following the first cycles of adjuvant therapy, patients will receive additional required MRI scans [MRI(N)]. Preliminary Radiographic Progression If the lesion size has increased ≥25% sum of bidirectional product or ≥40% in vol- ume between MRI Scan 1 and N, these patients should be categorized as Bpreliminary radiographic progression^. If the investigator believes the patient can safely continue on thera- py, then they should continue to treat and acquire a follow-up confirmatory scan [MRI(N + 1)] at the next scan interval (8 weeks ± 4 weeks from MRI Scan (N) or no less than 4 weeks minimum duration between preliminary PD and confirmed PD scans) to verify tumor growth and progression. For pa- tients with gross-total resection (GTR) and no measurable enhancing disease, preliminary radiographic progression is defined as a transition from no measurable disease to non- measureable (but present) disease (<10mm x 10mm) or mea- surable disease (>10mm x 10mm). If the investigator feels it is safe to keep the patient on, a confirmatory scan at MRI(N + 1) should be obtained to verify tumor progression. References 1. Ostrom QT, Gittleman H, Fulop J et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2008-2012. Neuro Oncol, 17 Suppl 4, iv1-iv62 (2015). 2. Stupp R, Mason WP, van den Bent MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 352(10), 987-996 (2005). 3. Stupp R, Hegi ME, Mason WP et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol, 10(5), 459-466 (2009). 4. Weller M, Cloughesy T, Perry JR, Wick W. Standards of care for treatment of recurrent glioblastoma—are we there yet? Neuro Oncol, 15(1), 4-27 (2013). Stable Disease If the lesion size has not increased or de- creased beyond the set thresholds between Scan 1 and N, the patient is considered Bstable.^ Such patients will con- tinue on therapy, and the date of preliminary progression is the time point of an increase ≥25% sum of bidirectional product or ≥40% in volume (from Nadir) during the re- mainder of the study. Upon preliminary progression the investigator can choose to either continue therapy and confirm progression or discontinue therapy. For cases with significant neurologic decline at the time of imaging progression as determined from MRI(N), a confirmatory scan at time point MRI(N + 1) may not be possible or neces- sary. For these cases, it is appropriate to define MRI(N) as the progression time point. 5. Chinot OL, Wick W, Mason W et al. Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma. N Engl J Med, 370(8), 709-722 (2014). 6. Gilbert MR, Dignam JJ, Armstrong TS et al. A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med, 370(8), 699-708 (2014). 7. Lamborn KR, Yung WK, Chang SM et al. Progression-free surviv- al: an important end point in evaluating therapy for recurrent high- grade gliomas. Neuro Oncol, 10(2), 162-170 (2008). 8. Provenzale JM, Ison C, Delong D. Bidimensional measurements in brain tumors: assessment of interobserver variability. AJR Am J Roentgenol, 193(6), W515-522 (2009). 9. Russell SM, Elliott R, Forshaw D, Golfinos JG, Nelson PK, Kelly PJ. Glioma vascularity correlates with reduced patient survival and increased malignancy. Surg Neurol, 72(3), 242-246; discussion 246-247 (2009). 10. Leon SP, Folkerth RD, Black PM. References Microvessel density is a prog- nostic indicator for patients with astroglial brain tumors. Cancer, 77(2), 362-372 (1996). Recurrent GBM (Fig. 4) Recurrent GBM patients will undergo a pre-entry MRI scan [MRI(0)] at the time of recurrence. At the time of study entry, two scans to confirm progression should be submitted consisting of at least one scan at the time of progression and one scan at Nadir or baseline. If the pa- tient undergoes surgery (optional), then the post-surgical, pre-treatment MRI can be used as the baseline [MRI(1)], assuming it is obtained < 72 hours from surgery to reduce post-operative reactive enhancement [91, 98]. (Note: If the post-operative MRI scan is used as the baseline refer- ence, the standardized MRI protocols must be used.) If the patient does not go to surgery or if the start of treatment is > 21 days from the start of therapy, the patient will undergo a pre-treatment MRI [MRI(1)] scan as the base- line scan for which response will be determined. Following the first cycles of therapy, patients will receive additional MRI scans [MRI(N)]. Confirmed Progression If the patient has an increase ≥25% sum of bidirectional product or ≥40% in volume between MRI Scan N and N + 1, this is BConfirmed Progression^, the pa- tient should stop therapy and the date of radiographic progres- sion is the date of suspected progression, MRI(N). If the pa- tient has SD/PR/CR on MRI(N + 1) with respect to MRI(N), PsP is confirmed and the patient should continue on therapy. Patients will then continue on therapy and receive additional follow-up MRI scans [MRI(M)]. If the lesion size has in- creased ≥25% sum of bidirectional product or ≥40% in vol- ume on MRI(M) relative to the smaller of Nadir or MRI(N + 1), then the patient has BConfirmed Progression^, the patient should stop therapy and the date of radiographic progression is the new date, MRI(M). For patients with no measurable disease at the Post-RT baseline, BConfirmed Progression^ will be defined as a transition from non-measurable (but present) 317 Modified RANO Criteria for Glioblastoma disease (<10mm x <10mm) on MRI(N) to measurable disease (>10mm x 10mm) on MRI(N + 1). For patients with confirmed PsP and no measurable disease at Nadir, BConfirmed Progression^ should be defined as a transition from no mea- surable disease to measurable disease (>10mm x 10mm). In all cases, patients with confirmed progression should stop therapy. efficacy of new treatments in GBM. Compliance with Ethical Standards Funding American Cancer Society (ACS) Research Scholar Grant (RSG-15-003-01-CCE) (Ellingson); National Brain Tumor Society (NBTS) Research Grant (Ellingson, Cloughesy); Art of the Brain (Cloughesy); Ziering Family Foundation in memory of Sigi Ziering (Cloughesy); Singleton Family Foundation (Cloughesy). Preliminary & Confirmed Radiographic Response If a measurable lesion has decreased ≥50% sum of bidirectional product or ≥65% in volume between MRI(1) and MRI(N), these patients should be categorized as Bpreliminary radio- graphic responders^ and will be monitored for an additional time point and/or treatment cycle. After an additional cycle of therapy (8 weeks ± 4 weeks from MRI(N)), patients will re- ceive a confirmatory MRI(N + 1). If the lesion(s) have in- creased ≥25% sum of bidirectional product or ≥40% in vol- ume from MRI(N) (indicating radiographic progression from MRI(N)), this is considered an Bunsustained radiographic response^ or Bpseudoresponse^. The date of radiographic pro- gression for these patients will be MRI(N + 1) and the patient should stop therapy. Alternatively, if the lesion has not in- creased from MRI(N), this is considered a Bdurable radio- graphic response,^ the patient will continue on therapy, and the date of preliminary radiographic progression is the time point of an increase ≥25% sum of bidirectional product or ≥40% in volume (from Nadir) during the remainder of the study. The investigator can then decide whether to continue safely on therapy until progression has been confirmed and at the subsequent time point stop therapy if they feel the patient cannot safely continue therapy. 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. Recurrent GBM (Fig. 4) The outlined modifica- tions in this report are meant to both build on the strengths of the current RANO criteria while providing potential solutions for many of the common challenges. Conclusions A phase 2 trial of irinotecan (CPT-11) in patients with recurrent malignant glioma: a North American Brain Tumor Consortium study. Neuro Oncol, 8(2), 189-193 (2006). 18. Macdonald DR, Cascino TL, Schold SC, Jr., Cairncross JG. Response criteria for phase II studies of supratentorial malignant glioma. J Clin Oncol, 8(7), 1277-1280 (1990). 39. 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https://openalex.org/W4295571832	https://link.springer.com/content/pdf/10.1007/s43630-022-00290-4.pdf	English	null	Rosa davurica inhibits skin photoaging via regulating MAPK/AP-1, NF-κB, and Nrf2/HO-1 signaling in UVB-irradiated HaCaTs	Photochemical & photobiological sciences	2,022	cc-by	9,301	Rosa davurica inhibits skin photoaging via regulating MAPK/AP‑1, NF‑κB, and Nrf2/HO‑1 signaling in UVB‑irradiated HaCaTs Minzhe Fang1 · Hyun‑Myung Lee1 · Sarang Oh2 · Shengdao Zheng1 · A. D. Bellere1 · Minseon Myeongju Kim1 · Duna Yu1 · Tae‑Hoo Yi1 Received: 18 May 2022 / Accepted: 16 August 2022 / Published online: 14 September 2022 © The Author(s) 2022 Photochemical & Photobiological Sciences (2022) 21:2217–2230 https://doi.org/10.1007/s43630-022-00290-4 Photochemical & Photobiological Sciences (2022) 21:2217–2230 https://doi.org/10.1007/s43630-022-00290-4 ORIGINAL PAPERS ORIGINAL PAPERS Abstract Rosa davurica is widely used to treat various kinds of diseases because of its high antioxidant, antiviral and anti-inflamma- tory activities. This use of plant-based materials as medicine is called phytomedicine and has been widely practiced since time immemorial. However, the pharmacological mechanism of R. davurica in skin photoaging is not yet fully understood. Therefore, this study was carried out to evaluate the recovery effects of R. davurica leaf extracts (RDE) in UVB-irradiated human skin keratinocytes (HaCaTs) and investigate whether RDE is a potential therapeutic agent against skin photoaging. The expression of aging-related markers including mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1), nuclear factor-κB (NF-κB), and nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) was evaluated using Western blot analysis. The reactive oxygen species (ROS) was also used by FACS in HaCaTs. Findings indicated that RDE is efficient in scavenging free radicals and dose-dependently reducing ROS generation. Furthermore, RDE notably decreased UVB-induced matrix metalloproteinase-1 (MMP-1) expression through inhibition of MAPK/AP-1 and NF-κB signaling pathways as well as induced blocking of extracellular matrix (ECM) degradation in UVB-irradiated HaCaTs. In addition, RDE improved Nrf2/ HO-1 signaling that increases oxidative defense capacity and enhances transforming growth factor-beta (TGF-β) signaling activation to promote procollagen type I synthesis, relieving UVB-induced skin cell damage. In conclusion, the protective effects of RDE on skin cellular components suggest that it has a high biological potential for skin protection from UVB- induced skin photoaging and is a good candidate for drug and cosmetic application. Minzhe Fang and Hyun-Myung Lee equally contributed to this work. Junhui Choi junhi4703@khu.ac.kr Myeongju Kim espritmj@khu.ac.kr Duna Yu dbensk0205@khu.ac.kr * Tae‑Hoo Yi drhoo@khu.ac.kr Minzhe Fang mincheol-bang1030@gmail.com Hyun‑Myung Lee leehm@khu.ac.kr Sarang Oh blazma1021@gmail.com Shengdao Zheng sdjeong0719@khu.ac.kr A. D. Bellere arcedbellere@khu.ac.kr Minseon Kim dbs03067@khu.ac.kr * Tae‑Hoo Yi drhoo@khu.ac.kr Minzhe Fang mincheol-bang1030@gmail.com Hyun‑Myung Lee leehm@khu.ac.kr Sarang Oh blazma1021@gmail.com Shengdao Zheng sdjeong0719@khu.ac.kr A. D. Bellere arcedbellere@khu.ac.kr Minseon Kim dbs03067@khu.ac.kr (0123456789) 1 3 :(0123 1 3456789) 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2218 Graphical abstract Keywords Rosa davurica leaf extracts (RDE) · Skin photoaging · MAPK/AP-1 · NF-kB · Nrf2/HO-1 Keywords Rosa davurica leaf extracts (RDE) · Skin photoaging · MAPK/AP-1 · NF-kB · Nrf2/HO- In the skin, impairment of type I procollagen is depend- ent on the expression of MMP-1, which is a zinc-dependent endopeptidase that degrades type I collagen specifically. Abstract It is mainly regulated by the upper signaling cascades, such as MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 [7]. After activation of the MAPKs by UV, transcription factor (AP-1) expression increases, leading to collagen fragmentation, inflammatory response, and cell death [8, 9]. In addition, NF-κB, another critical transcription factor of MMP in the dermis, is trans- located under photodamaged conditions into the nucleus by degradation of IκB and elevates MMP and pro-inflamma- tory cytokines such as TNF-α, IL-1β, and IL-6 [10, 11]. Interestingly, UVB alters not only the collagen degradation pathway but also the synthesis pathway, TGFβ/Smad. The TGFβ/Smad pathway involves reduction of TβRII level, and phosphorylation of Smad3 the downstream signaling is significantly decreased in photoaged human skin [12, 13]. Impairment of the TGFβ/Smad pathway is also one of the mechanisms causing the loss of collagen. Due to com- plicated changes in various pathways, the ECM collapses, resulting in wrinkle formation. 2.6 ABTS radical scavenging activity The 2,2-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS, PubChem CID: 5,464,076) assay was determined using reagent. The protocol was followed from the previous study with a slight modification. The reaction of the mixture of ABTS solution and diluted sample with different concen- trations (satisfied the standard curve) were processed at 37 ℃ for 10 min. The reduction of blue green ABTS+ resulting of hydrogen-donating antioxidant and were read using microplate 1 Introduction Skin aging is characterized by loss of structure, wrinkles, pigmentation, drying, and degradation of collagen, and physiological functions [1]. It occurs due to two biochemi- cal processes, intrinsic and extrinsic (photoaging) aging. Intrinsic aging is an inevitable process that occurs over time and is highly correlated with genetic factors. In con- trast, extrinsic aging is caused by several hazardous envi- ronmental factors [2]. Therefore, the skin serves as the first line of defense protecting the body against extrinsic factors and is continuously in contact with toxic chemicals, infectious agents, and ultraviolet radiation [3]. UVB exposure evokes complex and multifaceted signal networks that promote the intracellular formation of ROS, which in turn damages lipids, proteins, and nucleic acids in keratinocytes. In addition, UV exposure increases epi- dermal thickness, reduces collagen synthesis, and facili- tates collagenolysis by upregulating the expression of matrix metalloproteases (MMPs) [4]. UVB-induced ROS activates mitogen-activated protein kinase (MAPK) sign- aling that further induces MMPs and eventually promotes collagen degradation [5, 6]. Several studies on skin damage by UVB have been done focusing on anti-photoaging. Currently, research is focusing 1 3 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2219 based on the aluminum chloride (AlCl3) colorimetric method [19]. on identifying natural products and organic compounds. with anti-photoaging effects. Rosa davurica is a herbal plant occuring in Korea, China, and Japan [14]. It has long been used as a traditional Chinese herbal therapy for diseases because of its antioxidant activities, anti-HIV, anti-viral, anti-biotic, and hypoglycemic activities [15, 16]. In addi- tion, R. davurica improves DNCB-induced atopic dermatitis [17]. Although R. davurica is known for various efficacies, it’s anti-photoaging effect is not yet well understood. There- fore, we investigate R. davurica’s anti-photoaging effects and its molecular mechanism through regulating MAPK/ AP-1, NF-κB, and Nrf2/HO-1 Signaling on UVB-irradiated human keratinocytes (HaCaTs). 2.1 Chemicals and reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin were supplied by Gibco RBL (Grand Island, NY, USA). Standard apigenin, positive control ascorbic acid, dexamethasone, and 5-diphe- nyltetrazolium bromide (MTT) were purchased from Sigma- Aldrich (St. Louis, MO, USA). Human Total MMP-1 and Human Total MMP-3 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Organic solvents were purchased from Samchun Chemical (Seoul, Korea) and Daejung Chemical & Metal (Siheung, Korea). Inorganic salts were purchased from Sigma-Aldrich. Silica gel was purchased from Merck (Kenilworth, NJ, USA). The primary and secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnol- ogy (Santa Cruz, CA, USA), and Bio-Rad Laboratories, Inc. (Hercules, CA, USA). DPPH radical scavenging activity (%) = ODo −ODx ODo × 100 The leaf of R. davurica was provided from Wonsamrosehip Co., Ltd. (Gyeonggi-do, Korea) and was confirmed geneti- cally by DNA analysis. The voucher specimen is kept in the Snowwhitefactory Co, Ltd. laboratory. The dried leaf of R. davurica was etracted using 30% (v/v) aqueous ethanol for 24 h at room temperature. After being filtered through 5 μm filters, the extract was concentrated and lyophilized. The yield of extract was 20.9%. whereas: ODo negative control, ODx various tested RDE and ascorbic acid concentrations. 2.5 DPPH radical scavenging activity The 2,2-diphenyl-1-picrylhydrazyl (DPPH, PubChem CID: 2,375,032) assay was used for scavenging activity against free radical. The absorbance of 0.1 mM DPPH solution in 80% (v/v) methanol was set at 0.650 ± 0.02 at 517 nm. The reaction of the mixture of DPPH solution and diluted sample with different concentrations (satisfied the standard curve) were processed at 37 ℃ for 30 min. The descent of absorb- ance resulting of radical scavenging capacity and were read using microplate reader at 595 nm. The DPPH radical scav- enging activity of the sample was evaluated using the fol- lowing formula: 2.4 HPLC anaysis The phytochemical characteristics of RDE and the standard compounds ethyl gallate and ellagic acid were identified by high performance liquid chromatography (HPLC). The HPLC analysis was performed on Waters e2695 system with 2998 PDA detector (Millford, MA, USA) with an Atlantis T3 column (4.6 mm X 250 mm, 5 μm, Waters, Millford, MA, USA). The mobile phase consisted of (A) water and (B) acetonitrile both containing 0.1% formic acid with gradient elution: 8% B (0–10 min), 8–30% B (10–35 min), 30% B (35–40 min), 30–60% B (40–45 min), 60% B (45–50 min), 60–100% (50–60 min); then keeping 100% B for 5 min to clean the column, and the re-equilibrating step of the column was 8% B isocratic for 5 min. The flow rate was 1.0 mL/min, and the injection volume was 10 μl. The samples were ana- lyzed at a wavelength of 280 nm and the column temperature was maintained at 40 ℃. 2.2 Sample preparations DPPH radical scavenging activity (%) = ODo −ODx ODo × 100 2.12 Reverse transcriptase (RT)‑PCR After 24 h of incubation treatments total RNA was isolated from HaCaT cells according to the manufacturer’s instruc- tions by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). The RNA (3 µg) was reverse transcribed with 200 units of reverse transcriptase and 0.5 µg/µL oligo- (dT)15 primer (Bioneer Co., Daejeon, Korea). This reaction was performed at 70 ℃ for 5 min, 42 ℃ for 60 min, and was terminated at 94 ℃ for 5 min. Amplified products were observed by gel electrophoresis and detected by nucleic acid staining (NobleBio Inc., Korea) under UV illumination. GAPDH was used for normalization. 2.3 Total phenolic content and total flavonoid content The total phenolic content (TPC) of RDE, was quantified based on Folin–Ciocalteau colorimetric method [18]. Also, the total flavonoid content (TFC) of RDE was evaluated 1 3 2220 Photochemical & Photobiological Sciences (2022) 21:2217–2230 reader at 405 nm. The ABTS radical scavenging activity of the sample was evaluated using the following formula: reader at 405 nm. The ABTS radical scavenging activity of the sample was evaluated using the following formula: treatment, the cells were stained with 30 µM 2,7-dichloroflu- orescein diacetate (DCF-DA) (Sigma-Aldrich) for 30 min at 37 ℃ in a CO2 incubator. The cells were collected and then analyzed by flow cytometry (FACSCalibur™; Becton–Dick- inson, San Jose, CA, USA). ABTS radical scavenging activity (%) = ODO −ODX ODO × 100 ABTS radical scavenging activity (%) = ODO −ODX ODO × 100 whereas: ODo negative control, ODx various tested RDE and ascorbic acid concentrations. 2.13 Western blot analysis For western blot analysis, the total proteins were lysed using ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1.0% Triton X-100, 1.0% sodiumdexycholate, 0.1% SDS and so on) for 1 h and centrifuged at 12,000 g for 20 min. After centrifugation, the proteins were quantified with Bradford reagent (Bio-Rad, Hercules, CA, USA). 50 μg of proteins were separated on 10–15% sodium dodecyl sulfate–poly- acrylamide gels (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Subsequently, the membrane that contained the transferred proteins was blocked in 5% non- fat dry milk in TBST (50 mM Tris–HCL, 150 mM NaCl and 0.1% Tween 20, PH 7.4) for 1 h at room temperature and then incubated with appropriate primary antibody in TBST overnight at 4 ℃. After incubation with the primary antibody, the membrane was washed three times by TBST buffer and then incubated with the corresponding secondary antibody for 1 h at room temperature. Protein bands were detected using chemiluminescence detection electrochemi- luminescence (ECL) reagents (Fujifilm, LAS-4000, Tokyo, Japan). Densitometric measurements of bands were analyzed by ImageMasterTM 17 2D Elite software version 3.1 (Amer- sham Pharmacia Biotech, Piscataway, NJ, USA). 2.11 Enzyme‑linked immunosorbent assay After 24 h samples treatment, the supernatants were har- vested. The secretion of MMP-1, MMP-3 and IL-6 were detected with commercially available ELISA kits (R&D Sys- tems, Inc., Minneapolis, MN, USA) following the manufac- turer’s instruction. 2.9 UVB irradiation and samples treatment For UVB irradiation, the cells were rinsed twice with phos- phate buffered saline (PBS) and then were exposed to UVB light with a dose of 125 mJ/cm2 generated from a UVB radia- tion machine (Bio-Link BLX-312; Vilber Lourmat GmbH, France). After UVB irradiation, the cells were washed twice with PBS and immediately treated with or without samples in serum-free medium conditions. The normal/control group was kept in the same culture conditions without samples and undergo UVB irradiation. 10 µM ascorbic acid (positive con- trol) or three doses of RDE, fraction and isolation component were added to each plate for incubation. 2.8 Cell viability The effects of RDE on the viabilities of HaCaT cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTT, Sigma-Aldrich, St-Louis, MO, USA) assay. MTT is a colorimetric assay for evaluating cell viability. The cell survival was initiated through measuring the absorb- ance of the colored solution at a certain wavelength. After 48 h treatment supernatant of each well was removed entirely and a total of 100 µL MTT solution (0.1 mg/mL) was added to each well. After 4 h of incubation, the supernatants were aspirated and 150 µL dimethyl sulfoxides were added to each well. The absorbance at 570 nm was measured by a microplate reader (Molecular Devices FilterMax F5; San Francisco, CA, USA). 2.7 Cell culture HaCaT cells, originating from human keratinocytes, were obtained from Sciencell (Carlsbad, CA, USA). Cells were cultured with DMEM medium supplemented with 10% heat- inactivated FBS and 1% penicillin–streptomycin (Gibco BRL, Grand Island, NY, USA) at 37 ℃ in the atmosphere containing 5% CO2. When the population reached 90–95% confluency, the cells were seeded in 6/96-well plates and 35 mm/100 mm dishes. 3.4 Effect of RDE on intracellular ROS production by FACS RDE had high contents of total phenols, and flavonoid of 176.0 ± 0.6 mg gallic acid/g extract, 173.7 ± 1.2 mg catechin/g extract, respectively. And the HPLC analysis was performed as described in the Materials and Methods. Ethyl gallate and ellagic acid were used as authentic stand- ards, and their contents in the RDE were determined to be 25.20 ± 0.19 and 8.79 ± 0.2 (mg/g extract), respectively Fig. 1. ROS generation plays an essential role in UVB-irradiated cellular signaling [20]. The intracellular ROS production was measured using flow cytometry and ROS scavenging activities of RDE were assessed by the fluorescence dye, DCFH-DA assay. DCFH-DA is hydrolyzed by intracellular esterase and is changed to nonfluorescent DCFH, which is oxidized to highly fluorescent DCF in the presence of ROS. The fluorescent signal increased after UVB radiation (84.1% of UVB-irradiated group). However, treatment with RDE significantly reduced UVB-induced upregulation of fluores- cent intensity to 41.7%. 3.3 Effect of RDE on cell viability Results of cytoprotective effects of UVB-irradiated cells treated with RDE (0.1–50 µg/mL) indicated that UVB irradiation at 125 mJ/cm2 has slight cytotoxicity (Fig. 2). Furthermore, cells treated with RDE had lower cytotox- icity than UVB radiation group. Based on these results, a range of RDE of 1–50 µg/mL was used for the subsequent experiments. 2.10 Measurement of ROS production The 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS levels. After 24 h samples 1 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2221 Fig. 1 HPLC chromatogram of standard and R. davurica extract Fig. 1 HPLC chromatogram of standard and R. davurica extract scavenging activity is consistent with its high antioxidant activity. The antioxidant acts as a preventive agent or delays the mechanism of some types of cell damage. 2.14 Statistical analysis The data were analyzed using Statistical Analysis System (GraphPad Prism 5). All the quantitative data are repre- sented as the means ± SD. The significant differences were determined using a one-way analysis of variance (ANOVA) with the Student–Newman–Keuls test for the multiple com- parisons. p < 0.05, p < 0.01 and p < 0.001 were considered statistically significant for t test. All experiments were per- formed independently three times. 3.2 Antioxidative activities of RDE To evaluate the antioxidant potential of RDE, DPPH, and ABTS assays were performed. Ascorbic acid was used as a positive control because it has free radical scavenging activ- ity. Findings revealed that RDE at different concentrations (1, 10, 50, 100 and 250 µg/mL) has free radical scaveng- ing activities on DPPH (IC50 of 22.8 µg/mL) and ABTS (IC50 of 19.2 µg/mL), which congruently increases in a dose-dependent manner. The presence of RDE free radical 1 3 1 3 2222 Photochemical & Photobiological Sciences (2022) 21:2217–2230 Fig. 2 A DPPH and B ABTS graphical prism scavenging activity of RDE. The results were shown as the mean ± SD of three independent experiments Fig. 2 A DPPH and B ABTS graphical prism scavenging activity of RDE. The results were shown as the mean ± SD of three independent experiments TS graphical prism scavenging activity of RDE. The results were shown as the mean ± SD of three independent Fig. 3 Effect of RDE on cell viability of HaCaT cells. Data are pre- sented as the mean ± SD. # and * indicate significant differences between the non-treated cells and induced groups, respectively. #p < 0.05 vs. the non-treated group. p < 0.05 vs. the induced 3.6 Effect of RDE on mRNA expression of MMP‑1 and Type I procollagen analyzed by RT‑PCR An increase in MMPs and a decrease in procollagen are the primary causes of skin photoaging [21]. To make the photoprotective effects of RDE clear, the mRNA levels of MMP-1 and procollagen type Iwere further measured by RT-PCR. In agreement with ELISA test results, the mRNA level of MMP-1 in UVB-irradiated HaCaTs was also mark- edly elevated, compared with that of non-irradiated cells, whereas RDE decreased UVB-induced MMP-1 by 66.5%, compared with the UVB-irradiated group (Fig. 4A). In con- trast, UVB irradiation inhibited type I procollagen mRNA expression, compared with that of the normal group. Inter- estingly, RDE reversed UVB-induced type I procollagen reduction in a dose-dependent manner (Fig. 4B). Based on the above findings, RDE showed the best effect on UVB- induced photodamage. Therefore, RDE were used to further explored their biological potential. Fig. 3 Effect of RDE on cell viability of HaCaT cells. Data are pre- sented as the mean ± SD. # and indicate significant differences between the non-treated cells and induced groups, respectively. #p < 0.05 vs. the non-treated group. p < 0.05 vs. the induced 3.5 Effect of RDE on MMP‑1, MMP‑3 and IL‑6 production measured by ELISA The secretion of MMP-1, MMP-3, and IL-6 was measured to confirm the effects of RDE on cells anti-aging activity. Cells were exposed to UVB 125 mJ/cm2 radiation prior to treatment with indicated concentrations of RDE at 1, 10, and 50 μg/mL. After 24 h, supernatants were collected and measured using ELISA kits. UVB irradiation significantly increased MMP-1, MMP-3, and IL-6 secretion by 33.6, 32.9 and 1005.9%, respectively (Fig. 3). However, the presence of RDE significantly reversed these trends. Compared with UVB-irradiated cells, RDE inhibited MMP-1 secretion by 31.7% at 50 μg/mL. Also, RDE downregulated the levels of MMP-3 and IL-6 in a dose-dependent manner, leading to a decrease in release of MMP-3 and IL-6 by 46.5 and 44.7% at 50 μg/mL, respectively. 3.7 Effect of RDE on UV‑B induced AP‑1 signaling pathway The NF-κB pathway is another key intermediary in UVB- induced MMPs expression [24]. Effects of RDE on p-NF-κB in UVB-irradiated HaCaTs were explored. Results indicated that RDE effectively inhibited UVB-induced phospho- rylation of NF-κB. As shown in Fig. 5, RDE (10 µg/mL) decreased the p-NF-κB expression by 53.8%. 3.7 Effect of RDE on UV‑B induced AP‑1 signaling pathway AP-1 as a transcription factor could regulate MMPs gene expression in response to UVB radiation [22]. To explore the inhibitory mechanism of RDE on MMP-1 expression, the expression of the major components of AP-1 (c-fos and c-jun, as well as their phosphorylation) were quanti- fied. For the cells subjected to UVB radiation, the expres- sion of p–c-fos and p–c-jun were significantly higher than that of the normal group. The elevated p–c-fos and p–c-jun expression caused by UVB were significantly decreased using RDE treatment. RDE at 10 and 50 µg/mL suppressed UVB-induced p–c-fos by 10.4 and 49.8%, and p–c-jun by 23.7 and 56.4%, respectively (Fig. 5) as compared to the UVB-irradiated group. These significant findings suggested that RDE inhibited MMP-1 expression through regulation of AP-1 transcriptional factor. 1 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2223 3.8 Effect of RDE on UV‑B induced MAPK signaling pathway 3.9 Effect of RDE on UV‑B induced NF‑κB signaling pathway Fig. 4 Intracellular ROS on UVB‐irradiated HaCaT cells from RDE. The intracellular ROS levels were quantitatively determined via flow cytometry (A). Following UVB irradiation (125 mJ/cm2), HaCaTs were treated with or without the indicated concentration of RDE (1, 10, and 50 μg/mL) for 24 h. ROS levels were measured by DCFH-DA assay. The relative intensity of DCF fluorescence is shown in the bar graph. The results were shown as the mean ± SD of three independ- ent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group 3.9 Effect of RDE on UV‑B induced NF‑κB signaling pathway 3.8 Effect of RDE on UV‑B induced MAPK signaling pathway The MAPK is a ROS-sensitive signal pathway, which plays a key role in activation of AP-1 transcription factor and MMPs expression [23]. The expression of the phosphorylated forms of the MAPKs family, including p-JNK, p-p38, and p-ERK were explored to investigate the effects of RDE on UVB- induced photoaging. As shown in Fig. 6, UVB irradiation significantly increased the expression of p-JNK, p-p38, and p-ERK in HaCaTs, whereas RDE significantly reduced the activation of the MAPK signaling pathway. Compared with UVB-induced HaCaTs, RDE dose-dependently decreased the phosphorylation of ERK, JNK, and p38. Furthermore, at 50 µg/mL, RDE were able to reduce the expression of p-ERK, p-JNK and p-p38 by 37.7, 53.7 and 61.0%, respec- tively Fig. 7. 3.10 Effect of RDE on UV‑B induced TGF‑β signaling pathway HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (1, 10 and 50 μg/mL) for 24 h. MMP-1 and procollagen type I mRNA levels were deter- mined by RT-PCR (A). The band intensities were quantified by den- Fig. 6 MMP-1 and Procollagen type I mRNA expression on UVB- irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (1, 10 and 50 μg/mL) for 24 h. MMP-1 and procollagen type I mRNA levels were deter- mined by RT-PCR (A). The band intensities were quantified by den- sitometry and normalized to the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group sitometry and normalized to the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group Fig. 6 MMP-1 and Procollagen type I mRNA expression on UVB- irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (1, 10 and 50 μg/mL) for 24 h. MMP-1 and procollagen type I mRNA levels were deter- mined by RT-PCR (A). The band intensities were quantified by den- and NQO-1, which could be attributed to physiological self-protection. UVB-induced Nrf2 was further elevated by RDE treatment. As shown in Fig. 11, 50 µg/mL RDE could increase Nrf2 expression by 53.8% in UVB-irradi- ated HaCaTs, compared with the UVB group. The levels of HO-1 and NQO-1 were also enhanced when cells were treated with RDE. A concentration of 50 µg/mL RDE sig- nificantly promoted the expression of HO-1 and NQO-1 by 111.7 and 136.7%, respectively. TGF-β1 and procollagen type I proteins by 16.7 and 55.3%, respectively. On the other hand, it decreased the expres- sion of MMP-1 proteins by 50.4%, compared with the UVB group. These results suggest that RDE protected procollagen reduction by activation of the TGF-β signaling pathway. 3.10 Effect of RDE on UV‑B induced TGF‑β signaling pathway To determine if RDE promoted type I procollagen syn- thesis by activation of TGF-β signaling pathway, the effects of RDE on the levels of TGF-β1, procollagen type I and MMP-1 were further investigated in UVB-irradiated HaCaTs. Results show that UVB irradiation significantly decreased TGF-β1 and procollagen type I but increased MMP-1 expression. The RDE dose-dependently enhanced UVB-induced TGF-β1 and procollagen type I, attenuated MMP-1 through downregulation. As shown in Fig. 10, 50 µg/mL RDE significantly increased the expression of 1 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2224 Fig. 5 MMP-1, -3 and IL-6 secretion on UVB-irradiated HaCaT cells from RDE. Following UVB irradiation (125 mJ/cm2), HaCaTs were treated with or without the indicated concentration of RDE (1, 10, and 50 μg/mL) for 24 h. MMP-1 secretion (A), MMP-3 secretion (B), IL-6 secretion (C) in UVB-irradiated HaCaTs were measured by ELISA kits. The results were shown as the mean ± SD of three inde- pendent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 com- pared with only UVB-irradiated group Fig. 5 MMP-1, -3 and IL-6 secretion on UVB-irradiated HaCaT cells from RDE. Following UVB irradiation (125 mJ/cm2), HaCaTs were treated with or without the indicated concentration of RDE (1, 10, and 50 μg/mL) for 24 h. MMP-1 secretion (A), MMP-3 secretion (B), IL-6 secretion (C) in UVB-irradiated HaCaTs were measured by ELISA kits. The results were shown as the mean ± SD of three inde- pendent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 com- pared with only UVB-irradiated group sitometry and normalized to the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group sitometry and normalized to the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group Fig. 6 MMP-1 and Procollagen type I mRNA expression on UVB- irradiated HaCaT cells from RDE. 3.11 Effect of RDE on UV‑B induced Nrf2/ARE signaling pathway Another possible mechanism of anti-photoaging effects of RDE can be the regulation of the endogenous antioxi- dant system. Nrf2 is an important cellular defense system that induces numerous antioxidant-related gene expression such as HO-1 and NQO-1 [25]. Here, the effects of RDE on the protein levels of Nrf2, HO-1 and NQO-1 were eval- uated in UVB-irradiated HaCaTs. The results indicate that exposure of cells to UVB radiation significantly increased the expression of Nrf2 and promoted the levels of HO-1 4 Discussion Phytochemicals have been used by mankind in a wide vari- ety of ways since time immemorial. As stated by Yadav and Agarwala, the World Health Organization claimed that medicinal plants are an ideal source of drugs [26]. Around 80% of individuals in developed countries use traditional 1 3 3 2225 Photochemical & Photobiological Sciences (2022) 21:2217–2230 Fig. 7 AP-1 signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (10 and 50 μg/mL) for 4 h. Phosphorylated and non-phosphorylated forms of c-Fos and c-Jun were detected by Western blot (A). The band intensities were quantified by densitom- etry, normalized to the level of c-Jun (B) and c-fos (C), and calcu- lated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradi- ated group lated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradi- ated group lated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradi- ated group Fig. 7 AP-1 signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (10 and 50 μg/mL) for 4 h. Phosphorylated and non-phosphorylated forms of c-Fos and c-Jun were detected by Western blot (A). The band intensities were quantified by densitom- etry, normalized to the level of c-Jun (B) and c-fos (C), and calcu- Korea, many of which exhibit numerous pharmacologi- cal properties [15, 27]. As a traditional or folk medicine, R. davurica has long been used to treat various diseases, such as dyspepsia, gastroenterology, arteriosclerosis, medicines based on compounds derived from medicinal plants. One of the widely used plants for medicinal pur- poses is the R. davurica. The genus R. 4 Discussion However, there has been no study on the skin anti-photoaging effects of the R. davurica. Thus, this study was done determine if RDE inhibited UVB‐induced photoaging via inhibition of the MAPK/AP‐1, NF-κB, and Nrf2/HO-1 pathway. Nrf2 upregulated ARE-mediated gene expression in UVB exposed fibroblasts. Our results also show that activation of both Nrf2 and its target genes, such as NQO1 and HO-1, is due to UVB-triggered ROS signaling. In skin subjected to extrinsic aging by UVB irradiation, RDE led to decreased oxidative stress by regulating the Nrf2/ARE signaling path- way via the expression of antioxidant enzymes such as NQO1 and HO-1 (Fig. 8). Keratinocytes, the predominant cell type in the epidermis, absorb the majority of UVB irradiation, leading to dryness, loosing, and inflammation [34]. The UVB irradiation of the epidermis results in ROS generation, which increased MMP- 1, MMP-3, and IL-6 secretion. In this study, RDE treatment in HaCaTs while undergoing UVB irradiation resulted in decreases in MMP-1, MMP-3, and IL-6 secretion. Hwang et al. [17] reported low cytokine levels in groups treated for atopic dermatitis with leaf extracts of R. davurica. IL-6 plays a key role in the inflammatory responses of HaCaTs following UVB exposure, and as shown in our data, treat- ment with RDE can regulate IL-6 production. After UVB irradiation, RDE-treated cells showed TGF-β1 and type I procollagen (Fig. 3, 7). Oxidative stress caused by the factors considered in this study leads to increased ROS generation and reduced antioxidant capacity, resulting in aging and visible dete- rioration in skin condition [29, 30]. Our results indicate that the inhibitory activity of DPPH and ABTS free scav- enging radicals was detectable at IC50 values of 22.8 µg/ mL and 19.2 µg/mL, respectively (Fig. 9). After treatment with RDE, the level of ROS significantly dose-dependently decreased (Fig. 10). The ability of RDE to scavenge DPPH radicals was similar to that of ascorbic acid, a well-known skin brightener that was used as a positive control. Also, UV radiation-induced ROS triggers an increase in MMPs, degrades ECM proteins such as collagen and elastin, and leads to skin photoaging and wrinkle forma- tion [31]. To evaluate the effect of pre-and post-enzyme- treated RDE on UVB-induced photoaging, we investigated the mRNA expression of MMP-1 and procollagen type I in UVB-irradiated HaCaT cells using RT-PCR. Our results demonstrated that RDE recovered the expression of procol- lagen type I (Fig. 4 Discussion davurica, with about 100 species, occurs in the north of China, Japan, and Fig. 8 MAPK signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1 h. Phospho- rylated and non-phosphorylated forms of ERK, JNK and P38 were detected by Western blot (A). The band intensities were quantified by densitometry, normalized to the level of ERK, JNK and P38, and cal- culated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group culated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group Fig. 8 MAPK signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1 h. Phospho- rylated and non-phosphorylated forms of ERK, JNK and P38 were detected by Western blot (A). The band intensities were quantified by densitometry, normalized to the level of ERK, JNK and P38, and cal- culated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB- irradiated group Fig. 8 MAPK signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1 h. Phospho- rylated and non-phosphorylated forms of ERK, JNK and P38 were detected by Western blot (A). The band intensities were quantified by densitometry, normalized to the level of ERK, JNK and P38, and cal- 1 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2226 pulmonary tuberculosis, frostbite, and menoxenia. Modern pharmacological research has revealed that R. davurica exhibits antioxidant, anti-inflammatory, anti-angiogenic, anti-allergy, anti-nociceptive activities, anti-lipid peroxi- dation effect, and inhibitory activity against neutrophil migration [28]. 4 Discussion 4) and significantly decreased MMP-1 expression in UVB-irradiated HaCaTs. These findings sug- gest that RDE prevents collagen loss through the inhibition of UVB-induced MMP-1 expression and the enhancement of procollagen type I expression. The MAPK signaling pathway plays a key role in regu- lating MMPs expression, cell proliferation, differentiation, and death [35]. The major function of MAPK is transfer- ring extracellular signals to the nucleus, thereby activating transcription factors and inducing target gene expression. AP-1, as a MAPK downstream activator, is a well-known transcription factor that stimulates gene transcription of MMPs [36]. To explore the underlying mechanism of RDE, we performed further studies on the MAPK/AP-1 signal- ing pathway in HaCaTs (Fig. 5, 6). RDE showed that the deglycosylated products exhibited stronger activity than their respective counterparts in inhibiting UVB radiation- induced phosphorylation of ERK, JNK, p-38, as well as p–c-fos, and p–c-jun expression in HaCaT cells. Lu Li et al. [37] and Choi HJ et al. [38] reported UVB-induced MAPK/ The main factors that defend skin cells from oxida- tive stress are Nrf2 and several of its target genes, such as NQO1 and HO-1 [32]. Kannan and Jaiswal [33] found that 1 3 (A) (B) - + + + + 0 50 100 150 - - 10 - - - - - 10 50 UVB (125 mJ/cm2) R. davurica ( g/mL) Ascorbic acid ( M) # Protein relative density NF- B / -actin (% of Normal) * * Fig. 9 NF-κB signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 4 h. NF-κB pro- tein expression were detected by Western blot (A). The band inten- sities were quantified by densitometry, normalized to the level of β-actin, and calculated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non- irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group (A) Fig. 9 NF-κB signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 4 h. NF-κB pro- tein expression were detected by Western blot (A). 4 Discussion The band inten- sities were quantified by densitometry, normalized to the level of (B) - + + + + 0 50 100 150 - - 10 - - - - - 10 50 UVB (125 mJ/cm2) R. davurica ( g/mL) Ascorbic acid ( M) # Protein relative density NF- B / -actin (% of Normal) * β-actin, and calculated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non- irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group (A) (A) (B) - + + + + 0 50 100 150 - - 10 - - - - - 10 50 UVB (125 mJ/cm2) R. davurica ( g/mL) Ascorbic acid ( M) # Protein relative density NF- B / -actin (% of Normal) * (A) (B) Fig. 9 NF-κB signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 4 h. NF-κB pro- tein expression were detected by Western blot (A). The band inten- sities were quantified by densitometry, normalized to the level of β-actin, and calculated as the percentage of the non-irradiated group (B). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non- irradiated group, p < 0.05, p < 0.01, *p < 0.001 compared with only UVB-irradiated group 1 3 3 2227 Photochemical & Photobiological Sciences (2022) 21:2217–2230 Fig. 10 TGF-β1 signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1.5 h. TGF-β1, procollagen type I and MMP-1 protein expression was detected by Western blot (A). The band intensities were quantified by densitom- Fig. 10 TGF-β1 signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1.5 h. TGF-β1, procollagen type I and MMP-1 protein expression was detected by Western blot (A). The band intensities were quantified by densitom- etry, normalized to the level of β-actin, and calculated as the percent- age of the non-irradiated group (B, C). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group etry, normalized to the level of β-actin, and calculated as the percent- age of the non-irradiated group (B, C). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group etry, normalized to the level of β-actin, and calculated as the percent- age of the non-irradiated group (B, C). The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group Fig. 10 TGF-β1 signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), fol- lowed by treatment with RDE (10 and 50 μg/mL) for 1.5 h. TGF-β1, procollagen type I and MMP-1 protein expression was detected by Western blot (A). The band intensities were quantified by densitom- accumulation of Nrf2. Therefore, we conclude that the RDE have great potential for use against skin photoaging. AP-1 expression in HaCaTs, similarly our results indicate that RDE regulates MAPK and AP-1 signaling pathways. Aside from reducing collagen production in the skin, UVB-irradiated keratinocytes also overexpressed certain inflammatory cytokines [39]. NF-κB is responsible for acti- vating the transcription of inflammatory cytokines. 3 When combined with its inhibitory protein IκB-α, NF-κB com- monly exists in an inactivated state in the cytoplasm. Once stimulated, degraded IκB-α leads to the nuclear localization of NF-κB, inducing the expression of a variety of inflam- matory factors like IL-6 [40]. Moreover, inflammatory cytokine-mediated inflammation increases ROS genera- tion and further amplifies UV-induced skin damage [41]. Thus, the NF-κB pathway plays a central role in skin aging and the inflammatory generation. We further measured the expression of NF-κB and IκB-α by Western blot to verify the mechanisms of RDE. Hwang E et al. [42] and Zhang M et al. [43] reported that UVB-induced damage is controlled through the regulation of the NF-κB pathways. Similar to the findings of the above studies, we found that the RDE inhibited the UVB-induced promoter activity of NF-κB and degradation of IκB-α to varying degrees (Fig. 11). 5 Conclusions In summary, we demonstrated that RDE effectively pro- tected HaCaTs against UVB‐induced photodamage by decreasing ROS generation, MMPs, and IL-6. The rela- tive protection mechanism involved inhibition of the MAPK/AP‐1 and NF-κB pathways. Moreover, RDE extract improved Nrf2/HO-1 signaling that procollagen type I synthesis by increasing the level of TGF‐β1. All these results suggest that RDE on skin cellular compo- nents suggest that it has a high biological potential for skin protection from UVB-induced skin photoaging and is a good candidate for drug and cosmetic application. However, evaluation of the biological effect of RDE and its active compounds on in vivo models and clinical trials should be further clarified. i Supplementary Materials: Table S1: Oligonucleotide primers used for RT-PCR. Overall, we found that RDE exhibited a superior ability to inhibit MMPs and IL-6 production by regulating the MAPK/ AP-1, NF-κB/IκB-α pathways in UVB-irradiated HaCaTs. Moreover, RDE increases cytoprotective antioxidants such as HO-1 and NQ-O1 expression by facilitating the nuclear Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s43630-022-00290-4. 1 3 Photochemical & Photobiological Sciences (2022) 21:2217–2230 2228 Fig. 11 Nrf2/ARE signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (10 and 50 μg/mL) for 3 h. Total HO-1 and NQO-1 (A) and nucleus Nrf2 (B) expression were detected by Western blot. The band intensities of total HO-1 and NQO-1 (C), nucleus Nrf2 (D) were quantified by densitometry, normalized to the level of β-actin and histone, and calculated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group Fig. 11 Nrf2/ARE signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (10 and 50 μg/mL) for 3 h. Total HO-1 and NQO-1 (A) and nucleus Nrf2 (B) expression were detected by Western blot. The band intensities of total HO-1 and NQO-1 (C), nucleus Nrf2 (D) were quantified by densitometry, normalized to the level of β-actin and histone, and calculated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. 5 Conclusions #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, p < 0.001 compared with only UVB-irradiated group the level of β-actin and histone, and calculated as the percentage of the non-irradiated group. The results were shown as the mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with non-irradiated group, p < 0.05, p < 0.01, *p < 0.001 compared with only UVB-irradiated group Fig. 11 Nrf2/ARE signaling pathway on UVB-irradiated HaCaT cells from RDE. HaCaTs were irradiated with UVB (125 mJ/cm2), followed by treatment with RDE (10 and 50 μg/mL) for 3 h. Total HO-1 and NQO-1 (A) and nucleus Nrf2 (B) expression were detected by Western blot. The band intensities of total HO-1 and NQO-1 (C), nucleus Nrf2 (D) were quantified by densitometry, normalized to Acknowledgements This research was kindly supported by Snow White Factory. Acknowledgements This research was kindly supported by Snow White Factory. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Author contributions Conceptualization: THY and SO, methodology: MF, HML, and MSK, software: SZ and DY, validation: MF, HML and SO, formal analysis: MF, HML and JC investigation: THY, MF, HML, and SO, resources: THY, data curation: SZ, MSK, and MJK, writing—SO, and BAD, writing—review and editing: THY and SO, visualization: SZ, BAD, MSK, and MJK, supervision: THY, project administration: THY, All authors have read and agreed to the published version of the manuscript. Funding This study was carried out with the support of ´R&D Program for Forest Science Technology (Project No. 2021392B10-2223–0104) provided by Korea Forest Service (Korea Forestry Promotion Institute). References Data availability statement The data presented in this study are avail- able in this paper. 1. Zhang, S., & Duan, E. (2018). Fighting against skin aging: the way from bench to bedside. Cell Trans, 27(5), 729–738. https:// doi.org/10.1177/0963689717725755 Sample availability Samples are not available. g 2. 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https://openalex.org/W4396683357	https://digitalcommons.unomaha.edu/cgi/viewcontent.cgi?article=2121&context=jrf	English	null	Scare Me	The journal of religion and film	2,020	cc-by	1,039	Journal of Religion & Film Journal of Religion & Film Volume 24 Issue 1 April 2020 January 2020 January 2020 Recommended Citation Recommended Citation This Sundance Film Festival Review is brought to you for free and open access by DigitalCommons@UNO. It has been accepted for inclusion in Journal of Religion & Film by an authorized editor of DigitalCommons@UNO. For more information, please contact unodigitalcommons@unomaha.edu. Follow this and additional works at: https://digitalcommons.unomaha.edu/jrf Follow this and additional works at: https://digitalcommons.unomaha.edu/jrf Follow this and additional works at: https://digitalcommons.unomaha.edu/jrf Part of the Feminist, Gender, and Sexuality Studies Commons, and the Film and Media Studies Commons Please take our feedback survey at: https://unomaha.az1.qualtrics.com/jfe/form/ SV_8cchtFmpDyGfBLE Scare Me Scare Me Jodi McDavid Cape Breton University, jodi@mcdavidbrodie.com Scare Me Scare Me Abstract Abstract This is a film review of Scare Me (2020), directed by Josh Ruben. Keywords Keywords Horror, #Metoo Creative Commons License Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. Author Notes Author Notes Jodi McDavid is an instructor in folklore and gender and women's studies at Cape Breton University. This sundance film festival review is available in Journal of Religion & Film: https://digitalcommons.unomaha.edu/jrf/ vol24/iss1/12 This sundance film festival review is available in Journal of Religion & Film: https://digitalcommons.u Keywords Keywords Horror, #Metoo This work is licensed under a Creative Commons Attribution 4.0 License. Author Notes Author Notes Jodi McDavid is an instructor in folklore and gender and women's studies at Cape Breton University. Jodi McDavid is an instructor in folklore and gender and women's studies at Cape Breton University. This sundance film festival review is available in Journal of Religion & Film: https://digitalcommons.unomaha.edu/jrf/ vol24/iss1/12 McDavid: Scare Me Scare Me (2019) dir. Josh Ruben Josh Ruben is the writer, director and one of the actors in Scare Me. He got his chops in CollegeHumor, creating thousands of videos for the website. While reviews were mixed among my unofficial early-reactions-survey of my nearby seatmates, I think this film will resonate well with the generation that CollegeHumor speaks to. CollegeHumor tends to take on complex topics in a flippant way, satirizing them and sometimes sacrificing some aspects of execution, rather preferring to ensure their topic gets into the limelight. The take more of a sketch comedy form rather than creating a cinematic world. Another stylistic influence is 1980s episodic horror shows (such as Tales from the Crypt) and comedy horror such as the Scary Movie series. One could be critical of the style, however, that doesn’t really serve much of a purpose; my thoughts are that this style will ring true for those who are familiar with those forms. In most ways Scare Me is not scary But in a very important way it is Fred (Josh Ruben) Scare Me (2019) dir. Josh Ruben Scare Me (2019) dir. Josh Ruben Josh Ruben is the writer, director and one of the actors in Scare Me. He got his chops in CollegeHumor, creating thousands of videos for the website. While reviews were mixed among my unofficial early-reactions-survey of my nearby seatmates, I think this film will resonate well with the generation that CollegeHumor speaks to. CollegeHumor tends to take on complex topics in a flippant way, satirizing them and sometimes sacrificing some aspects of execution, rather preferring to ensure their topic gets into the limelight. The take more of a sketch comedy form rather than creating a cinematic world. Another stylistic influence is 1980s episodic horror shows (such as Tales from the Crypt) and comedy horror such as the Scary Movie series. Published by DigitalCommons@UNO, 2020 Keywords Keywords Horror, #Metoo One could be critical of the style, however, that doesn’t really serve much of a purpose; my thoughts are that this style will ring true for those who are familiar with those forms. In most ways, Scare Me is not scary. But in a very important way, it is. Fred (Josh Ruben) goes to a remote cabin, ostensibly to work on his writing. He meets Fanny (Aya Cash) while he is out for a jog, and discovers that she is a successful writer. Later that night Published by DigitalCommons@UNO, 2020 Published by DigitalCommons@UNO, 2020 1 Journal of Religion & Film, Vol. 24 [2020], Iss. 1, Art. 12 Journal of Religion & Film, Vol. 24 [2020], Iss. 1, Art. 12 Journal of Religion & Film, Vol. 24 [2020], Iss. 1, Art. 12 the power goes out and Fanny shows up at this cabin and suggests they tell scary stories, an exchange that goes back and forth throughout the night. Fanny gives Fred some critical advice, and he seems conflicted about her, somewhat intrigued but also intimidated. Eventually, Fred confronts Fanny about her notebook and says he wants to give her the best scare. That he will chase her and if he catches her, its implied he will beat her with a fireplace poker. If he can’t find her, she will keep the notebook. This is clearly too real for Fanny, and the viewer is also left to wonder, is Fred serious or is he joking? Has he misread the dynamic of the night and does be believe Fanny thinks him to be harmless? Or does he really want to terrify her? We are left wondering. The director’s notes confirm that Ruben wrote this script in response to the #MeToo movement and from hearing stories from his female friends about things they had experienced. While Ruben makes an interesting attempt to bring this idea into the comedy horror genre, there are also some missed opportunities. One story broaches the idea that the two will kiss, which does not create the tension it could. At another point a second man shows up, at which point Fanny is also calm and easy going about the change in balance. As a female watching this film, those were moments of tension for me, and concern for the character of Fanny, which the director didn’t develop. Keywords Keywords Horror, #Metoo As #MeToo is a recurring subject of films, and more films are devoted to the experiences of marginalised people, it is important that authentic voice is used and that these stories are developed from a place of experience and knowledge. https://digitalcommons.unomaha.edu/jrf/vol24/iss1/12 DOI: https://doi.org/10.32873/uno.dc.jrf.24.01.12 https://digitalcommons.unomaha.edu/jrf/vol24/iss1/12 DOI: https://doi.org/10.32873/uno.dc.jrf.24.01.12 https://digitalcommons.unomaha.edu/jrf/vol24/iss1/12 DOI: https://doi.org/10.32873/uno.dc.jrf.24.01.12 2 2
https://openalex.org/W4200146900	https://www.degruyter.com/document/doi/10.1515/9783110747614-001/pdf	German	null	Editorial	De Gruyter eBooks	2,021	cc-by	948	OpenAccess. © 2021 Matthias Bruhn, Bildwelten des Wissens 17, publiziert von De Gruyter. Dieses Werk ist lizenziert unter der Creative Commons Attribution 4.0 International Lizenz. https://doi.org/10.1515/9783110747614-002 enAccess. © 2021 Matthias Bruhn, Bildwelten des Wissens 17, publiziert von De Gruyter. Editorial Der moderne Mensch ist ein kaltes Wesen. Nachdem die Beherrschung des Feuers seit mythischen Zeiten die Macht des Menschen über die Natur versinnbildlicht hatte, ist im Industriezeitalter auch die Kälte in ihrer wissenschaftlichen Bedeutung erkannt und als technische Herausforderung angenommen worden. Nach Galvanismus und organischer Chemie versprechen neue Verfahren zur Herstellung und Kontrolle künst licher Kälte eine weitere Stufe in der Höherentwicklung des Lebendigen. Sie wecken Hoffnungen, das Leben verlängern oder den Tod überwinden zu können, und werden ein fester Bestandteil der utopischen Literatur und Science-Fiction. Auch in kaliforni scher Wendung ist die Kryotechnik neben Elektrizität und Raumfahrt weiterhin ein Schlüssel im transhumanistischen Plan. Jenseits der Fantastik gehört künstlich erzeugte Kälte tatsächlich zu den folgen reichsten Errungenschaften der jüngeren Geschichte: Mitte des 19. Jahrhunderts stellte der englische Bierbrauer James Prescott Joule fest, dass sich bestimmte Gasmischungen unter Druck erwärmen und bei nachlassendem Druck wieder abkühlen – ein Phä nomen, das seither auf unterschiedlichste Weise zur Anwendung gekommen ist. Zur Jahrhundertwende entstanden Laboratorien, in denen unter extremer Kälte Gase ver flüssigt werden konnten, mit Effekten, die den Naturgesetzen zu widersprechen schie nen. Im Jahr 1911 beobachtete Heike Kamerlingh Onnes in seinem Leidener Labor, dass leitfähige Materialien bei extremer Kälte keinen elektrischen Widerstand mehr aufweisen. Diese Supraleitung wurde von Lew Wassiljewitsch Schubnikow, einem Pionier der sowjetischen Kryogenik, in Charkiw weiter untersucht, aber erst später in ihrer vollen Bedeutung erkannt. So beruht beispielsweise die Magnetresonanzto mografie auf starken Magnetfeldern, die mit Hilfe von Supraleitern erzeugt werden können. Sie ist damit ebenfalls und auf indirekte Weise ein bildgebendes Verfahren, das aus der Kälte kam. Die Kältelabore in Leiden, Berlin oder Charkiw entstanden ungefähr zeitgleich mit der Jagd nach dem kältesten natürlichen Punkt der Erde. Onnes machte seine Beobachtung im selben Jahr, in dem auch eine norwegische Expedition den geografi schen Südpol erreichte. Diese entbehrungsreichen Reisen in das angeblich ewige Eis waren von großer Medienaufmerksamkeit begleitet, Fotografien schwarzer Silhouetten vor absolutem Weiß wurden zu Ikonen männlicher Abhärtung und eines unerbittli chen Pioniergeistes, dem am Ende nur noch der Aufbruch in die Leere des Weltraums bleibt. Mit den neuen Verfahren veränderten sich die Lebensgewohnheiten, und den Wissenschaften erwuchsen ungeahnte Möglichkeiten, weil sich biologische Prozesse durch Kälte verlangsamen, neue Werkstoffe per Gefriertechnik formen oder prüfen lassen. Quantenrechner setzen wie die medizinische Diagnostik auf Supraleitung. OpenAccess. © 2021 Matthias Bruhn, Bildwelten des Wissens 17, publiziert von De Gruyter. Editorial Dieses Werk ist lizenziert unter der Creative Commons Attribution 4.0 International Lizenz. https://doi.org/10.1515/9783110747614-002 Editorial Editorial 8 Auch in epistemologischer Hinsicht ist Kälte damit so fundamental wie die Einfüh rung optischer Systeme zur Mikro- oder Fernbeobachtung, und in vielen Fällen ist sie mit dieser direkt verbunden, etwa in Gestalt von Gefrierschnitten für die mikro- und makroskopische Beobachtung, deren zeitgenössische Form der Buchdeckel dieses Heftes vor Augen führt. Aber Kälte ist nicht gleich Kälte. Neben der Temperatur entscheiden auch die Geschwindigkeit und das Verfahren über ihren erfolgreichen Einsatz: Eisbildung kann organische Strukturen zum Platzen bringen. Schockfrostung hinterlässt andere Muster als eine allmähliche Abkühlung oder eine stabil gehaltene Dauerkälte. Einen besonders drastischen Fall liefern die anatomischen Schnittbilder nach gefrorenen menschlichen Kadavern, die der Leipziger Anatomie-Professor Christian Wilhelm Braune im Jahre 1867 in einem aufwendigen chromolithografischen Atlas herausgebracht hat. Braune musste sich noch mit winterlichen Temperaturen und mit herkömmlichen Kühlkammern behelfen. Sein Werk blieb lange Zeit unübertroffen wegen einer Darstellungsweise, deren Radikalität im Zeitalter strahlenbasierter Verfah ren leicht übersehen werden kann, denn es bestand dem Titel entsprechend aus farbigen Wiedergaben vollständig durchgesägter menschlicher Leichname. ◊ Abb. 1 Die sprich wörtlich gewordene Kälte oder Coolness des wissenschaftlichen Labors artikuliert sich hier gleichermaßen technisch wie symbolisch. Erst das Visual Human Project, bei dem ein männlicher und ein weiblicher Leichnam in Hunderte feiner Lagen zerschnitten wurden – was entsprechende ethische Vorwürfe mit sich brachte –, ist in Sachen formaler Gnadenlosigkeit noch einen Schritt weiter gegangen, und zwar gerade weil bildge bende Verfahren zur Verfügung standen, die aber noch nicht dieselbe Feinheit boten. Zuletzt ist die Bedeutung von Kühlprozessen in allgemeine Erinnerung gerufen worden durch komplexe Impfstoffe auf Basis von mRNA, die bei -70° C in Trockeneis gelagert werden müssen. Es gehört zur Dialektik der Kühltechnik, dass sie Leben abtöten und verlängern kann oder dass ihr steigender Energiehunger den menschen gemachten Klimawandel begleitet und mitverschuldet: eine überkochende Erde aus Verbrennungs- und Klimaanlagen, deren Atmosphäre in ein Treibhaus verwandelt wird, während dahinter der absolute Nullpunkt wartet. Die Bilder für diesen Pro zess – Diagramme oder Aufnahmen schmelzender Gletscher und Polkappen – schei nen daran wenig ändern zu können. Im vorliegenden Band soll es nicht um moralische, sondern um technische Kälte gehen. Es sind darin aus gutem Grunde die künstlerischen Formen der Beobachtung und Darstellung eingeschlossen, die ohne sie nicht zu denken sind. Kälte ist ästhetische Haltung oder kristalline Form. Ein weißer Papiergrund ist mit wenigen Strichen in eine Winterlandschaft aus imaginärem Schnee verwandelt. Editorial Und für die bildenden Editorial 9 1: Wilhelm Braune: Topographisch-anatomischer Atlas nach Durchschnitten an gefrornen Cadavern, Tab. XXIII Fig. II, Oberschenkel, 1867, kolorierter Holzschnitt, lebensgroß. 1: Wilhelm Braune: Topographisch-anatomischer Atlas nach Durchschnitten an gefrornen Cadavern, Tab. XXIII Fig. II, Oberschenkel, 1867, kolorierter Holzschnitt, lebensgroß. Künste der Gegenwart ist der Klimawandel mehr als nur Thema, er ist ein ernsthaftes konservatorisches Problem, das die eigene Arbeit betrifft, denn Museen sind in der Regel Kühlräume zur Aufbewahrung fragiler Gebilde. Es braucht daher auch den Blick auf die Kälte und ihre Bedeutung für das Entstehen und Verschwinden von Bildern, um die globale Entwicklung ganz zu erfassen. Matthias Bruhn mit Claudia Blümle, Horst Bredekamp und Katja Müller-Helle
https://openalex.org/W2308920253	https://eprints.gla.ac.uk/118402/1/118402.pdf	English	null	Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence	Journal of virology	2,016	cc-by	22,432	JVI Accepted Manuscript Posted Online 23 March 2016 J. Virol. doi:10.1128/JVI.00422-16 Copyright © 2016 Ratinier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Bluetongue virus NS4 protein is an interferon antagonist and a 1 determinant of virus virulence 2 Maxime Ratinier1,2, Andrew E. Shaw1, Gerald Barry1,3, Quan Gu1, Luigina Di Gialleonardo4, 3 Anna Janowicz1, Mariana Varela1, Richard E. Randall5, Marco Caporale1,4 and Massimo 4 Palmarini1# 5 6 1MRC–University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom. 7 2Current address: Univ Lyon, Institut National de la Recherche Agronomique, EPHE, 8 UMR754, Infections virales et pathologie comparée (IVPC), Laboratoire "Biologie des 9 Phlébovirus", F-69007, Lyon, France. 10 3Veterinary Sciences Centre, School of Veterinary Medicine,University College Dublin, 11 Dublin, Ireland. 12 4Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise “G. Caporale”, Teramo, Italy. 13 5Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St 14 Andrews, United Kingdom. 15 16 17 #To whom correspondence should be addressed. MRC-University of Glasgow Centre for 18 JVI Accepted Manuscript Posted Online 23 March 2016 J. Virol. doi:10.1128/JVI.00422-16 Copyright © 2016 Ratinier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Bluetongue virus NS4 protein is an interferon antagonist and a 1 determinant of virus virulence 2 Maxime Ratinier1,2, Andrew E. Shaw1, Gerald Barry1,3, Quan Gu1, Luigina Di Gialleonardo4, 3 Anna Janowicz1, Mariana Varela1, Richard E. Randall5, Marco Caporale1,4 and Massimo 4 Palmarini1# 5 6 1MRC–University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom. 7 2Current address: Univ Lyon, Institut National de la Recherche Agronomique, EPHE, 8 UMR754, Infections virales et pathologie comparée (IVPC), Laboratoire "Biologie des 9 Phlébovirus", F-69007, Lyon, France. 10 3Veterinary Sciences Centre, School of Veterinary Medicine,University College Dublin, 11 Dublin, Ireland. 12 4Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise “G. Caporale”, Teramo, Italy. 13 5Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St 14 Andrews, United Kingdom. 15 16 17 #To whom correspondence should be addressed. MRC-University of Glasgow Centre for 18 Virus Research, 464 Bearsden Road, Glasgow G61 1QH, United Kingdom. Phone: +44 (0) 141 19 330 2541. massimo.palmarini@glasgow.ac.uk 20 21 Running title: Modulation of the IFN response by BTV 22 23 Abstract: 234 words; Importance: 129 words; Main text: 5469 24 3Veterinary Sciences Centre, School of Veterinary Medicine,University College Dublin, 11 y , y , y g , Dublin, Ireland. 12 1 \| P a g e ABSTRACT 25 Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of 26 ruminants with serious consequences to both animal health and the economy. The clinical 27 outcome of BTV infection is highly variable and dependent on a variety of factors related to 28 both the virus and the host. In this study, we show that the BTV non-structural protein NS4 29 favours viral replication in sheep, the animal species most affected by bluetongue. In 30 addition, NS4 confers a replication advantage to this virus in primary sheep endothelial cells 31 and in IFN-competent immortalized cell lines. We determined that in cells infected with a 32 NS4 deletion mutant (BTV8ΔNS4) there is an increased synthesis of type I interferon 33 (IFN) compared to cells infected with wild type BTV-8. In addition, using RNAseq, we show 34 that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and 35 interferon stimulated genes. Moreover, using reporter assays and protein synthesis assays, 36 we show that NS4 downregulates the activity of a variety of promoters such as the 37 cytomegalovirus immediate early promoter, the IFN-β promoter and a promoter containing 38 interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory 39 activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does 40 not affect mRNA splicing or cellular translation. The data obtained in this study strongly 41 suggest that BTV NS4 is an IFN-antagonist and a key determinant of viral virulence. 42 43 IMPORTANCE 44 l i f h i i f i di f i d i d b bl 4 Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of 26 ruminants with serious consequences to both animal health and the economy. The clinical 27 outcome of BTV infection is highly variable and dependent on a variety of factors related to 28 both the virus and the host. In this study, we show that the BTV non-structural protein NS4 29 favours viral replication in sheep, the animal species most affected by bluetongue. and the host immune responses. In this study we show that a non-structural protein of BTV 49 (NS4) is critical to counteract the innate immune response of the host. Infection of cells with 50 a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of 51 interferon stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by 52 favouring viral replication in sheep, the animal species most susceptible to bluetongue. 53 In 30 addition, NS4 confers a replication advantage to this virus in primary sheep endothelial cells 31 and in IFN-competent immortalized cell lines. We determined that in cells infected with a 32 NS4 deletion mutant (BTV8ΔNS4) there is an increased synthesis of type I interferon 33 (IFN) compared to cells infected with wild type BTV-8. In addition, using RNAseq, we show 34 that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and 35 interferon stimulated genes. Moreover, using reporter assays and protein synthesis assays, 36 we show that NS4 downregulates the activity of a variety of promoters such as the 37 cytomegalovirus immediate early promoter, the IFN-β promoter and a promoter containing 38 interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory 39 activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does 40 not affect mRNA splicing or cellular translation. The data obtained in this study strongly 41 suggest that BTV NS4 is an IFN-antagonist and a key determinant of viral virulence. 42 2 \| P a g e Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue 45 virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by 46 Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to 47 both virus and host factors. It is therefore critical to understand the interplay between BTV 48 and the host immune responses. In this study we show that a non-structural protein of BTV 49 (NS4) is critical to counteract the innate immune response of the host. Infection of cells with 50 a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of 51 interferon stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by 52 favouring viral replication in sheep, the animal species most susceptible to bluetongue. 53 and the host immune responses. In this study we show that a non-structural protein of BTV 49 (NS4) is critical to counteract the innate immune response of the host. Infection of cells with 50 a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of 51 interferon stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by 52 favouring viral replication in sheep, the animal species most susceptible to bluetongue. 53 54 3 \| P a g e 3 \| P a g e INTRODUCTION INTRODUCTION 55 Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of 56 ruminants with serious consequences to both animal health and the economy (1). 57 Throughout the twentieth century, bluetongue has occurred almost exclusively in tropical 58 and sub-tropical geographical areas (2). However, in the last two decades the geographical 59 limits of the disease have expanded and BTV is now endemic in more temperate areas such 60 as Southern and Central Europe (3). 61 Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of 56 ruminants with serious consequences to both animal health and the economy (1). 57 Throughout the twentieth century, bluetongue has occurred almost exclusively in tropical 58 and sub-tropical geographical areas (2). However, in the last two decades the geographical 59 limits of the disease have expanded and BTV is now endemic in more temperate areas such 60 as Southern and Central Europe (3). 61 BTV is an arbovirus transmitted by Culicoides spp. biting midges and belongs to the Orbivirus 62 genus within the Reoviridae (4, 5). The BTV genome consists of 10 dsRNA genome segments 63 encoding for seven structural and four, possibly five, non-structural proteins (6-9). The core 64 particle, formed by VP3 and VP7, encapsidates the viral genome segments, each associated 65 with a replicase complex comprising VP1, VP4 and VP6 (6, 10-12). VP2 and VP5 constitute 66 the outer capsid of the BTV virion and are responsible for cell attachment and entry (13-15). 67 VP2 is the most variable BTV protein and determines the virus serotype, of which there are 68 27 described to date (16-19). 69 BTV is an arbovirus transmitted by Culicoides spp. biting midges and belongs to the Orbivirus 62 genus within the Reoviridae (4, 5). The BTV genome consists of 10 dsRNA genome segments 63 encoding for seven structural and four, possibly five, non-structural proteins (6-9). The core 64 particle, formed by VP3 and VP7, encapsidates the viral genome segments, each associated 65 with a replicase complex comprising VP1, VP4 and VP6 (6, 10-12). VP2 and VP5 constitute 66 the outer capsid of the BTV virion and are responsible for cell attachment and entry (13-15). 67 VP2 is the most variable BTV protein and determines the virus serotype, of which there are 68 27 described to date (16-19). 69 The BTV non-structural proteins play fundamental roles in virus replication. NS1 forms 70 tubules in the cytoplasm of BTV infected cells and favours viral protein synthesis (20-22). 71 NS2 is the major component of viral inclusion bodies (23-25), while NS3/NS3A play a critical 72 role in virus intracellular trafficking and egress (26, 27). NS3 has also been shown to 73 downregulate transcription from the IFN-β promoter in reporter assays (28). A putative fifth 74 non-structural protein may be expressed from a conserved small open reading frame in 75 segment 10 (29). 76 NS4 is a small protein (77-79 amino acid residues) encoded by an open reading frame (ORF) 77 in genome segment 9 overlapping the larger ORF encoding for VP6 and with nucleolar 78 localization. Although NS4 has been shown to confer a replication advantage to BTV in cells 79 treated with interferon, it is dispensable for BTV replication in IFN incompetent cell lines 80 and has no impact on pathogenicity in IFNAR-/- mice (8, 9). 81 The clinical outcome of BTV infection is highly variable and dependent upon a variety of 82 viral, host and likely environmental factors (30-35). Understanding the interplay between 83 BTV and the host immune response will be central to identifying the virus and host 84 determinants of disease susceptibility. 85 The clinical outcome of BTV infection is highly variable and dependent upon a variety of 82 viral, host and likely environmental factors (30-35). Understanding the interplay between 83 BTV and the host immune response will be central to identifying the virus and host 84 determinants of disease susceptibility. 85 The clinical outcome of BTV infection is highly variable and dependent upon a variety of 82 viral, host and likely environmental factors (30-35). Understanding the interplay between 83 BTV and the host immune response will be central to identifying the virus and host 84 determinants of disease susceptibility. 85 In order to establish a successful infection, BTV must overcome both physical and innate 86 immune barriers. One of the key innate immune mechanisms to fight viral infections is the 87 IFN system (36). Mammalian cells possess pattern-recognition receptors that detect 88 incoming pathogen signatures and induce the synthesis and secretion of IFN-β. Secreted IFN 89 signals in both an autocrine and paracrine fashion, leading to the transcription of hundreds 90 of IFN-stimulated genes (ISGs), some of which have a direct or indirect antiviral effect (37). 91 BTV is known to induce an IFN response, both in vitro and in vivo, and consequently it must 92 possess countermeasures that allow the virus to replicate in the face of this host response 93 (8, 38-43). Evidence to date suggests that BTV NS3 and NS4 are the viral proteins most likely 94 responsible for the disruption of the cellular innate immune response to BTV infection (8, 95 28). 96 In order to establish a successful infection, BTV must overcome both physical and innate 86 immune barriers. One of the key innate immune mechanisms to fight viral infections is the 87 IFN system (36). Mammalian cells possess pattern-recognition receptors that detect 88 incoming pathogen signatures and induce the synthesis and secretion of IFN-β. Secreted IFN 89 signals in both an autocrine and paracrine fashion, leading to the transcription of hundreds 90 of IFN-stimulated genes (ISGs), some of which have a direct or indirect antiviral effect (37). 91 BTV is known to induce an IFN response, both in vitro and in vivo, and consequently it must 92 possess countermeasures that allow the virus to replicate in the face of this host response 93 (8, 38-43). Evidence to date suggests that BTV NS3 and NS4 are the viral proteins most likely 94 responsible for the disruption of the cellular innate immune response to BTV infection (8, 95 28). 96 In this study, we investigated the interaction of BTV and the IFN response of the host. We 97 demonstrate that NS4 is an important virulence factor in sheep, a natural host of BTV 98 infection, and acts as an interferon antagonist. 99 In this study, we investigated the interaction of BTV and the IFN response of the host. We 97 demonstrate that NS4 is an important virulence factor in sheep, a natural host of BTV 98 infection, and acts as an interferon antagonist. 99 5 \| P a g e 5 \| P a g e MATERIALS AND METHODS MATERIALS AND METHODS 100 101 Cell cultures. Vero, BHK21 and BSR cells (a clone of BHK21, kindly provided by Karl K. 102 Conzelmann) (44) were grown in Dulbecco’s modified Eagle’s medium (DMEM) 103 supplemented with 10% fetal bovine serum (FBS). CPT-Tert cells are sheep choroid plexus 104 cells immortalized with the simian virus 40 (SV40) T antigen and human telomerase reverse 105 transcriptase (hTERT) (45). CPT-Tert cells were grown in Iscove’s modified Dulbecco’s 106 medium (IMDM), supplemented with 10% FBS. A549 cells are derived from a human lung 107 adenocarcinoma and were grown in DMEM supplemented with 10% FBS. All cell lines were 108 cultured at 37°C in a 5% CO2 humidified atmosphere. 109 Primary ovine endothelial (ovEC) cells were obtained as previously described (46). ovEC 110 were seeded in 12-well plates and maintained in a low oxygen incubator (37°C, 5% CO2 and 111 3% O2). In this study, ovEC were passaged once before being used and cultured at 37°C in a 112 5% CO2 humidified atmosphere. 113 Primary ovine endothelial (ovEC) cells were obtained as previously described (46). ovEC 110 were seeded in 12-well plates and maintained in a low oxygen incubator (37°C, 5% CO2 and 111 3% O2). In this study, ovEC were passaged once before being used and cultured at 37°C in a 112 5% CO2 humidified atmosphere. 113 Viruses. Wild type BTV8 (BTV8wt) and a NS4 deletion mutant (BTV8ΔNS4) viruses used in 114 this study were obtained by reverse genetics as described previously (8, 47). Virus stock 115 titres were determined by standard plaque assays using CPT-Tert cells (48). The defective 116 interfering-rich (DI-rich) preparations of Sendai virus (SeV, Cantell strain) was generated by 117 sequentially passaging the virus at high MOI as previously described (49). 118 Encephalomyocarditis virus (EMCV) was used in interferon protection assays (see below) 119 and was prepared as previously described (50). 120 Virus replication curves. Replication curves were carried out in either ovEC or A549 cells. 121 Cells were infected with the appropriate virus (MOI = 0.01) and supernatants were collected 122 at 24, 48 and 72h post-infection (pi). Supernatants were subsequently titrated by endpoint 123 Virus replication curves. Replication curves were carried out in either ovEC or A549 cells. 121 Cells were infected with the appropriate virus (MOI = 0.01) and supernatants were collected 122 at 24, 48 and 72h post-infection (pi). MATERIALS AND METHODS Supernatants were subsequently titrated by endpoint 123 6 \| P a g e dilution analysis on BSR cells using the method of Reed and Muench and expressed as log10 124 (TCID50/ml) (51). Each experiment was performed independently in triplicate using at least 125 two different stocks of each virus. 126 Ethical Statement. All animal experimental procedures carried out in this study were 127 approved by the ethical committee of the Istituto Zooprofilattico Sperimentale dell'Abruzzo 128 e Molise “G. Caporale” (Teramo, Italy) (protocol no. 11427/2012) and further approved by 129 the Italian Ministry of Health (Ministero della Salute) in accordance with Council Directive 130 86/609/EEC of the European Union and the Italian D.Igs 116/92. 131 Ethical Statement. All animal experimental procedures carried out in this study were 127 approved by the ethical committee of the Istituto Zooprofilattico Sperimentale dell'Abruzzo 128 e Molise “G. Caporale” (Teramo, Italy) (protocol no. 11427/2012) and further approved by 129 the Italian Ministry of Health (Ministero della Salute) in accordance with Council Directive 130 86/609/EEC of the European Union and the Italian D.Igs 116/92. 131 In vivo pathogenicity studies. Experiments were carried out using 15 sheep (Italian mixed- 132 breed) in an insect-proof isolation unit. Before inoculation, all animals were confirmed to 133 have no antibodies against BTV using a blocking enzyme-linked immunosorbent assay 134 (ELISA) as previously described (52). The absence of BTV genome in blood samples of each 135 animal was also confirmed by qRT-PCR as already described (30). Animals (n=5 per group) 136 were infected with 5 ml of either BTV8wt or BTV8ΔNS4 (2×106 PFU in total) by multiple 137 intradermal inoculations in the inner leg and prescapular areas. Negative controls were 138 inoculated with 5 ml of mock-infected cell supernatants. Body temperature was recorded 139 daily, beginning a week before inoculation, until day 14 pi and subsequently at days 17, 21, 140 and 28. Fever was defined as rectal temperature above 40°C. EDTA blood samples were 141 collected daily from all animals for 14 days pi and thereafter at days 17, 21, and 28, when 142 the experiment was terminated. Blood samples were analyzed for the presence of viremia 143 by qRT-PCR as previously described (30). Sera were collected from each animal on the day of 144 the inoculation (day 0) and then at days 7, 14, 21, and 28 pi. MATERIALS AND METHODS The presence of neutralizing 145 antibodies in infected sheep against BTV8 was assessed by virus neutralization assay as 146 previously described (53). 147 In vivo pathogenicity studies. Experiments were carried out using 15 sheep (Italian mixed- 132 breed) in an insect-proof isolation unit. Before inoculation, all animals were confirmed to 133 have no antibodies against BTV using a blocking enzyme-linked immunosorbent assay 134 (ELISA) as previously described (52). The absence of BTV genome in blood samples of each 135 animal was also confirmed by qRT-PCR as already described (30). Animals (n=5 per group) 136 were infected with 5 ml of either BTV8wt or BTV8ΔNS4 (2×106 PFU in total) by multiple 137 intradermal inoculations in the inner leg and prescapular areas. Negative controls were 138 inoculated with 5 ml of mock-infected cell supernatants. Body temperature was recorded 139 daily, beginning a week before inoculation, until day 14 pi and subsequently at days 17, 21, 140 and 28. Fever was defined as rectal temperature above 40°C. EDTA blood samples were 141 collected daily from all animals for 14 days pi and thereafter at days 17, 21, and 28, when 142 the experiment was terminated. Blood samples were analyzed for the presence of viremia 143 by qRT-PCR as previously described (30). Sera were collected from each animal on the day of 144 the inoculation (day 0) and then at days 7, 14, 21, and 28 pi. The presence of neutralizing 145 antibodies in infected sheep against BTV8 was assessed by virus neutralization assay as 146 previously described (53). 147 7 \| P a g e 7 \| P a g e Plasmids and antisera. The open reading frame encoding NS4 was either amplified by PCR 148 (BTV8 NET2006/04 (GenBank Accession number JX680455), BTV1 RSArrrr/01 (JX680465), 149 BTV-2IT(L) (JN255870)), or synthesised commercially (Genscript), ((BTV-2RSA(WT) 150 (JN255930), BTV-9IT(L) (JN255910), BTV1SASEG9 (D10905), BTV25 (EU839845), BTV26 151 (JN255161)) and cloned into the pCI mammalian expression vector (Promega). BTV10 152 segment 8 (NC006007) was synthesized commercially (Genscript) and cloned into pCI. All 153 plasmids used in this study were sequenced before use. Plasmid pCMV-luc was obtained by 154 inserting the firefly luciferase (FLuc) open reading frame into pCDNA3.1 (Invitrogen) as 155 previously described (54). pRL-CMV (Promega) is similar to pCMV-luc but contains an intron 156 before the Renilla luciferase gene. p125Luc expresses FLuc under the control of the IFN-β 157 promoter (55). MATERIALS AND METHODS Total RNA was then extracted using the TRIzol method and further purified using 180 the RNeasy mini spin columns (Qiagen), including an on-column DNase I digestion step 181 (Qiagen) according to the manufacturer’s protocol. 182 Labelling and extraction of nascent RNA for transcriptomic analyses. A549 cells were 176 seeded in 6-well plates, incubated at 37°C for 24h and mock-infected or infected with 177 BTV8wt or BTV8ΔNS4 at a MOI of 4. At 8, 12 and 16h post-infection (pi), nascent RNA was 178 labelled with 0.4 mM EU for 90 min. Cells were lysed with 1 ml of TRIzol® spiked with 1 ng of 179 EU-Luc-pA. Total RNA was then extracted using the TRIzol method and further purified using 180 the RNeasy mini spin columns (Qiagen), including an on-column DNase I digestion step 181 (Qiagen) according to the manufacturer’s protocol. 182 RNAseq. 4.5 μg of total RNA was enriched by selectively depleting rRNA using the 183 RiboMinus™ Eukaryote Kit v2 (Ambion, Life Technologies). The EU-labelled RNA was 184 specifically linked to an azide-modified biotin by a chemical reaction and then captured on 185 streptavidin magnetic beads using the Click-iT® Nascent RNA Capture Kit (Molecular probes, 186 Life Technologies). Biotin-labelled RNA (attached to the magnetic beads) was fragmented 187 and subsequently used to construct libraries using the Ion Total RNAseq v2 Kit (Life 188 Technologies) as described by the manufacturer. Amplified libraries were size-selected using 189 the E-Gel size select system (Life Technologies) and assessed using a Tapestation (Agilent). 190 Libraries were quantified using the Qubit HS dsDNA assay (Life Technologies) and sequenced 191 using the Ion Proton sequencer (Life Technologies). The sequence reads were processed 192 according to the Tuxedo pipeline (57). Read quality was first assessed using FastQC (58). 193 Tophat2 and Bowtie2 were used to map short reads against the Homo Sapiens genome 194 (UCSC hg19) and a list of differentially expressed genes was generated using CuffDiff2 195 \| RNAseq. 4.5 μg of total RNA was enriched by selectively depleting rRNA using the 183 RiboMinus™ Eukaryote Kit v2 (Ambion, Life Technologies). The EU-labelled RNA was 184 specifically linked to an azide-modified biotin by a chemical reaction and then captured on 185 streptavidin magnetic beads using the Click-iT® Nascent RNA Capture Kit (Molecular probes, 186 Life Technologies). MATERIALS AND METHODS pISRE-Luc (Promega) contains five copies of the ISRE-binding sequence (56) 158 located upstream of the TATA like promoter region from the herpes simplex virus thymidine 159 kinase (HSV-TK) promoter. 160 Antisera used in this study included polyclonal rabbit antisera raised against the BTV NS4 161 and NS2 proteins as previously described (8). Antibodies against B23 (Sigma), α-tubulin 162 (Sigma), IRF-3 (clone FL-425, Santa Cruz) and NF-κB p65 (clone D14E12, cell signalling) were 163 obtained commercially. 164 In vitro RNA transcription. Capped and polyadenylated RNA for use in luciferase assays was 165 generated using the mMESSAGE mMACHINE® T7 Ultra Kit as recommended by the 166 manufacturers (Ambion, Life Technologies), using linearised pCMV-luc or pRL-CMV as a 167 template. 168 8 \| P a g e An RNA control (named EU-Luc-pA) for use in RNAseq and qRT-PCR experiments was 169 obtained as follows. Linearized pCMV-luc was transcribed using the Megascript® T7 kit 170 (Ambion, Life Technologies) according to the manufacturer’s instructions, with the 171 8 \| P a g e An RNA control (named EU-Luc-pA) for use in RNAseq and qRT-PCR experiments was 169 obtained as follows. Linearized pCMV-luc was transcribed using the Megascript® T7 kit 170 (Ambion, Life Technologies) according to the manufacturer’s instructions, with the 171 exception that the transcription reaction mix was supplemented with an analogue of uridine 172 (5-Ethynyl Uridine [EU], Invitrogen, Life Technologies). The RNA was then polyadenylated 173 following the polyA tailing procedure of the mMESSAGE mMACHINE® T7 Ultra Kit. All in vitro 174 transcribed RNA was recovered using the RNeasy Mini Kit (Qiagen). 175 exception that the transcription reaction mix was supplemented with an analogue of uridine 172 (5-Ethynyl Uridine [EU], Invitrogen, Life Technologies). The RNA was then polyadenylated 173 following the polyA tailing procedure of the mMESSAGE mMACHINE® T7 Ultra Kit. All in vitro 174 transcribed RNA was recovered using the RNeasy Mini Kit (Qiagen). 175 Labelling and extraction of nascent RNA for transcriptomic analyses. A549 cells were 176 seeded in 6-well plates, incubated at 37°C for 24h and mock-infected or infected with 177 BTV8wt or BTV8ΔNS4 at a MOI of 4. At 8, 12 and 16h post-infection (pi), nascent RNA was 178 labelled with 0.4 mM EU for 90 min. Cells were lysed with 1 ml of TRIzol® spiked with 1 ng of 179 EU-Luc-pA. MATERIALS AND METHODS Biotin-labelled RNA (attached to the magnetic beads) was fragmented 187 and subsequently used to construct libraries using the Ion Total RNAseq v2 Kit (Life 188 Technologies) as described by the manufacturer. Amplified libraries were size-selected using 189 the E-Gel size select system (Life Technologies) and assessed using a Tapestation (Agilent). 190 Libraries were quantified using the Qubit HS dsDNA assay (Life Technologies) and sequenced 191 using the Ion Proton sequencer (Life Technologies). The sequence reads were processed 192 according to the Tuxedo pipeline (57). Read quality was first assessed using FastQC (58). 193 Tophat2 and Bowtie2 were used to map short reads against the Homo Sapiens genome 194 (UCSC hg19) and a list of differentially expressed genes was generated using CuffDiff2 195 9 \| P a g e (genes with Benjamini Hochberg P-value ≤ 0.05 were considered significant) (59, 60). 196 Canonical pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) 197 software (Qiagen) by submitting the differentially expressed (DE) gene datasets (Tables S1- 198 3) and using the default parameters. 199 (genes with Benjamini Hochberg P-value ≤ 0.05 were considered significant) (59, 60). 196 Canonical pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) 197 software (Qiagen) by submitting the differentially expressed (DE) gene datasets (Tables S1- 198 3) and using the default parameters. 199 qRT-PCR For qRT-PCR analysis, mRNA was purified from the total RNA fraction using the 200 Dynabeads® mRNA DIRECT™ Kit (Ambion, Life Technologies) according to the 201 manufacturer’s instructions, before capturing EU-labelled RNA as described above. RNA 202 captured on the Streptavidin beads was used directly as a template for cDNA synthesis using 203 the SuperScript® VILO™ cDNA synthesis Kit (Invitrogen, Life Technologies). qPCR was 204 performed using the Brilliant III Ultra Fast QPCR Master Mix (Agilent) to detect a selection of 205 ISGs, housekeeping genes and the spiked EU-Luc-pA. Sequences of primers and probes are 206 available upon request. Samples were run on an Mx3005P (Stratagene) PCR machine and 207 analyzed using the MxPro software (Stratagene). Mock-infected cells were used as a 208 calibrator against which the infected cells were compared. The EU-Luc-pA RNA was used as 209 an exogenous control to normalise the results. 210 qRT-PCR For qRT-PCR analysis, mRNA was purified from the total RNA fraction using the 200 Dynabeads® mRNA DIRECT™ Kit (Ambion, Life Technologies) according to the 201 manufacturer’s instructions, before capturing EU-labelled RNA as described above. MATERIALS AND METHODS For quantitative 221 western blotting, primary antibodies were detected using fluorescently labelled secondary 222 antibodies (DyLight, Thermo Scientific) in a LI-COR Odyssey scanner. Bands were quantified 223 with the Odyssey software (LI-COR Biosciences). 224 Western blotting. Protein expression was assessed from total cell lysates by sodium 219 dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using 220 the various antisera indicated above and as previously described (61). For quantitative 221 western blotting, primary antibodies were detected using fluorescently labelled secondary 222 antibodies (DyLight, Thermo Scientific) in a LI-COR Odyssey scanner. Bands were quantified 223 with the Odyssey software (LI-COR Biosciences). 224 Western blotting. Protein expression was assessed from total cell lysates by sodium 219 dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using 220 the various antisera indicated above and as previously described (61). For quantitative 221 western blotting, primary antibodies were detected using fluorescently labelled secondary 222 antibodies (DyLight, Thermo Scientific) in a LI-COR Odyssey scanner. Bands were quantified 223 with the Odyssey software (LI-COR Biosciences). 224 Metabolic radiolabeling. ovEC cells were mock-infected or infected with BTV8wt or 225 BTV8ΔNS4 at a MOI of 4. At the time points indicated, cells were incubated at 37°C for 30 226 min in media lacking methionine. Nascent proteins were labelled for 2 h with 35S 227 methionine/cysteine (0.8 MBq/ml; PerkinElmer) after which they were resuspended in 228 sample buffer (60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol, 229 0.01% bromophenol blue) and the proteins separated by SDS-PAGE as described above. 230 Visualisation of labelled proteins was achieved by exposure to a Storm840 Phospho-imager 231 (Molecular Dynamics) or to X-ray film. Band intensities were determined using ImageQuant 232 software (Amersham). 233 Metabolic radiolabeling. ovEC cells were mock-infected or infected with BTV8wt or 225 BTV8ΔNS4 at a MOI of 4. At the time points indicated, cells were incubated at 37°C for 30 226 min in media lacking methionine. Nascent proteins were labelled for 2 h with 35S 227 methionine/cysteine (0.8 MBq/ml; PerkinElmer) after which they were resuspended in 228 sample buffer (60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol, 229 0.01% bromophenol blue) and the proteins separated by SDS-PAGE as described above. 230 Visualisation of labelled proteins was achieved by exposure to a Storm840 Phospho-imager 231 (Molecular Dynamics) or to X-ray film. Band intensities were determined using ImageQuant 232 software (Amersham). 233 Luciferase assays. MATERIALS AND METHODS RNA 202 captured on the Streptavidin beads was used directly as a template for cDNA synthesis using 203 the SuperScript® VILO™ cDNA synthesis Kit (Invitrogen, Life Technologies). qPCR was 204 performed using the Brilliant III Ultra Fast QPCR Master Mix (Agilent) to detect a selection of 205 ISGs, housekeeping genes and the spiked EU-Luc-pA. Sequences of primers and probes are 206 available upon request. Samples were run on an Mx3005P (Stratagene) PCR machine and 207 analyzed using the MxPro software (Stratagene). Mock-infected cells were used as a 208 calibrator against which the infected cells were compared. The EU-Luc-pA RNA was used as 209 an exogenous control to normalise the results. 210 Interferon protection assays. Interferon protection assays were performed as previously 211 described (46). Briefly, OvEC cells were first seeded in 12-well plates and incubated for 2 212 days. Cells were then mock-infected or infected with the indicated virus at a MOI of 4 and 213 the medium was collected 16h post-infection. Cell culture supernatants were treated with 214 UV light to inactivate any virus. CPT-Tert cells were incubated with two-fold dilutions of the 215 medium for 24h, before infecting them with EMCV for 72h. The level of IFN (expressed as 216 “international units”, IU) was calculated by monitoring wells that were protected from cell 217 death induced by EMCV and comparing them to known amounts of universal IFN. 218 Interferon protection assays. Interferon protection assays were performed as previously 211 described (46). Briefly, OvEC cells were first seeded in 12-well plates and incubated for 2 212 days. Cells were then mock-infected or infected with the indicated virus at a MOI of 4 and 213 the medium was collected 16h post-infection. Cell culture supernatants were treated with 214 UV light to inactivate any virus. CPT-Tert cells were incubated with two-fold dilutions of the 215 medium for 24h, before infecting them with EMCV for 72h. The level of IFN (expressed as 216 “international units”, IU) was calculated by monitoring wells that were protected from cell 217 death induced by EMCV and comparing them to known amounts of universal IFN. 218 10 \| P a g e 10 \| P a g e Western blotting. Protein expression was assessed from total cell lysates by sodium 219 dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using 220 the various antisera indicated above and as previously described (61). MATERIALS AND METHODS CPT-Tert cells grown in 24-well plates were co-transfected using 234 Lipofectamine 2000 (Invitrogen) with 100ng of pCMV-luc or pRL-CMV along with 100 to 400 235 ng of the indicated pCI plasmids expressing NS4. A similar procedure has been performed to 236 co-transfect 200 ng of in vitro transcribed RNA expressing firefly luciferase (see above) and 237 the appropriate expression plasmids as indicated (100 to 400 ng). When using p125Luc (50 238 ng) and pISRE-luc (400 ng), 293T cells were either infected with DI-rich preparations of SeV 239 or treated with 200 U/ml of Universal Interferon (UIFN, PBL InterferonSource) 4h post 240 transfection, before incubating for a further for 18h. 241 Luciferase assays. CPT-Tert cells grown in 24-well plates were co-transfected using 234 Lipofectamine 2000 (Invitrogen) with 100ng of pCMV-luc or pRL-CMV along with 100 to 400 235 ng of the indicated pCI plasmids expressing NS4. A similar procedure has been performed to 236 co-transfect 200 ng of in vitro transcribed RNA expressing firefly luciferase (see above) and 237 the appropriate expression plasmids as indicated (100 to 400 ng). When using p125Luc (50 238 ng) and pISRE-luc (400 ng), 293T cells were either infected with DI-rich preparations of SeV 239 or treated with 200 U/ml of Universal Interferon (UIFN, PBL InterferonSource) 4h post 240 transfection, before incubating for a further for 18h. 241 11 \| P a g e 11 \| P a g e The total amount of plasmid DNA transfected used was 500ng for all experiments, using the 242 empty pCI plasmid to balance each transfection. Luciferase activity was detected 22h post- 243 transfection using the Luciferase or Dual Luciferase Assay System (Promega) as described by 244 the manufacturer. The percentage of luciferase was determined by assigning the luciferase 245 activity or luminescence (expressed as relative light unit, RLU) detected in control cells (i.e. 246 transfected with pCMV-luc and an empty pCI plasmid only) as 100%. Constitutive promoters 247 as transfection controls could not be used as NS4 affects gene expression. However, each 248 experiment was repeated independently at least three times with each samples assessed in 249 in triplicate. For each experiment, at least two independent plasmid preparations were 250 used. Relative lights units for each sample were always at least 100 fold above background. 251 In addition, in some experiments, 5 ng of in vitro transcribed RNA encoding renilla luciferase 252 were used as additional internal control. MATERIALS AND METHODS BTV RNA was below the detection levels in 4 of the 5 inoculated sheep at 4 weeks 275 pi when the experiment was stopped (Fig. 1, middle panels). Between days 4 and 28 pi, the 276 average levels of BTV RNA were between 102 and 105 fold higher in sheep infected with 277 BTV8wt compared to those infected with BTV8ΔNS4. As expected, all BTV infected animals 278 developed neutralizing antibodies from day 7 pi (Fig. 1, lower panels). Altogether, these 279 data show that BTV8ΔNS4 is attenuated in sheep and suggest that NS4 is a virulence factor 280 in vivo. 281 BTV8ΔNS4 is attenuated in experimentally infected sheep. In a previous study, we showed 266 that a BTV8 NS4 deletion mutant (BTV8ΔNS4) is as virulent as BTV8 wt in experimental 267 mouse models of infection (8). Here we wanted to determine whether NS4 influenced 268 virulence in sheep, the natural host of BTV infection. Therefore, we experimentally infected 269 sheep with BTV8wt or BTV8ΔNS4. Sheep infected with BTV8wt showed an elevation of body 270 temperature from day 6 to day 8 pi (Fig. 1, top panels). BTV RNA was detectable at day 4 pi, 271 reached a plateau from day 6 to 10 pi and was followed by a slow decrease. Viremia 272 remained detectable until the end of the experiment. In contrast, animals infected by 273 BTV8ΔNS4 did not develop pyrexia and displayed a delayed onset and lower levels of 274 viremia. BTV RNA was below the detection levels in 4 of the 5 inoculated sheep at 4 weeks 275 pi when the experiment was stopped (Fig. 1, middle panels). Between days 4 and 28 pi, the 276 average levels of BTV RNA were between 102 and 105 fold higher in sheep infected with 277 BTV8wt compared to those infected with BTV8ΔNS4. As expected, all BTV infected animals 278 developed neutralizing antibodies from day 7 pi (Fig. 1, lower panels). Altogether, these 279 data show that BTV8ΔNS4 is attenuated in sheep and suggest that NS4 is a virulence factor 280 in vivo. 281 13 \| P a g e Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 human A549 cells. MATERIALS AND METHODS 253 Confocal microscopy. Experiments were performed using CPT-Tert or A549 cells cultured in 254 two-well glass chamber slides (Lab-Tek, Nalge Nunc International). Cells were either 255 transfected with the appropriate plasmids or infected with the indicated viruses (MOI = 4) 256 for 16-22h. Cells were washed with PBS and fixed with 5% formaldehyde for 15 min. Fixed 257 cells were then processed as described previously (62) and incubated with the appropriate 258 antisera. Secondary antibodies were conjugated with either Alexa Fluor 488 or Alexa Fluor 259 594 (Invitrogen, Molecular Probes). Slides were mounted using Vectashield mounting 260 medium with DAPI (4’,6-diamidino-2-phenylindole, Vector Laboratories). Slides were 261 analysed and images collected using a Leica TCS SP2 confocal microscope. 262 Confocal microscopy. Experiments were performed using CPT-Tert or A549 cells cultured in 254 two-well glass chamber slides (Lab-Tek, Nalge Nunc International). Cells were either 255 transfected with the appropriate plasmids or infected with the indicated viruses (MOI = 4) 256 for 16-22h. Cells were washed with PBS and fixed with 5% formaldehyde for 15 min. Fixed 257 cells were then processed as described previously (62) and incubated with the appropriate 258 antisera. Secondary antibodies were conjugated with either Alexa Fluor 488 or Alexa Fluor 259 594 (Invitrogen, Molecular Probes). Slides were mounted using Vectashield mounting 260 medium with DAPI (4’,6-diamidino-2-phenylindole, Vector Laboratories). Slides were 261 analysed and images collected using a Leica TCS SP2 confocal microscope. 262 12 \| P a g e 12 \| P a g e RESULTS 265 BTV8ΔNS4 is attenuated in experimentally infected sheep. In a previous study, we showed 266 that a BTV8 NS4 deletion mutant (BTV8ΔNS4) is as virulent as BTV8 wt in experimental 267 mouse models of infection (8). Here we wanted to determine whether NS4 influenced 268 virulence in sheep, the natural host of BTV infection. Therefore, we experimentally infected 269 sheep with BTV8wt or BTV8ΔNS4. Sheep infected with BTV8wt showed an elevation of body 270 temperature from day 6 to day 8 pi (Fig. 1, top panels). BTV RNA was detectable at day 4 pi, 271 reached a plateau from day 6 to 10 pi and was followed by a slow decrease. Viremia 272 remained detectable until the end of the experiment. In contrast, animals infected by 273 BTV8ΔNS4 did not develop pyrexia and displayed a delayed onset and lower levels of 274 viremia. MATERIALS AND METHODS We showed previously that BTV8ΔNS4 replicates as efficiently as BTV8wt 283 in immortalized cell lines, such as hamster BSR and sheep CPT-Tert cells (8) but these cell 284 lines do not possess an intact IFN response to viral infections (44, 45). Hence, we compared 285 the replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells (ovEC). 286 BTV8wt reached titers approximately 20 fold higher than BTV8ΔNS4 at 72h pi (Fig. 2A). 287 Similarly, BTV8wt reached titers nearly 60 fold higher than BTV8ΔNS4 in the IFN-competent 288 Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 14 \| P a g e human cell line A549 (Fig. 2A). These data demonstrate that the presence of NS4 confers a 289 replication advantage to BTV8 in cells that are capable of mounting an antiviral response. 290 NS4 inhibits IFN release, but not virus sensing, in infected cells. Given the data obtained 291 above and the previously published observation that NS4 confers a replication advantage to 292 BTV in cells pre-treated with IFN (8), we next sought to determine whether NS4 impacted 293 the level of IFN released into the supernatant of infected cells. Primary ovEC were infected 294 at a MOI of 4 with either BTV8wt or BTV8ΔNS4. 16 h pi the level of IFN in cell culture media 295 was measured using an IFN protection assay. BTV8ΔNS4 infected cells had, on average, 13 296 times more IFN (p<0.001) in their supernatants than BTV8wt infected cells (Fig. 2B). 297 These results could potentially be explained by NS4 affecting either the sensing of viral 298 infection, or transcription and/or the synthesis/secretion of IFN into the supernatant. The 299 first step in IFN-β expression is the recognition of pathogen-associated molecular patterns 300 (PAMPs) by various host pattern recognition receptors, which results in the translocation of 301 the transcription factors IRF-3 and NFκB into the nucleus (37). In order to determine 302 whether NS4 impacted supernatant levels of IFN by interfering with this process, we 303 infected A549 cells at a MOI of 4 for 16h and assessed the nuclear translocation of IRF-3 and 304 NFκB by confocal microscopy. The cellular localization of IRF-3 and NFκB in mock-infected 305 cells was exclusively cytoplasmic (Fig. 3) while stimulation with TNFα (tumor necrosis factor 306 α) used as positive control resulted in nuclear translocation of these proteins in essentially 307 100% of the cells (not shown). In contrast, between 25 and 35% of cells infected by BTV8wt 308 showed translocation of IRF-3 and NFκB into the nucleus. BTV8ΔNS4 was found to induce 309 similar levels of translocation as BTV8wt, suggesting that NS4 does not prevent either PAMP 310 recognition by the host cells or translocation of IRF-3 and NFκB. NS2 immunolabelling 311 confirmed similar levels of infection in cells infected with BTV8wt or BTV8∆NS4 (Fig. 3). 312 NS4 inhibits IFN release, but not virus sensing, in infected cells. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Given the data obtained 291 above and the previously published observation that NS4 confers a replication advantage to 292 BTV in cells pre-treated with IFN (8), we next sought to determine whether NS4 impacted 293 the level of IFN released into the supernatant of infected cells. Primary ovEC were infected 294 at a MOI of 4 with either BTV8wt or BTV8ΔNS4. 16 h pi the level of IFN in cell culture media 295 was measured using an IFN protection assay. BTV8ΔNS4 infected cells had, on average, 13 296 times more IFN (p<0.001) in their supernatants than BTV8wt infected cells (Fig. 2B). 297 These results could potentially be explained by NS4 affecting either the sensing of viral 298 infection, or transcription and/or the synthesis/secretion of IFN into the supernatant. The 299 first step in IFN-β expression is the recognition of pathogen-associated molecular patterns 300 (PAMPs) by various host pattern recognition receptors, which results in the translocation of 301 the transcription factors IRF-3 and NFκB into the nucleus (37). In order to determine 302 whether NS4 impacted supernatant levels of IFN by interfering with this process, we 303 infected A549 cells at a MOI of 4 for 16h and assessed the nuclear translocation of IRF-3 and 304 NFκB by confocal microscopy. The cellular localization of IRF-3 and NFκB in mock-infected 305 cells was exclusively cytoplasmic (Fig. 3) while stimulation with TNFα (tumor necrosis factor 306 α) used as positive control resulted in nuclear translocation of these proteins in essentially 307 100% of the cells (not shown). In contrast, between 25 and 35% of cells infected by BTV8wt 308 showed translocation of IRF-3 and NFκB into the nucleus. BTV8ΔNS4 was found to induce 309 similar levels of translocation as BTV8wt, suggesting that NS4 does not prevent either PAMP 310 recognition by the host cells or translocation of IRF-3 and NFκB. NS2 immunolabelling 311 confirmed similar levels of infection in cells infected with BTV8wt or BTV8∆NS4 (Fig. 3). 312 These results could potentially be explained by NS4 affecting either the sensing of viral 298 infection, or transcription and/or the synthesis/secretion of IFN into the supernatant. The 299 first step in IFN-β expression is the recognition of pathogen-associated molecular patterns 300 (PAMPs) by various host pattern recognition receptors, which results in the translocation of 301 the transcription factors IRF-3 and NFκB into the nucleus (37). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 In order to determine 302 whether NS4 impacted supernatant levels of IFN by interfering with this process, we 303 infected A549 cells at a MOI of 4 for 16h and assessed the nuclear translocation of IRF-3 and 304 NFκB by confocal microscopy. The cellular localization of IRF-3 and NFκB in mock-infected 305 cells was exclusively cytoplasmic (Fig. 3) while stimulation with TNFα (tumor necrosis factor 306 α) used as positive control resulted in nuclear translocation of these proteins in essentially 307 100% of the cells (not shown). In contrast, between 25 and 35% of cells infected by BTV8wt 308 showed translocation of IRF-3 and NFκB into the nucleus. BTV8ΔNS4 was found to induce 309 similar levels of translocation as BTV8wt, suggesting that NS4 does not prevent either PAMP 310 recognition by the host cells or translocation of IRF-3 and NFκB. NS2 immunolabelling 311 confirmed similar levels of infection in cells infected with BTV8wt or BTV8∆NS4 (Fig. 3). 312 14 \| P a g e 14 \| P a g e 15 \| P a g e NS4 downregulates IFN-β and interferon stimulated genes (ISGs) mRNA levels. Next, we 313 carried out RNAseq analysis on IFN-competent A549 cells infected with either BTV8wt or 314 BTV8ΔNS4 in order to further characterize the activity of NS4. Mock-infected A549 cells 315 were used as negative controls. Libraries were prepared from nascent RNA metabolically 316 labelled with a uracil analogue for 90 minutes at 12h pi (Fig. 4A), and sequenced on an Ion 317 Proton sequencer (Life Technologies, Thermo Fisher). On average, 64.58 million reads per 318 sample were generated (Phred quality > 20). We found 2863 differentially expressed genes 319 in cells infected by BTV8wt compared to mock-infected cells, out of which 1055 were 320 downregulated and 1808 were upregulated (Fig. 4B). In comparison, fewer genes (n=2292) 321 were found to be differentially expressed in cells infected by BTV8ΔNS4 compared to mock- 322 infected cells, with 752 of them being downregulated and 1540 upregulated. The entire list 323 of differentially expressed genes in BTV8wt and BTV8ΔNS4 infected cells is presented in 324 Tables S1-S3. CH25H (cholesterol 25-hydroxylase, a known ISG) was found to be the most 325 upregulated gene in BTV8ΔNS4 infected cells. 117 genes were upregulated in BTV8ΔNS4- 326 infected cells compared to BTV8wt-infected cells (Table S3). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Most of the pathways 343 were involved in the cellular immune response, cytokine signalling, inflammatory response, 344 apoptosis and pathogen related signalling. Consistent with the RNAseq dataset, pathways 345 relating to the innate immune response were particularly evident when comparing 346 BTV8ΔNS4 with BTV8wt (Fig. 5C). 347 The availability of the transcriptome of cells infected by BTV8wt and BTV8ΔNS4 also 348 provided additional information regarding the possible influence of NS4 on mRNA 349 maturation. Comparable proportions of reads containing intron sequences were found in 350 mock-infected cells or in cells infected with BTV8wt or BTV8ΔNS4 (data not shown). In 351 addition, we also quantified how many reads finished with 8 or more adenines, assuming 352 these reads as representative of polyadenylated mRNAs. Comparable percentages of polyA 353 reads were obtained in the three conditions tested, suggesting that NS4 was not associated 354 with a global defect in mRNA polyadenylation (data not shown). 355 The availability of the transcriptome of cells infected by BTV8wt and BTV8ΔNS4 also 348 provided additional information regarding the possible influence of NS4 on mRNA 349 maturation. Comparable proportions of reads containing intron sequences were found in 350 mock-infected cells or in cells infected with BTV8wt or BTV8ΔNS4 (data not shown). In 351 addition, we also quantified how many reads finished with 8 or more adenines, assuming 352 these reads as representative of polyadenylated mRNAs. Comparable percentages of polyA 353 reads were obtained in the three conditions tested, suggesting that NS4 was not associated 354 with a global defect in mRNA polyadenylation (data not shown). 355 \| NS4 modulates the activity of a wide range of promoters. We next assessed the ability of 356 BTV-8 NS4 to reduce the activity of basal promoters, such as the CMV immediate early 357 promoter, and promoters of genes involved in the host innate immune response (IFN-β and 358 ISRE-containing promoters). 293T cells were co-transfected with a plasmid expressing either 359 BTV NS4 or NS2, and a FLuc reporter plasmid driven by either the CMV promoter, the IFN-β 360 NS4 modulates the activity of a wide range of promoters. We next assessed the ability of 356 BTV-8 NS4 to reduce the activity of basal promoters, such as the CMV immediate early 357 promoter, and promoters of genes involved in the host innate immune response (IFN-β and 358 ISRE-containing promoters). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Of these 117 genes, 102 were 327 either IFN genes or ISGs according to the Interferome database (63). IFN-β, IFN-λ1, IFN-λ2 328 and IFN-λ3 were among the 6 highest upregulated genes in BTV8ΔNS4 infected cells (87 to 329 136 fold higher in comparison to mock-infected cells) (Table 1). Interestingly, more genes 330 were strongly upregulated (fold change >32) in BTV8ΔNS4 (n=33) compared to BTV8wt 331 infected cells (n= 7). These genes included MX1 and -2, IFIT1, -2 and -3, OASL and other well 332 characterised ISGs. However, all of the 33 genes highly upregulated in BTV8ΔNS4-infected 333 cells were also upregulated (albeit at a lower level) in BTV8wt infected cells (Table 1). IFN-β 334 for example, was also upregulated (17 fold) in BTV8wt infected cells. No major differences in 335 the levels of expression in BTV8wt and BTV8ΔNS4 infected cells were observed in those 336 15 \| P a g e genes with the highest levels of downregulation (Table 2). We validated the RNAseq analysis 337 by qRT-PCR on selected genes found equally or differentially expressed in mock- and 338 infected cells. Relative mRNA levels of IFNB1, IFIT1, B2M, ACTB, ANXA1 and TBP determined 339 by RT-qPCR reflected the patterns of expression observed in the RNAseq analyses (Fig. 4C). 340 The Ingenuity Pathway Analysis software (IPA; Qiagen) was used to compare the 341 representation of canonical pathways comparisons between infected vs uninfected cells (Fig 342 5A-B), and cells infected with either BTV-8wt or BTV8ΔNS4 (Fig 5C). Most of the pathways 343 were involved in the cellular immune response, cytokine signalling, inflammatory response, 344 apoptosis and pathogen related signalling. Consistent with the RNAseq dataset, pathways 345 relating to the innate immune response were particularly evident when comparing 346 BTV8ΔNS4 with BTV8wt (Fig. 5C). 347 genes with the highest levels of downregulation (Table 2). We validated the RNAseq analysis 337 by qRT-PCR on selected genes found equally or differentially expressed in mock- and 338 infected cells. Relative mRNA levels of IFNB1, IFIT1, B2M, ACTB, ANXA1 and TBP determined 339 by RT-qPCR reflected the patterns of expression observed in the RNAseq analyses (Fig. 4C). 340 The Ingenuity Pathway Analysis software (IPA; Qiagen) was used to compare the 341 representation of canonical pathways comparisons between infected vs uninfected cells (Fig 342 5A-B), and cells infected with either BTV-8wt or BTV8ΔNS4 (Fig 5C). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 293T cells were co-transfected with a plasmid expressing either 359 BTV NS4 or NS2, and a FLuc reporter plasmid driven by either the CMV promoter, the IFN-β 360 16 \| P a g e promoter or a promoter containing ISRE elements (Fig. 5). 4h post-transfection, cells 361 transfected with the IFN-β promoter were stimulated with Sendai virus while cells 362 transfected with ISRE-containing promoter were stimulated with universal IFN (200 U/ml). 363 NS4, unlike NS2, was able to reduce between 40 and 60% the activity of both CMV, IFN-β 364 and ISRE promoters (Fig. 6). 365 promoter or a promoter containing ISRE elements (Fig. 5). 4h post-transfection, cells 361 transfected with the IFN-β promoter were stimulated with Sendai virus while cells 362 transfected with ISRE-containing promoter were stimulated with universal IFN (200 U/ml). 363 NS4, unlike NS2, was able to reduce between 40 and 60% the activity of both CMV, IFN-β 364 and ISRE promoters (Fig. 6). 365 promoter or a promoter containing ISRE elements (Fig. 5). 4h post-transfection, cells 361 transfected with the IFN-β promoter were stimulated with Sendai virus while cells 362 transfected with ISRE-containing promoter were stimulated with universal IFN (200 U/ml). 363 NS4, unlike NS2, was able to reduce between 40 and 60% the activity of both CMV, IFN-β 364 and ISRE promoters (Fig. 6). 365 To further investigate the ability of NS4 to block host gene expression, sheep cells were co- 366 transfected with an expression plasmid for FLuc, under the control of the CMV immediate- 367 early promoter along with a variety of expression plasmids expressing BTV NS4, or an empty 368 plasmid (also containing a CMV promoter) as a control (Fig. 7). NS4 is well conserved among 369 the BTV serotypes/strains identified to date (8). The only exception was a strain of BTV-1 370 (GenBank accession number D10905 submitted in 1992) and the more divergent BTV-25 and 371 BTV-26 strains (EU839845 and JN255161) which showed only 77.9%, 76.6% and 75.3% 372 identity to BTV-8 NS4, respectively (Fig. 7A). All of the NS4 proteins tested were able to 373 reduce FLuc expression with the notable exception of the NS4 from the BTV/D10905 (Fig. 374 7B). Interestingly, the NS4 protein of BTV-1 D10905 displayed a different mobility from 375 other NS4 proteins by SDS-PAGE that could be explained by the presence of considerable 376 differences in its basic residues in the N-terminus. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 In addition, using BTV-8 NS4 we showed 377 that gene expression inhibition by NS4 was dose dependent (Fig. 7C). 378 To further investigate the ability of NS4 to block host gene expression, sheep cells were co- 366 transfected with an expression plasmid for FLuc, under the control of the CMV immediate- 367 early promoter along with a variety of expression plasmids expressing BTV NS4, or an empty 368 plasmid (also containing a CMV promoter) as a control (Fig. 7). NS4 is well conserved among 369 the BTV serotypes/strains identified to date (8). The only exception was a strain of BTV-1 370 (GenBank accession number D10905 submitted in 1992) and the more divergent BTV-25 and 371 BTV-26 strains (EU839845 and JN255161) which showed only 77.9%, 76.6% and 75.3% 372 identity to BTV-8 NS4, respectively (Fig. 7A). All of the NS4 proteins tested were able to 373 reduce FLuc expression with the notable exception of the NS4 from the BTV/D10905 (Fig. 374 7B). Interestingly, the NS4 protein of BTV-1 D10905 displayed a different mobility from 375 other NS4 proteins by SDS-PAGE that could be explained by the presence of considerable 376 differences in its basic residues in the N-terminus. In addition, using BTV-8 NS4 we showed 377 that gene expression inhibition by NS4 was dose dependent (Fig. 7C). 378 In a previous study, we showed that NS4 localizes in the nucleoli of infected or transfected 379 cells (8). Interestingly, BTV-1/D10905 NS4 was the only variant which failed to localise to the 380 nucleoli of sheep CPT-Tert cells (Fig. 7D), suggesting that nucleolar localization may be 381 critical for the activity of this non-structural protein. 382 \| NS4 is not the only viral protein involved in host cell protein shutoff. It is well established 383 that BTV induces protein synthesis shutoff in infected cells (64-66). Conceivably, a reduced 384 17 \| P a g e level of IFN (both mRNA and proteins) observed in cells infected with BTV8wt could reflect 385 the general virus-induced shut off of protein synthesis. In order to test this hypothesis, we 386 metabolically radiolabelled nascent proteins in ovEC cells mock-infected, or infected with 387 either BTV8wt or BTV8ΔNS4. As expected, we observed decreased levels of 35S-labelled 388 methionine/cysteine proteins in BTV8wt-infected cells compared to mock infected cells 389 (particularly evident at 18 and 26h pi), confirming previously published data (Fig. 8) (65, 66). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 390 Protein synthesis shutoff was also evident in BTV8ΔNS4 infected cells, although at 391 somewhat reduced levels compared to BTV8wt. In order to quantify the reduction in protein 392 synthesis during viral infection we used phosphoimaging and measured the signal intensity 393 of a prominent band present in all samples (Fig. 8A, black arrow). The signal intensity of 394 actin in BTV8wt-infected cells relative to mock-infected cells (taken as 100%) reduced 395 progressively to 69% at 10h pi, 37% at 18h pi and 2% at 24h pi, by which point cytopathic 396 effect was apparent. On the other hand, the signal intensity of actin was 82%, 56% and 12%. 397 Hence, these data suggest that host protein shutoff induced by BTV occurs largely 398 independently of NS4, although the latter may contribute to this phenomenon. 399 level of IFN (both mRNA and proteins) observed in cells infected with BTV8wt could reflect 385 the general virus-induced shut off of protein synthesis. In order to test this hypothesis, we 386 metabolically radiolabelled nascent proteins in ovEC cells mock-infected, or infected with 387 either BTV8wt or BTV8ΔNS4. As expected, we observed decreased levels of 35S-labelled 388 methionine/cysteine proteins in BTV8wt-infected cells compared to mock infected cells 389 (particularly evident at 18 and 26h pi), confirming previously published data (Fig. 8) (65, 66). 390 Protein synthesis shutoff was also evident in BTV8ΔNS4 infected cells, although at 391 somewhat reduced levels compared to BTV8wt. In order to quantify the reduction in protein 392 synthesis during viral infection we used phosphoimaging and measured the signal intensity 393 of a prominent band present in all samples (Fig. 8A, black arrow). The signal intensity of 394 actin in BTV8wt-infected cells relative to mock-infected cells (taken as 100%) reduced 395 progressively to 69% at 10h pi, 37% at 18h pi and 2% at 24h pi, by which point cytopathic 396 effect was apparent. On the other hand, the signal intensity of actin was 82%, 56% and 12%. 397 Hence, these data suggest that host protein shutoff induced by BTV occurs largely 398 independently of NS4, although the latter may contribute to this phenomenon. 399 18 \| P BTV-8 NS4 does not influence mRNA splicing nor translation. We also assessed the impact 400 of NS4 on RNA transcript splicing. interfere with the level of FLuc activity driven by RNA, as opposed to the activity driven by 409 plasmid DNA used as control, suggesting that the inhibition mediated by NS4 is happening 410 most likely at transcriptional and not at the translational level. 411 Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 CPT-Tert cells were transfected with the pRL-CMV vector, 401 which is driven by the CMV immediate early promoter and contains the RLuc gene 402 downstream of an intron. In this assay, NS4 retained the ability to inhibit the expression of 403 the reporter gene in a dose-dependent manner (Fig. 8B), indicating that NS4 does not affect 404 mRNA splicing and confirming the data obtained by RNAseq described above. Cells were 405 also co-transfected with in vitro transcribed RNA encoding FLuc and an expression plasmid 406 for the BTV8 NS4, in order to determine whether NS4-induced inhibition of protein 407 expression could also occur at translational level (Fig. 8C). Increasing levels of NS4 did not 408 18 \| P BTV-8 NS4 does not influence mRNA splicing nor translation. We also assessed the impact 400 of NS4 on RNA transcript splicing. CPT-Tert cells were transfected with the pRL-CMV vector, 401 which is driven by the CMV immediate early promoter and contains the RLuc gene 402 downstream of an intron. In this assay, NS4 retained the ability to inhibit the expression of 403 the reporter gene in a dose-dependent manner (Fig. 8B), indicating that NS4 does not affect 404 mRNA splicing and confirming the data obtained by RNAseq described above. Cells were 405 also co-transfected with in vitro transcribed RNA encoding FLuc and an expression plasmid 406 for the BTV8 NS4, in order to determine whether NS4-induced inhibition of protein 407 expression could also occur at translational level (Fig. 8C). Increasing levels of NS4 did not 408 18 \| P a g e interfere with the level of FLuc activity driven by RNA, as opposed to the activity driven by 409 plasmid DNA used as control, suggesting that the inhibition mediated by NS4 is happening 410 most likely at transcriptional and not at the translational level. 411 412 19 \| P a g e 19 \| P a g e 413 DISCUSSION In this study we showed that NS4 is an IFN-antagonist and a virulence factor for BTV. BTV 414 NS4 deletion mutants replicate as efficiently as wild type viruses in cell lines that lack a 415 competent innate immune system and are lethal to IFNAR-/- mice (8). In contrast, here we 416 demonstrated that NS4 facilitates BTV replication in vitro in IFN competent cells, and in vivo 417 in sheep, the animal species most affected by bluetongue. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 418 In this study we showed that NS4 is an IFN-antagonist and a virulence factor for BTV. BTV 414 NS4 deletion mutants replicate as efficiently as wild type viruses in cell lines that lack a 415 competent innate immune system and are lethal to IFNAR-/- mice (8). In contrast, here we 416 demonstrated that NS4 facilitates BTV replication in vitro in IFN competent cells, and in vivo 417 in sheep, the animal species most affected by bluetongue. 418 BTV induces a type 1 IFN response in vivo and in vitro (38-42). Our data strongly suggest that 419 NS4 is required by the virus to help overcome the cellular innate antiviral responses. 420 Reporter assays revealed that NS4 can inhibit transcription from a variety of mammalian 421 and viral promoters, whilst having little if any direct impact on mRNA editing or translation. 422 However, NS4 does appear to influence the extent of the host IFN response as supernatants 423 of primary sheep endothelial cells infected with BTV8ΔNS4 contained more IFN than 424 supernatants collected from BTV8wt infected cells; a result which may explain the more 425 efficient growth of BTVwt in IFN competent cells. These results were supported by RNAseq 426 analyses of nascent RNA isolated from infected and mock-infected cells. Genes relating to 427 the innate immune system were among the highest upregulated genes in BTV8ΔNS4 428 infected cells, even if some of them were also found upregulated in BTV8wt infected cells, 429 suggesting that NS4 is not by itself sufficient to inhibit entirely the host IFN response. Most 430 of the genes that were specifically upregulated in BTV8ΔNS4 infected cells, as compared to 431 BTV8wt infected cells, were ISGs including well characterised antiviral factors. 432 BTV induces a type 1 IFN response in vivo and in vitro (38-42). Our data strongly suggest that 419 NS4 is required by the virus to help overcome the cellular innate antiviral responses. 420 Reporter assays revealed that NS4 can inhibit transcription from a variety of mammalian 421 and viral promoters, whilst having little if any direct impact on mRNA editing or translation. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 422 However, NS4 does appear to influence the extent of the host IFN response as supernatants 423 of primary sheep endothelial cells infected with BTV8ΔNS4 contained more IFN than 424 supernatants collected from BTV8wt infected cells; a result which may explain the more 425 efficient growth of BTVwt in IFN competent cells. These results were supported by RNAseq 426 analyses of nascent RNA isolated from infected and mock-infected cells. Genes relating to 427 the innate immune system were among the highest upregulated genes in BTV8ΔNS4 428 infected cells, even if some of them were also found upregulated in BTV8wt infected cells, 429 suggesting that NS4 is not by itself sufficient to inhibit entirely the host IFN response. Most 430 of the genes that were specifically upregulated in BTV8ΔNS4 infected cells, as compared to 431 BTV8wt infected cells, were ISGs including well characterised antiviral factors. 432 O ll th d t bt i d i thi t d i di t th t NS4 t it ff t th ti i l 433 20 \| P a g e Overall, the data obtained in this study indicate that NS4 exerts its effect upon the antiviral 433 host response at a point upstream of RNA editing and translation. Interestingly, NS4 434 possesses features of a transcription factor of the bZip family, with a basic domain followed 435 by a leucine zipper motif (67) and it has been suggested to have the ability to bind DNA 436 20 \| P a g e Overall, the data obtained in this study indicate that NS4 exerts its effect upon the antiviral 433 host response at a point upstream of RNA editing and translation. Interestingly, NS4 434 possesses features of a transcription factor of the bZip family, with a basic domain followed 435 by a leucine zipper motif (67) and it has been suggested to have the ability to bind DNA 436 using DNase protection assays (9). Our reporter assays, showing that NS4 inhibits 437 transcription from a variety of promoters, including IFN-β and an ISRE-containing promoter, 438 suggest that this protein may inhibit cellular transcription. In a previous study we showed 439 that NS4 displays a nucleolar localization in infected cells (8). Thus, a direct interaction of 440 NS4 with transcription factors and/or chromatin, resulting in the downregulation of cellular 441 transcription is a possible mechanism of action for this protein. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 In support of this 442 hypothesis, we found that the NS4 of a BTV-1 strain that did not show nucleolar localization 443 was not able to modulate gene expression in reporter assays. It is also possible that NS4 444 inhibits IFN-β activation before transcription. Nuclear translocation of IRF-3 and NFκB 445 appeared to occur at similar levels in both BTVwt and BTV∆NS4 infected cells, suggesting 446 that NS4 does not prevent PAMP recognition by the host cells. However, it is possible that 447 IRF-3 or NF-kB could be inhibited by NS4 in the nucleus. It is important to note that, in a 448 previous study, we showed that NS4 confers a replication advantage to BTV in cells pre- 449 treated with type I IFN, highlighting its ability to counteract the innate immune response 450 after IFN activation. Hence, it is less that NS4 blocks PAMP recognition and subsequent 451 signalling. More studies will be necessary to fully dissect how the NS4 counteracts the host 452 innate immune system. Obviously NS4, like other non-structural proteins of different 453 viruses, might have different functions and antagonise the IFN response via multiple 454 mechanisms. For example, the NS1 protein of Influenza A viruses inhibits IFN production by 455 blocking IRF3 and NF-κB activation, and mRNA maturation (68, 69). 456 using DNase protection assays (9). Our reporter assays, showing that NS4 inhibits 437 transcription from a variety of promoters, including IFN-β and an ISRE-containing promoter, 438 suggest that this protein may inhibit cellular transcription. In a previous study we showed 439 that NS4 displays a nucleolar localization in infected cells (8). Thus, a direct interaction of 440 NS4 with transcription factors and/or chromatin, resulting in the downregulation of cellular 441 transcription is a possible mechanism of action for this protein. In support of this 442 hypothesis, we found that the NS4 of a BTV-1 strain that did not show nucleolar localization 443 was not able to modulate gene expression in reporter assays. It is also possible that NS4 444 inhibits IFN-β activation before transcription. Nuclear translocation of IRF-3 and NFκB 445 appeared to occur at similar levels in both BTVwt and BTV∆NS4 infected cells, suggesting 446 that NS4 does not prevent PAMP recognition by the host cells. However, it is possible that 447 IRF-3 or NF-kB could be inhibited by NS4 in the nucleus. 21 \| P a g e Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 In addition, it 469 has also been shown that BTV inhibits the IFN signalling pathway by downregulating key 470 components of the JAK/STAT pathway: JAK1 and TYK2 (43). The downregulation of 471 JAK1/TYK2 may be the result of a specific interaction with a BTV protein and/or due to the 472 general host protein synthesis shutdown induced by this virus. We did not find either of 473 these two genes to be differentially expressed in BTV8wt or BTV8ΔNS4 infected cells. 474 (also members of the Reoviridae) acts in a similar fashion and outcompetes the polyA 461 binding protein of cells, thus biasing translation of viral transcripts (70, 71). It remains 462 possible that a similar mechanism may exist for BTV. Our data, obtained in metabolically 463 labelled cells, confirm that BTV induces host protein synthesis shutdown and also 464 establishes that this occurs largely independently of NS4. Hence, other BTV proteins might 465 also play a similar role in the shutoff of host cell protein synthesis or function in different 466 ways as IFN antagonists. For example, NS3 is believed to modulate IFN induction 467 downstream of RIG-I and upstream of IKKε activation. Therefore, NS3 and NS4 may 468 potentially act synergistically to counteract the innate immune system (28). In addition, it 469 has also been shown that BTV inhibits the IFN signalling pathway by downregulating key 470 components of the JAK/STAT pathway: JAK1 and TYK2 (43). The downregulation of 471 JAK1/TYK2 may be the result of a specific interaction with a BTV protein and/or due to the 472 general host protein synthesis shutdown induced by this virus. We did not find either of 473 these two genes to be differentially expressed in BTV8wt or BTV8ΔNS4 infected cells. 474 In this study, we showed that BTV8ΔNS4 infected sheep display lower levels of viremia, 475 lasting for shorter periods of time, compared to the viremia observed in animals infected 476 with BTV8wt. The levels and duration of viremia in infected animals are essential factors for 477 successful transmission between the mammalian hosts and the intermediate arthropod 478 vectors. As mentioned above, BTV infection can result in a lethal hemorrhagic fever in some 479 animals while in others the virus induces a mild febrile illness or even a clinically unapparent 480 infection. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 It is important to note that, in a 448 previous study, we showed that NS4 confers a replication advantage to BTV in cells pre- 449 treated with type I IFN, highlighting its ability to counteract the innate immune response 450 after IFN activation. Hence, it is less that NS4 blocks PAMP recognition and subsequent 451 signalling. More studies will be necessary to fully dissect how the NS4 counteracts the host 452 innate immune system. Obviously NS4, like other non-structural proteins of different 453 viruses, might have different functions and antagonise the IFN response via multiple 454 mechanisms. For example, the NS1 protein of Influenza A viruses inhibits IFN production by 455 blocking IRF3 and NF-κB activation, and mRNA maturation (68, 69). 456 21 \| P a g e It has been known for at least three decades that BTV induces host protein synthesis 457 shutdown (22, 64, 65). Recent studies suggest that BTV NS1 favours viral RNA translation 458 and therefore competes with cellular protein synthesis (22). A key difference between 459 mammalian and BTV transcripts is the lack of a polyA tail. The NSP3 protein of Rotaviruses 460 21 \| P a g e It has been known for at least three decades that BTV induces host protein synthesis 457 shutdown (22, 64, 65). Recent studies suggest that BTV NS1 favours viral RNA translation 458 and therefore competes with cellular protein synthesis (22). A key difference between 459 mammalian and BTV transcripts is the lack of a polyA tail. The NSP3 protein of Rotaviruses 460 (also members of the Reoviridae) acts in a similar fashion and outcompetes the polyA 461 binding protein of cells, thus biasing translation of viral transcripts (70, 71). It remains 462 possible that a similar mechanism may exist for BTV. Our data, obtained in metabolically 463 labelled cells, confirm that BTV induces host protein synthesis shutdown and also 464 establishes that this occurs largely independently of NS4. Hence, other BTV proteins might 465 also play a similar role in the shutoff of host cell protein synthesis or function in different 466 ways as IFN antagonists. For example, NS3 is believed to modulate IFN induction 467 downstream of RIG-I and upstream of IKKε activation. Therefore, NS3 and NS4 may 468 potentially act synergistically to counteract the innate immune system (28). Acknowledgements This study was supported by a programme grant from the Wellcome Trust. The authors are 487 This study was supported by a programme grant from the Wellcome Trust. The authors are 487 grateful to Meredith Stewart for useful suggestions. 488 Acknowledgements 486 grateful to Meredith Stewart for useful suggestions. 488 grateful to Meredith Stewart for useful suggestions. 488 Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 The data obtained in this study provide evidence that viral proteins modulating 481 the host innate immune response play a significant role in viral pathogenesis. This study lays 482 the foundations for a deeper understanding of the mechanisms by which BTV antagonises 483 the IFN system, in turn helping us to define the molecular determinants of BTV virulence. 484 In this study, we showed that BTV8ΔNS4 infected sheep display lower levels of viremia, 475 lasting for shorter periods of time, compared to the viremia observed in animals infected 476 with BTV8wt. The levels and duration of viremia in infected animals are essential factors for 477 successful transmission between the mammalian hosts and the intermediate arthropod 478 vectors. As mentioned above, BTV infection can result in a lethal hemorrhagic fever in some 479 animals while in others the virus induces a mild febrile illness or even a clinically unapparent 480 infection. The data obtained in this study provide evidence that viral proteins modulating 481 the host innate immune response play a significant role in viral pathogenesis. This study lays 482 the foundations for a deeper understanding of the mechanisms by which BTV antagonises 483 the IFN system, in turn helping us to define the molecular determinants of BTV virulence. 484 22 \| P a g e 485 23 \| P a g e 23 \| P a g e 486 This study was supported by a programme grant from the Wellcome Trust. The authors are 487 grateful to Meredith Stewart for useful suggestions. 488 24 \| P a g e REFERENCES 489 1. Mellor, P. S., M. 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K. Reddy, and P. Roy. 2002. The membrane trafficking 555 protein calpactin forms a complex with bluetongue virus protein NS3 and mediates 556 virus release. Proc Natl Acad Sci U S A 99:13154-13159. 557 Reassortment between two serologically unrelated bluetongue virus strains is 535 flexible and can involve any genome segment. J Virol 87:543-557. 536 19. Zientara, S., C. Sailleau, C. Viarouge, D. Hoper, M. Beer, M. Jenckel, B. Hoffmann, A. 537 Romey, L. Bakkali-Kassimi, A. Fablet, D. Vitour, and E. Breard. 2014. Novel 538 bluetongue virus in goats, Corsica, France, 2014. Emerg Infect Dis 20:2123-2125. 539 20. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Virus and host factors 568 affecting the clinical outcome of bluetongue virus infection. J Virol 88:10399-10411. 569 31. Janowicz, A., M. Caporale, A. Shaw, S. Gulletta, L. Di Gialleonardo, M. Ratinier, and 570 M. Palmarini. 2015. Multiple genome segments determine the virulence of 571 bluetongue virus serotype 8. Journal of Virology. 572 32. Caporale, M., R. Wash, A. Pini, G. Savini, P. Franchi, M. Golder, J. Patterson-Kane, 573 P. Mertens, L. Di Gialleonardo, G. Armillotta, R. Lelli, P. Kellam, and M. Palmarini. 574 2011. Determinants of bluetongue virus virulence in murine models of disease. J 575 Virol 85:11479-11489. 576 33. Maclachlan, N. J., C. P. Drew, K. E. Darpel, and G. Worwa. 2009. The pathology and 577 pathogenesis of bluetongue. J Comp Pathol 141:1-16. 578 34. Brenner, J., C. Oura, I. Asis, S. Maan, D. Elad, N. Maan, O. Friedgut, K. Nomikou, D. 579 Rotenberg, V. Bumbarov, P. Mertens, H. Yadin, and C. Batten. Multiple serotypes of 580 bluetongue virus in sheep and cattle, Israel. Emerg Infect Dis 16:2003-2004. 581 27. Celma, C. C., and P. Roy. 2009. A viral nonstructural protein regulates bluetongue 558 virus trafficking and release. J Virol 83:6806-6816. 559 28. Chauveau, E., V. Doceul, E. Lara, E. Breard, C. Sailleau, P. O. Vidalain, E. F. Meurs, S. 560 Dabo, I. Schwartz-Cornil, S. Zientara, and D. Vitour. 2013. NS3 of bluetongue virus 561 interferes with the induction of type I interferon. J Virol 87:8241-8246. 562 29. Stewart, M., A. Hardy, G. Barry, R. M. Pinto, M. Caporale, E. Melzi, J. Hughes, A. 563 Taggart, A. Janowicz, M. Varela, M. Ratinier, and M. Palmarini. 2015. 564 Characterization of a second open reading frame in genome segment 10 of 565 bluetongue virus. J Gen Virol 96:3280-3293. 566 30. Caporale, M., L. Di Gialleonorado, A. Janowicz, G. Wilkie, A. Shaw, G. Savini, P. A. 567 Van Rijn, P. Mertens, M. Di Ventura, and M. Palmarini. 2014. Virus and host factors 568 affecting the clinical outcome of bluetongue virus infection. J Virol 88:10399-10411. 569 30. Caporale, M., L. Di Gialleonorado, A. Janowicz, G. Wilkie, A. Shaw, G. Savini, P. A. 567 Van Rijn, P. Mertens, M. Di Ventura, and M. Palmarini. 2014. 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Interplay between ovine bone marrow stromal cell antigen 613 2/tetherin and endogenous retroviruses. J Virol 84:4415-4425. 614 46. Varela, M., E. Schnettler, M. Caporale, C. Murgia, G. Barry, M. McFarlane, E. 615 McGregor, I. M. Piras, A. Shaw, C. Lamm, A. Janowicz, M. Beer, M. Glass, V. Herder, 616 K. Hahn, W. Baumgartner, A. Kohl, and M. Palmarini. 2013. Schmallenberg virus 617 pathogenesis, tropism and interaction with the innate immune system of the host. 618 PLoS Pathog 9:e1003133. 619 47. Boyce, M., C. C. Celma, and P. Roy. 2008. Development of reverse genetics systems 620 for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J 621 Virol 82:8339-8348. 622 48. Dulbecco, R., and M. Vogt. 1953. Some problems of animal virology as studied by 623 the plaque technique. Cold Spring Harb Symp Quant Biol 18:273-279. 624 49. Killip, M. J., D. F. Young, B. L. Precious, S. Goodbourn, and R. E. Randall. 2012. 625 Activation of the beta interferon promoter by paramyxoviruses in the absence of 626 virus protein synthesis. The Journal of general virology 93:299-307. 627 43. Doceul, V., E. Chauveau, E. Lara, E. Breard, C. Sailleau, S. Zientara, and D. Vitour. 605 2014. Dual modulation of type I interferon response by bluetongue virus. J Virol 606 88:10792-10802. 607 44. Buchholz, U. J., S. Finke, and K. K. Conzelmann. 1999. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Killip, M. J., D. F. Young, C. S. Ross, S. Chen, S. Goodbourn, and R. E. Randall. 2011. 628 Failure to activate the IFN-beta promoter by a paramyxovirus lacking an interferon 629 antagonist. Virology 415:39-46. 630 51. Reed, L. J., and H. A. Muench. 1938. A simple method for estimating fifty percent 631 endpoints. American Journal of Hygiene 27:493-497. 632 52. Lelli, R., M. Di Ventura, M. T. Mercante, M. Tittarelli, O. Mangana-Vougiouka, K. 633 Nomikou, A. Conte, B. Di Emidio, O. Portanti, G. Giovannucci, B. Bonfini, M. 634 Zaghini, and V. Caporale. 2004. Bluetongue laboratory diagnosis: a ring test to 635 evaluate serological results using a competitive ELISA kit. Vet Ital 40:577-580. 636 53. Polci, A., C. Camma, S. Serini, L. Di Gialleonardo, F. Monaco, and G. Savini. 2007. 637 Real-time polymerase chain reaction to detect bluetongue virus in blood samples. 638 Vet Ital 43:77-88. 639 54. Palmarini, M., C. Hallwirth, D. York, C. Murgia, T. de Oliveira, T. Spencer, and H. 640 Fan. 2000. Molecular cloning and functional analysis of three type D endogenous 641 retroviruses of sheep reveal a different cell tropism from that of the highly related 642 exogenous jaagsiekte sheep retrovirus. J Virol 74:8065-8076. 643 55. Hale, B. G., J. Steel, R. A. Medina, B. Manicassamy, J. Ye, D. Hickman, R. Hai, M. 644 Schmolke, A. C. Lowen, D. R. Perez, and A. Garcia-Sastre. 2010. Inefficient control of 645 host gene expression by the 2009 pandemic H1N1 influenza A virus NS1 protein. J 646 Virol 84:6909-6922. 647 56. Tanaka, N., T. Kawakami, and T. Taniguchi. 1993. Recognition DNA sequences of 648 interferon regulatory factor 1 (IRF-1) and IRF-2, regulators of cell growth and the 649 interferon system. Mol Cell Biol 13:4531-4538. 650 50. Killip, M. J., D. F. Young, C. S. Ross, S. Chen, S. Goodbourn, and R. E. Randall. 2011. 628 Failure to activate the IFN-beta promoter by a paramyxovirus lacking an interferon 629 antagonist. Virology 415:39-46. 630 51. Reed, L. J., and H. A. Muench. 1938. A simple method for estimating fifty percent 631 endpoints. American Journal of Hygiene 27:493-497. 632 52. Lelli, R., M. Di Ventura, M. T. Mercante, M. Tittarelli, O. Mangana-Vougiouka, K. 633 Nomikou, A. Conte, B. Di Emidio, O. Portanti, G. Giovannucci, B. Bonfini, M. 634 Zaghini, and V. Caporale. 2004. Bluetongue laboratory diagnosis: a ring test to 635 evaluate serological results using a competitive ELISA kit. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Generation of bovine 608 respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus 609 replication in tissue culture, and the human RSV leader region acts as a functional 610 BRSV genome promoter. J Virol 73:251-259. 611 45. Arnaud, F., S. G. Black, L. Murphy, D. J. Griffiths, S. J. Neil, T. E. Spencer, and M. 612 Palmarini. 2010. Interplay between ovine bone marrow stromal cell antigen 613 2/tetherin and endogenous retroviruses. J Virol 84:4415-4425. 614 45. Arnaud, F., S. G. Black, L. Murphy, D. J. Griffiths, S. J. Neil, T. E. Spencer, and M. 612 Palmarini. 2010. Interplay between ovine bone marrow stromal cell antigen 613 2/tetherin and endogenous retroviruses. J Virol 84:4415-4425. 614 46. Varela, M., E. Schnettler, M. Caporale, C. Murgia, G. Barry, M. McFarlane, E. 615 McGregor, I. M. Piras, A. Shaw, C. Lamm, A. Janowicz, M. Beer, M. Glass, V. Herder, 616 K. Hahn, W. Baumgartner, A. Kohl, and M. Palmarini. 2013. Schmallenberg virus 617 pathogenesis, tropism and interaction with the innate immune system of the host. 618 PLoS Pathog 9:e1003133. 619 46. Varela, M., E. Schnettler, M. Caporale, C. Murgia, G. Barry, M. McFarlane, E. 615 McGregor, I. M. Piras, A. Shaw, C. Lamm, A. Janowicz, M. Beer, M. Glass, V. Herder, 616 K. Hahn, W. Baumgartner, A. Kohl, and M. Palmarini. 2013. Schmallenberg virus 617 pathogenesis, tropism and interaction with the innate immune system of the host. 618 PLoS Pathog 9:e1003133. 619 47. Boyce, M., C. C. Celma, and P. Roy. 2008. Development of reverse genetics systems 620 for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J 621 Virol 82:8339-8348. 622 47. Boyce, M., C. C. Celma, and P. Roy. 2008. Development of reverse genetics systems 620 for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J 621 Virol 82:8339-8348. 622 48. Dulbecco, R., and M. Vogt. 1953. Some problems of animal virology as studied by 623 the plaque technique. Cold Spring Harb Symp Quant Biol 18:273-279. 624 49. Killip, M. J., D. F. Young, B. L. Precious, S. Goodbourn, and R. E. Randall. 2012. 625 49. Killip, M. J., D. F. Young, B. L. Precious, S. Goodbourn, and R. E. Randall. 2012. 625 Activation of the beta interferon promoter by paramyxoviruses in the absence of 626 virus protein synthesis. The Journal of general virology 93:299-307. 627 30 \| P a g e 50. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Association of 660 RON tyrosine kinase with the Jaagsiekte sheep retrovirus envelope glycoprotein. 661 Virology 350:347-357. 662 62. Mura, M., P. Murcia, M. Caporale, T. E. Spencer, K. Nagashima, A. Rein, and M. 663 Palmarini. 2004. Late viral interference induced by transdominant Gag of an 664 endogenous retrovirus. Proc Natl Acad Sci U S A 101:11117-11122. 665 63. Rusinova, I., S. Forster, S. Yu, A. Kannan, M. Masse, H. Cumming, R. Chapman, and 666 P. J. Hertzog. 2013. Interferome v2.0: an updated database of annotated interferon- 667 regulated genes. Nucleic Acids Res 41:D1040-1046. 668 64. Huismans, H. 1979. Protein synthesis in bluetongue virus-infected cells. Virology 669 92:385-396. 670 65. Huismans, H. 1971. Host cell protein synthesis after infection with bluetongue virus 671 and reovirus. Virology 46:500-503. 672 57. Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. 651 Salzberg, J. L. Rinn, and L. Pachter. 2012. Differential gene and transcript expression 652 analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562-578. 653 58. FastQC, posting date. [Online.] 654 57. Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. 651 Salzberg, J. L. Rinn, and L. Pachter. 2012. Differential gene and transcript expression 652 analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562-578. 653 58. FastQC, posting date. [Online.] 654 59. Trapnell, C., D. G. Hendrickson, M. Sauvageau, L. Goff, J. L. Rinn, and L. Pachter. 655 2013. Differential analysis of gene regulation at transcript resolution with RNA-seq. 656 Nat Biotechnol 31:46-53. 657 60. Langmead, B., and S. L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. 658 Nat Methods 9:357-359. 659 61. Varela, M., Y. H. Chow, C. Sturkie, P. Murcia, and M. Palmarini. 2006. Association of 660 RON tyrosine kinase with the Jaagsiekte sheep retrovirus envelope glycoprotein. 661 Virology 350:347-357. 662 62. Mura, M., P. Murcia, M. Caporale, T. E. Spencer, K. Nagashima, A. Rein, and M. 663 Palmarini. 2004. Late viral interference induced by transdominant Gag of an 664 endogenous retrovirus. Proc Natl Acad Sci U S A 101:11117-11122. 665 59. Trapnell, C., D. G. Hendrickson, M. Sauvageau, L. Goff, J. L. Rinn, and L. Pachter. 655 2013. Differential analysis of gene regulation at transcript resolution with RNA-seq. 656 Nat Biotechnol 31:46-53. 657 60. Langmead, B., and S. L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Vet Ital 40:577-580. 636 53. Polci, A., C. Camma, S. Serini, L. Di Gialleonardo, F. Monaco, and G. Savini. 2007. 637 Real-time polymerase chain reaction to detect bluetongue virus in blood samples. 638 Vet Ital 43:77-88. 639 53. Polci, A., C. Camma, S. Serini, L. Di Gialleonardo, F. Monaco, and G. Savini. 2007. 637 Real-time polymerase chain reaction to detect bluetongue virus in blood samples. 638 Vet Ital 43:77-88. 639 54. Palmarini, M., C. Hallwirth, D. York, C. Murgia, T. de Oliveira, T. Spencer, and H. 640 Fan. 2000. Molecular cloning and functional analysis of three type D endogenous 641 retroviruses of sheep reveal a different cell tropism from that of the highly related 642 exogenous jaagsiekte sheep retrovirus. J Virol 74:8065-8076. 643 55. Hale, B. G., J. Steel, R. A. Medina, B. Manicassamy, J. Ye, D. Hickman, R. Hai, M. 644 Schmolke, A. C. Lowen, D. R. Perez, and A. Garcia-Sastre. 2010. Inefficient control of 645 host gene expression by the 2009 pandemic H1N1 influenza A virus NS1 protein. J 646 Virol 84:6909-6922. 647 55. Hale, B. G., J. Steel, R. A. Medina, B. Manicassamy, J. Ye, D. Hickman, R. Hai, M. 644 Schmolke, A. C. Lowen, D. R. Perez, and A. Garcia-Sastre. 2010. Inefficient control of 645 host gene expression by the 2009 pandemic H1N1 influenza A virus NS1 protein. J 646 Virol 84:6909-6922. 647 56. Tanaka, N., T. Kawakami, and T. Taniguchi. 1993. Recognition DNA sequences of 648 interferon regulatory factor 1 (IRF-1) and IRF-2, regulators of cell growth and the 649 interferon system. Mol Cell Biol 13:4531-4538. 650 31 \| P a g e 31 \| P a g e 31 \| P a g e 57. Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. 651 Salzberg, J. L. Rinn, and L. Pachter. 2012. Differential gene and transcript expression 652 analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562-578. 653 58. FastQC, posting date. [Online.] 654 59. Trapnell, C., D. G. Hendrickson, M. Sauvageau, L. Goff, J. L. Rinn, and L. Pachter. 655 2013. Differential analysis of gene regulation at transcript resolution with RNA-seq. 656 Nat Biotechnol 31:46-53. 657 60. Langmead, B., and S. L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. 658 Nat Methods 9:357-359. 659 61. Varela, M., Y. H. Chow, C. Sturkie, P. Murcia, and M. Palmarini. 2006. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 658 Nat Methods 9:357-359. 659 60. Langmead, B., and S. L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. 658 Nat Methods 9:357-359. 659 61. Varela, M., Y. H. Chow, C. Sturkie, P. Murcia, and M. Palmarini. 2006. Association of 660 RON tyrosine kinase with the Jaagsiekte sheep retrovirus envelope glycoprotein. 661 Virology 350:347-357. 662 61. Varela, M., Y. H. Chow, C. Sturkie, P. Murcia, and M. Palmarini. 2006. Association of 660 RON tyrosine kinase with the Jaagsiekte sheep retrovirus envelope glycoprotein. 661 Virology 350:347-357. 662 62. Mura, M., P. Murcia, M. Caporale, T. E. Spencer, K. Nagashima, A. Rein, and M. 663 Palmarini. 2004. Late viral interference induced by transdominant Gag of an 664 endogenous retrovirus. Proc Natl Acad Sci U S A 101:11117-11122. 665 62. Mura, M., P. Murcia, M. Caporale, T. E. Spencer, K. Nagashima, A. Rein, and M. 663 Palmarini. 2004. Late viral interference induced by transdominant Gag of an 664 endogenous retrovirus. Proc Natl Acad Sci U S A 101:11117-11122. 665 63. Rusinova, I., S. Forster, S. Yu, A. Kannan, M. Masse, H. Cumming, R. Chapman, and 666 P. J. Hertzog. 2013. Interferome v2.0: an updated database of annotated interferon- 667 regulated genes. Nucleic Acids Res 41:D1040-1046. 668 63. Rusinova, I., S. Forster, S. Yu, A. Kannan, M. Masse, H. Cumming, R. Chapman, and 666 P. J. Hertzog. 2013. Interferome v2.0: an updated database of annotated interferon- 667 regulated genes. Nucleic Acids Res 41:D1040-1046. 668 64. Huismans, H. 1979. Protein synthesis in bluetongue virus-infected cells. Virology 669 92:385-396. 670 65. Huismans, H. 1971. Host cell protein synthesis after infection with bluetongue virus 671 and reovirus. Virology 46:500-503. 672 65. Huismans, H. 1971. Host cell protein synthesis after infection with bluetongue virus 671 and reovirus. Virology 46:500-503. 672 32 \| P a g e 32 \| P a g e 32 \| P a g e 66. Huismans, H., A. A. van Dijk, and A. R. Bauskin. 1987. In vitro phosphorylation and 673 purification of a nonstructural protein of bluetongue virus with affinity for single- 674 stranded RNA. J Virol 61:3589-3595. 675 67. Vinson, C., M. Myakishev, A. Acharya, A. A. Mir, J. R. Moll, and M. Bonovich. 2002. 676 Classification of human B-ZIP proteins based on dimerization properties. Mol Cell 677 Biol 22:6321-6335. 678 68. Hale, B. G. 2014. Conformational plasticity of the influenza A virus NS1 protein. J Gen 679 Virol 95:2099-2105. 680 69. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Krug, R. M. 2015. Functions of the influenza A virus NS1 protein in antiviral defense. 681 Curr Opin Virol 12C:1-6. 682 70. Poncet, D., C. Aponte, and J. Cohen. 1993. Rotavirus protein NSP3 (NS34) is bound 683 to the 3' end consensus sequence of viral mRNAs in infected cells. J Virol 67:3159- 684 3165. 685 71. Poncet, D., S. Laurent, and J. Cohen. 1994. Four nucleotides are the minimal 686 requirement for RNA recognition by rotavirus non-structural protein NSP3. EMBO J 687 13:4165-4173. 688 689 690 66. Huismans, H., A. A. van Dijk, and A. R. Bauskin. 1987. In vitro phosphorylation and 673 purification of a nonstructural protein of bluetongue virus with affinity for single- 674 stranded RNA. J Virol 61:3589-3595. 675 67. Vinson, C., M. Myakishev, A. Acharya, A. A. Mir, J. R. Moll, and M. Bonovich. 2002. 676 Classification of human B-ZIP proteins based on dimerization properties. Mol Cell 677 Biol 22:6321-6335. 678 Biol 22:6321-6335. 678 68. Hale, B. G. 2014. Conformational plasticity of the influenza A virus NS1 protein. J Gen 679 Virol 95:2099-2105. 680 68. Hale, B. G. 2014. Conformational plasticity of the influenza A virus NS1 protein. J Gen 679 Virol 95:2099-2105. 680 69. Krug, R. M. 2015. Functions of the influenza A virus NS1 protein in antiviral defense. 681 Curr Opin Virol 12C:1-6. 682 70. Poncet, D., C. Aponte, and J. Cohen. 1993. Rotavirus protein NSP3 (NS34) is bound 683 to the 3' end consensus sequence of viral mRNAs in infected cells. J Virol 67:3159- 684 3165. 685 70. Poncet, D., C. Aponte, and J. Cohen. 1993. Rotavirus protein NSP3 (NS34) is bound 683 to the 3' end consensus sequence of viral mRNAs in infected cells. J Virol 67:3159- 684 3165. 685 71. Poncet, D., S. Laurent, and J. Cohen. 1994. Four nucleotides are the minimal 686 requirement for RNA recognition by rotavirus non-structural protein NSP3. EMBO J 687 13:4165-4173. 688 71. Poncet, D., S. Laurent, and J. Cohen. 1994. Four nucleotides are the minimal 686 requirement for RNA recognition by rotavirus non-structural protein NSP3. EMBO J 687 13:4165-4173. 688 33 \| P a g e 33 \| P a g e 691 Table 1. Rank of upregulated genes. The associated number (e.g. U1, U2 etc) refers to the rank of the specified gene. 694 For example, CH25H is the gene found to be the most upregulated in BTV8ΔNS4 infected cells compared to 695 mock-infected cells. 696 697 Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Top 33 upregulated genes in BTV infected compared to mock-infected A549 cell 692 BTV8ΔNS4 vs Mock BTV8wt vs Mock Gene Rank Fold change Rank Fold change CH25H U1 142.3 U18 23.6 IFNB1 U2 136.4 U41 17.5 IFNL1 U3 105.6 U11 28.8 IFNL2 U4 91.0 U68 14.1 MX1 U5 88.6 U38 17.8 IFNL3 U6 87.7 U98 12.0 OASL U7 87.6 U13 28.3 IFIT2 U8 84.7 U14 25.9 RSAD2 U9 78.7 U50 16.3 IFIT1 U10 75.9 U36 18.2 CXCL11 U11 71.6 U24 21.5 IFIT3 U12 71.0 U40 17.6 CMPK2 U13 64.1 U112 11.2 IFI44 U14 63.4 U25 21.4 MX2 U15 61.8 U76 13.6 CCR4 U16 56.3 U17 24.0 IFI27 U17 49.7 U170 8.9 DDX58 U18 48.4 U115 11.0 OAS2 U19 46.1 U5 33.6 IFIH1 U20 45.5 U22 21.9 CA1 U21 45.0 U9 30.9 ISG15 U22 43.7 U78 13.4 FOSB U23 43.6 U3 35.0 EGR2 U24 42.5 U8 31.8 FOS U25 39.9 U15 25.3 IL8 U26 39.3 U29 19.8 KLRC2 U27 36.8 U10 29.0 EGR1 U28 36.1 U6 32.9 SLC1A3 U29 35.9 U46 16.7 EGR4 U30 35.7 U59 14.9 BATF2 U31 33.5 U548 4.3 HERC5 U32 32.4 U69 14.0 IFI6 U33 32.3 U303 6.2 693 Rank of upregulated genes. The associated number (e.g. U1, U2 etc) refers to the rank of the specified gene 694 For example, CH25H is the gene found to be the most upregulated in BTV8ΔNS4 infected cells compared to 695 mock-infected cells. 696 Table 1. Top 33 upregulated genes in BTV infected compared to mock-infected A549 cells. 692 34 \| P a g e Table 2. Top 20 downregulated genes in BTV infected compared to mock-infected A549 700 cells. 701 BTV8ΔNS4 vs Mock BTV8wt vs Mock Gene Rank Fold change Rank Fold change CLDND2 D1 0.13 D157 0.30 CPN1 D2 0.13 D24 0.20 DHRS4L1 D3 0.14 D425 0.38 EMID1 D4 0.14 NA** 1.0 LOC100506 D5 0.16 D3 0.15 HMOX1 D6 0.16 D4 0.15 KRT82 D7 0.16 D73 0.26 SHH D8 0.17 D5 0.15 NUPR1 D9 0.18 D11 0.18 KRT4 D10 0.18 D142 0.30 SFRP4 D11 0.19 D9 0.17 NRTN D12 0.19 NA 1.0 EMILIN1 D13 0.20 D18 0.20 LOC113230 D14 0.20 NA 1.0 LINC00086 D15 0.20 D329 0.36 OSGIN1 D16 0.21 D10 0.18 KLHDC7A D17 0.21 D1 0.12 ATOH8 D18 0.22 D2 0.14 MIR210HG D19 0.22 D106 0.28 KRTAP4-1 D20 0.22 D27 0.21 702 Rank of down-regulated genes. The numbers D1, D2 etc. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 refers to the rank of the specified gene (the most 703 down-regulated gene in a given condition being ranked first). 704 NA= not applicable as these genes have not been found to be differentially expressed in the samples 705 considered. 706 707 708 709 710 711 Table 2. Top 20 downregulated genes in BTV infected compared to mock-infected A549 700 cells. 701 BTV8ΔNS4 vs Mock BTV8wt vs Mock Gene Rank Fold change Rank Fold change CLDND2 D1 0.13 D157 0.30 CPN1 D2 0.13 D24 0.20 DHRS4L1 D3 0.14 D425 0.38 EMID1 D4 0.14 NA** 1.0 LOC100506 D5 0.16 D3 0.15 HMOX1 D6 0.16 D4 0.15 KRT82 D7 0.16 D73 0.26 SHH D8 0.17 D5 0.15 NUPR1 D9 0.18 D11 0.18 KRT4 D10 0.18 D142 0.30 SFRP4 D11 0.19 D9 0.17 NRTN D12 0.19 NA 1.0 EMILIN1 D13 0.20 D18 0.20 LOC113230 D14 0.20 NA 1.0 LINC00086 D15 0.20 D329 0.36 OSGIN1 D16 0.21 D10 0.18 KLHDC7A D17 0.21 D1 0.12 ATOH8 D18 0.22 D2 0.14 MIR210HG D19 0.22 D106 0.28 KRTAP4-1 D20 0.22 D27 0.21 702 R k f d l t d Th b D1 D2 t f t th k f th ifi d (th t 703 Table 2. Top 20 downregulated genes in BTV infected compared to mock-infected A549 700 cells. 701 Table 2. Top 20 downregulated genes in BTV infected compared to mock-infected A549 35 \| P a g e 35 \| P a g e 712 FIGURES Figure 1. Experimental infection of sheep with BTV8wt and BTV8ΔNS4. Three groups of 713 sheep (n=5 per group) were mock-infected (grey circle) or infected with either BTV8wt 714 (black square) or BTV8ΔNS4 (black triangle). (Top panel) Body temperature (average per 715 group) of experimentally infected sheep. Rectal temperature in sheep is normally between 716 38.3 and 39.9°C (indicated by plain black lines). (Middle panel) BTV RNA in blood samples of 717 experimentally infected sheep. Viral RNA was detected by qRT-PCR targeting Seg-5, and 718 values are expressed as log10 (copy number per μg of total RNA). (Bottom panel) Box and 719 whisker plot representing neutralizing antibodies in experimentally infected sheep at 7, 14, 720 21 and 28 days post-infection as indicated in Materials and Methods. Grey stars (Mock 721 versus BTV8wt) and black stars (BTV8wt versus BTV8ΔNS4). * = p<0.01; * = p<0.001 722 (two-way Anova, Bonferroni post-tests). 723 Figure 1. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Experimental infection of sheep with BTV8wt and BTV8ΔNS4. Three groups of 713 sheep (n=5 per group) were mock-infected (grey circle) or infected with either BTV8wt 714 (black square) or BTV8ΔNS4 (black triangle). (Top panel) Body temperature (average per 715 group) of experimentally infected sheep. Rectal temperature in sheep is normally between 716 38.3 and 39.9°C (indicated by plain black lines). (Middle panel) BTV RNA in blood samples of 717 experimentally infected sheep. Viral RNA was detected by qRT-PCR targeting Seg-5, and 718 values are expressed as log10 (copy number per μg of total RNA). (Bottom panel) Box and 719 whisker plot representing neutralizing antibodies in experimentally infected sheep at 7, 14, 720 21 and 28 days post-infection as indicated in Materials and Methods. Grey stars (Mock 721 versus BTV8wt) and black stars (BTV8wt versus BTV8ΔNS4). = p<0.01; *** = p<0.001 722 (two-way Anova, Bonferroni post-tests). 723 Figure 2. NS4 modulates IFN synthesis in infected cells. (A) In vitro replication kinetics of 724 BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells (ovEC) and human A549 cells. Cells 725 were infected by BTV8wt (red) and BTV8ΔNS4 (blue) at a MOI of 0.01 and supernatants 726 titrated at the indicated time points as described in Materials and Methods. Experiments 727 were performed independently three times. Two-way ANOVA test, p<0.01. * = p<0.05; ** = 728 p<0.01 (Bonferroni post-tests). (B) Upregulation of IFN-β synthesis in BTV8 infected cells. 729 IFN protection assay. Primary ovine endothelial cells (ovEC) were mock-infected or infected 730 with the indicated viruses (MOI=4). Supernatants were then collected at 16h pi, inactivated 731 by UV treatment and the amount of IFN released in the supernatants determined in 732 biological assays as described in Material and Methods. Bars in the figure represent 733 Figure 2. NS4 modulates IFN synthesis in infected cells. (A) In vitro replication kinetics of 724 BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells (ovEC) and human A549 cells. Cells 725 were infected by BTV8wt (red) and BTV8ΔNS4 (blue) at a MOI of 0.01 and supernatants 726 titrated at the indicated time points as described in Materials and Methods. Experiments 727 were performed independently three times. Two-way ANOVA test, p<0.01. * = p<0.05; ** = 728 p<0.01 (Bonferroni post-tests). (B) Upregulation of IFN-β synthesis in BTV8 infected cells. 729 IFN protection assay. Primary ovine endothelial cells (ovEC) were mock-infected or infected 730 with the indicated viruses (MOI=4). Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 A549 cells were 736 infected at a MOI of 4 for 16h, and fixed before being processed for immunofluorescence 737 using antibodies against NS2, IRF-3 and NFκB with an Alexa Fluor 488 secondary antibody 738 (shown in green). Nuclei have been stained with DAPI (blue). Average values corresponding 739 to the percentage of translocation have been indicated for each experimental condition +/- 740 standard deviation. Scale bars correspond to 47.62 μm. 741 Figure 3. IRF-3 and NFκB nuclear translocation in BTV infected cells. A549 cells were 736 infected at a MOI of 4 for 16h, and fixed before being processed for immunofluorescence 737 using antibodies against NS2, IRF-3 and NFκB with an Alexa Fluor 488 secondary antibody 738 (shown in green). Nuclei have been stained with DAPI (blue). Average values corresponding 739 to the percentage of translocation have been indicated for each experimental condition +/- 740 standard deviation. Scale bars correspond to 47.62 μm. 741 Figure 4. RNAseq of BTV8wt-, BTV8ΔNS4- and mock-infected cells. A. Schematic 742 representation of RNA sequencing workflow. A549 cells were mock-infected or infected 743 with BTV8wt or BTV8ΔNS4 at a MOI of 4. At 12h p.i., nascent RNA was metabolically labelled 744 with 0.4 mM of an analogue of uridine (5-ethynyl uridine, EU) for 90 m. Total RNA was 745 extracted and enriched by selectively depleting rRNA transcripts. The EU-labelled RNA were 746 chemically linked to an azide-modified biotin and then captured on streptavidin magnetic 747 beads. Biotin labelled RNA attached to the magnetic beads was used directly to construct a 748 library and subsequently sequenced using the Ion Proton sequencer. Sequence reads were 749 processed as described in Materials and Methods. B. Plots representing differentially 750 expressed (DE) genes according to their fold change values. Log2 fold change values >1 are 751 regarded as upregulated (q-value < 0.05), whereas <1 is regarded as statistically 752 downregulated (q-value < 0.05). C. Validation of RNAseq by qRT-PCR. mRNA levels of β-actin 753 (ACTB), Annexin A1 (ANXA1), TATA-binding protein (TBP), beta-2 microglobulin (B2M), IFN-β 754 (IFNB1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were 755 measured by qRT-PCR at 8 and 16h pi as described in Materials and Methods. * = p<0.05; 756 = p< 0.01; * = p<0.001 (One-way Anova, Tukey's Multiple Comparison Test). 757 Figure 4. RNAseq of BTV8wt-, BTV8ΔNS4- and mock-infected cells. A. Schematic 742 representation of RNA sequencing workflow. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 Supernatants were then collected at 16h pi, inactivated 731 by UV treatment and the amount of IFN released in the supernatants determined in 732 biological assays as described in Material and Methods. Bars in the figure represent 733 36 \| P a g e 36 \| P a g e 36 \| P a g e 37 \| P a g e standard deviation. One-way Anova p=0.002. *** = p<0.001 (Tukey's Multiple Comparison 734 Test). 735 Figure 3. IRF-3 and NFκB nuclear translocation in BTV infected cells. A549 cells were 736 infected at a MOI of 4 for 16h, and fixed before being processed for immunofluorescence 737 using antibodies against NS2, IRF-3 and NFκB with an Alexa Fluor 488 secondary antibody 738 (shown in green). Nuclei have been stained with DAPI (blue). Average values corresponding 739 to the percentage of translocation have been indicated for each experimental condition +/- 740 standard deviation. Scale bars correspond to 47.62 μm. 741 Figure 4. RNAseq of BTV8wt-, BTV8ΔNS4- and mock-infected cells. A. Schematic 742 representation of RNA sequencing workflow. A549 cells were mock-infected or infected 743 with BTV8wt or BTV8ΔNS4 at a MOI of 4. At 12h p.i., nascent RNA was metabolically labelled 744 with 0.4 mM of an analogue of uridine (5-ethynyl uridine, EU) for 90 m. Total RNA was 745 extracted and enriched by selectively depleting rRNA transcripts. The EU-labelled RNA were 746 chemically linked to an azide-modified biotin and then captured on streptavidin magnetic 747 beads. Biotin labelled RNA attached to the magnetic beads was used directly to construct a 748 library and subsequently sequenced using the Ion Proton sequencer. Sequence reads were 749 processed as described in Materials and Methods. B. Plots representing differentially 750 expressed (DE) genes according to their fold change values. Log2 fold change values >1 are 751 regarded as upregulated (q-value < 0.05), whereas <1 is regarded as statistically 752 downregulated (q-value < 0.05). C. Validation of RNAseq by qRT-PCR. mRNA levels of β-actin 753 (ACTB), Annexin A1 (ANXA1), TATA-binding protein (TBP), beta-2 microglobulin (B2M), IFN-β 754 (IFNB1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were 755 measured by qRT-PCR at 8 and 16h pi as described in Materials and Methods. * = p<0.05; 756 = p< 0.01; * = p<0.001 (One-way Anova, Tukey's Multiple Comparison Test). 757 Figure 3. IRF-3 and NFκB nuclear translocation in BTV infected cells. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 A549 cells were mock-infected or infected 743 with BTV8wt or BTV8ΔNS4 at a MOI of 4. At 12h p.i., nascent RNA was metabolically labelled 744 with 0.4 mM of an analogue of uridine (5-ethynyl uridine, EU) for 90 m. Total RNA was 745 extracted and enriched by selectively depleting rRNA transcripts. The EU-labelled RNA were 746 chemically linked to an azide-modified biotin and then captured on streptavidin magnetic 747 beads. Biotin labelled RNA attached to the magnetic beads was used directly to construct a 748 library and subsequently sequenced using the Ion Proton sequencer. Sequence reads were 749 processed as described in Materials and Methods. B. Plots representing differentially 750 expressed (DE) genes according to their fold change values. Log2 fold change values >1 are 751 regarded as upregulated (q-value < 0.05), whereas <1 is regarded as statistically 752 downregulated (q-value < 0.05). C. Validation of RNAseq by qRT-PCR. mRNA levels of β-actin 753 (ACTB), Annexin A1 (ANXA1), TATA-binding protein (TBP), beta-2 microglobulin (B2M), IFN-β 754 (IFNB1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were 755 measured by qRT-PCR at 8 and 16h pi as described in Materials and Methods. * = p<0.05; 756 = p< 0.01; * = p<0.001 (One-way Anova, Tukey's Multiple Comparison Test). 757 37 \| P a g e 37 \| P a g e Figure 5. Cellular pathways of BTV8wt- and BTV8ΔNS4-infected cells. A-C. Canonical 758 cellular pathways were analysed using Ingenuity Pathways Analysis. Only canonical 759 pathways with a p-value < 6.3x105 are shown. Ratio corresponds to the number of genes 760 found DE in a given pathway divided by the total number of genes contained in that 761 pathway. A specific pathway is coloured in orange or blue depending on whether it has been 762 predicted to be activated (positive z-scores) or inhibited (negative z-scores), respectively. 763 Grey bars are pathways where no prediction can currently be made. PRR: Pattern 764 Recognition Receptors, AII: Antiviral Innate Immunity, DC: dendritic dell, MΦ: macrophages, 765 EC: endothelial cells, RA: rheumatoid arthritis, GC: gastric cells, NKC: natural killer cells, AR: 766 antiviral response, OSR: oxydative stress response, DM: diabetes mellitus. A. BTV8ΔNS4 767 versus mock-infected cells. B. BTV8wt versus mock-infected cells. C. BTV8ΔNS4- versus 768 BTV8wt-infected cells. 769 Figure 6. Effects of NS4 on expression driven by various promoters. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 CPT-Tert cells were co- 770 transfected with 100 ng of a DNA plasmid expressing either BTV10 NS2 (GenBank # 771 NC006007), or BTV8 NS4 and a DNA plasmid expressing FLuc under the control of either 772 CMV (50 ng), IFN-β (50 ng) or IFN-stimulated response element-containing (ISRE, 400 ng) 773 promoter. After transfection (4h), cells transfected with the IFN-β promoter were 774 stimulated with Sendai virus while cells transfected with ISRE-containing promoter were 775 stimulated with universal IFN. FLuc activity was assessed 22h post-transfection in a 776 luminometer. 5ng of capped and polyadenylated RNA made in vitro were also co- 777 transfected as a control of transfection efficiency. One-way Anova p=0.0002 (CMV), 778 p=0.0015 (IFN-β) and p=0.0006 (ISRE). * = p< 0.05; * = p<0.001 (Dunnett's Multiple 779 Comparison Test). 780 38 \| P a g e 38 \| P a g e Figure 7. NS4 from various BTV strains display inhibitory activity on gene expression. A. 781 Multiple sequence alignment of representative NS4 sequences. Identical residues compared 782 to BTV8 (GenBank accession # JX680455) are shown as dots. Accession numbers refer to the 783 BTV genome segment 9 containing the NS4 open reading frame. B. CPT-Tert cells were co- 784 transfected with a plasmid expressing the indicated BTV NS4 protein (named according to 785 the accession number corresponding to the BTV segment 9) and a plasmid expressing FLuc 786 under the control of a CMV promoter. FLuc activity was assessed 22h post-transfection, and 787 in parallel by western blotting analysis of the same cell lysates using anti-NS4 or anti- 788 αtubulin antibodies. One-way Anova p<0.0001. * = p<0.001 (Dunnett's Multiple 789 Comparison Test). C. CPT-Tert cells were co-transfected with increasing amounts of an 790 expression plasmid for the BTV8 NS4 and a plasmid expressing the firefly luciferase (FLuc) 791 under the control of the CMV immediate early promoter. FLuc activity was assessed 22h 792 post-transfection in a luminometer and in parallel by western blotting of the same cell 793 lysates using antibodies towards the NS4 or tubulin antibodies. One-way Anova p<0.0001. 794 = p< 0.01; * = p<0.001 (Dunnett's Multiple Comparison Test). D. Confocal microscopy 795 of CPT-Tert cells transfected with pCI-NS4 plasmids. Replication kinetics of BTV8wt and BTV8ΔNS4 in primary ovine endothelial cells and 282 At 22h post-transfection, cells were 796 fixed and analysed by immunofluorescence using antibodies against the nucleolar marker 797 B23 (in red) and NS4 (in green), with the appropriate conjugated secondary antibodies as 798 described in Materials and Methods. Scale bars correspond to 33.21 μm. 799 Figure 8. NS4 expression does not affect significantly host cellular protein shutoff, mRNA 800 39 \| P a g e Figure 8. NS4 expression does not affect significantly host cellular protein shutoff, mRNA 800 splicing or translation. A. Primary ovEC were mock-infected or infected with BTV8wt and 801 BTV8ΔNS4 and nascent proteins were metabolically labelled with 35S-methionine/cysteine 802 for 2 h at the time points indicated. Cell extracts were fractionated by SDS-PAGE and gels 803 were stained with Coomassie blue. Dried gels were analysed by phosphoimaging. Black 804 39 \| P a g e Figure 8. NS4 expression does not affect significantly host cellular protein shutoff, mRNA 800 splicing or translation. A. Primary ovEC were mock-infected or infected with BTV8wt and 801 BTV8ΔNS4 and nascent proteins were metabolically labelled with 35S-methionine/cysteine 802 for 2 h at the time points indicated. Cell extracts were fractionated by SDS-PAGE and gels 803 were stained with Coomassie blue. Dried gels were analysed by phosphoimaging. Black 804 39 \| P a g e arrow indicates cellular actin. Signal intensity quantified using the ImageQuant software. 805 Red arrows indicate BTV proteins. B. CPT-Tert cells were co-transfected with variable 806 amounts of plasmids expressing BTV8 NS4 protein and either a plasmid expressing FLuc 807 under the control of a CMV promoter or a plasmid expressing renilla luciferase (RLuc) 808 downstream an intron and the CMV promoter. FLuc or RLuc expression was assessed 22h 809 post-transfection. Cell lysates were analysed by western blotting using antibodies towards 810 NS4. One-way Anova p<0.0001. = p< 0.01; * = p<0.001 (Dunnett's Multiple Comparison 811 Test). C. CPT-Tert cells were co-transfected with variable amount of a DNA plasmid 812 expressing BTV8 NS4 and either a plasmid expressing the firefly luciferase (FLuc) under the 813 control of a CMV promoter, or capped and polyadenylated RNA made in vitro. FLuc activity 814 was assessed 22h post-transfection. Cell lysates were also analysed by western blotting 815 using antibodies towards NS4. One-way Anova p<0.0001. * = p< 0.05; * = p<0.001 816 (Dunnett's Multiple Comparison Test). 817 arrow indicates cellular actin. Signal intensity quantified using the ImageQuant software. 805 Red arrows indicate BTV proteins. B. CPT-Tert cells were co-transfected with variable 806 amounts of plasmids expressing BTV8 NS4 protein and either a plasmid expressing FLuc 807 under the control of a CMV promoter or a plasmid expressing renilla luciferase (RLuc) 808 downstream an intron and the CMV promoter. FLuc or RLuc expression was assessed 22h 809 post-transfection. Figure 8. NS4 expression does not affect significantly host cellular protein shutoff, mRNA 800 Cell lysates were analysed by western blotting using antibodies towards 810 NS4. One-way Anova p<0.0001. = p< 0.01; *** = p<0.001 (Dunnett's Multiple Comparison 811 Test). C. CPT-Tert cells were co-transfected with variable amount of a DNA plasmid 812 expressing BTV8 NS4 and either a plasmid expressing the firefly luciferase (FLuc) under the 813 control of a CMV promoter, or capped and polyadenylated RNA made in vitro. FLuc activity 814 was assessed 22h post-transfection. Cell lysates were also analysed by western blotting 815 using antibodies towards NS4. One-way Anova p<0.0001. * = p< 0.05; *** = p<0.001 816 (Dunnett's Multiple Comparison Test). 817 40 \| P a g e 40 \| P a g e
https://openalex.org/W4213218527	https://link.springer.com/content/pdf/10.1007/s10653-022-01218-8.pdf	English	null	Short- and long-term exposure to trace metal(loid)s from the production of ferromanganese alloys by personal sampling and biomarkers	Environmental geochemistry and health	2,022	cc-by	14,294	Short‑ and long‑term exposure to trace metal(loid)s from the production of ferromanganese alloys by personal sampling and biomarkers B. Markiv · L. Ruiz‑Azcona · A. Expósito · M. Santibáñez · I. Fernández‑Olmo Received: 15 November 2021 / Accepted: 27 January 2022 / Published online: 22 February 2022 © The Author(s) 2022 Abstract The environmental exposure to trace metal(loid)s (As, Cd, Cu, Fe, Mn, Pb, and Zn) was assessed near a ferromanganese alloy plant using fil- ters from personal particulate matter (PM) samplers (bioaccessible and non-bioaccessible fine and coarse fractions) and whole blood as short-term exposure markers, and scalp hair and fingernails as long-term biomarkers, collected from volunteers (n = 130) liv- ing in Santander Bay (northern Spain). Bioaccessi- ble and non-bioaccessible metal(loid) concentrations in coarse and fine PM from personal samplers were determined by ICP-MS after extraction/digestion. Metal(loid) concentration in biomarkers was meas- ured after alkaline dilution (whole blood) and acid digestion (fingernails and scalp hair) by ICP-MS as well. Results were discussed in terms of exposure, considering the distance to the main Mn source, and sex. In terms of exposure, significant differences were found for Mn in all the studied fractions of PM, As in whole blood, Mn and Cu in scalp hair and Mn and Pb in fingernails, with all concentrations being higher for those living closer to the Mn source, with the excep- tion of Cu in scalp hair. Furthermore, the analysis of the correlation between Mn levels in the studied biomarkers and the wind-weighted distance to the main source of Mn allows us to conclude that scalp hair and mainly fingernails are appropriate biomark- ers of long-term airborne Mn exposure. This was also confirmed by the significant positive correla- tions between scalp hair Mn and bioaccessible Mn in coarse and fine fractions, and between fingernails Mn and all PM fractions. This implies that people living closer to a ferromanganese alloy plant are exposed to higher levels of airborne metal(loid)s, mainly Mn, leading to higher levels of this metal in scalp hair and fingernails, which according to the literature, might affect some neurological outcomes. According to sex, significant differences were observed for Fe, Cu and Pb in whole blood, with higher concentrations of Fe and Pb in males, and higher levels of Cu in females; and for Mn, Cu, Zn, Cd and Pb in scalp hair, with higher concentrations in males for all metal(loid)s except Cu. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10653-022-01218-8. Environ Geochem Health (2022) 44:4595–4618 https://doi.org/10.1007/s10653-022-01218-8 Environ Geochem Health (2022) 44:4595–4618 https://doi.org/10.1007/s10653-022-01218-8 ORIGINAL PAPER ORIGINAL PAPER Introduction Exposure to elevated levels of metal(loid)s that are present in the environment is concerning due to the resulting adverse health effects (carcinogenic, neuro- toxic, etc.), even if some of them can be considered as essential, required at trace levels for metabolism, such as copper (Cu), iron (Fe), manganese (Mn) or zinc (Zn) (Maret, 2016; Zoroddu et al., 2019). Three main routes of exposure to metal(loid)s are known: (i) ingestion from foods and water; (ii) dermal contact; and (iii) inhalation. The assessment of the exposure to metal(loid)s by the inhalation route can be done directly by measur- ing or modelling their levels in ambient air, or indi- rectly by measuring their concentration in selected biomarkers. Stationary samplers have been widely used in extensive PM sampling campaigns for the analysis of metal(loid)s concentration in filters, pro- viding information about the long-term exposure to such pollutants, but with the limitation of obtaining information only from specific sites where these sam- plers were located (Fulk et al., 2016). This limitation is addressed using personal PM samplers, which are easy to wear by exposed individuals, with the added advantage of accounting for changes in exposure dur- ing short sampling periods. Filters collected from these samplers can be analysed for total metal(loid)s content or for the bioaccessible concentration, which reflects the amount of each pollutant to be solubilised by a human synthetic fluid. The inhalation route of exposure is particu- larly important for people living close to airborne metal(loid)s emission sources in urban and indus- trial areas (Bauer et al., 2020), such as metallurgy, steelworks, combustion (including transportation) and incineration. Airborne metal(loid)s are bound to particulate matter (PM), its particle size being a key parameter on the fate of such pollutants in the human body (Kelly & Fussell, 2012). Thus, the thoracic frac- tion (equivalent to PM10) includes the tracheobron- chial fraction (i.e. inhaled particles that penetrate beyond the larynx but do not reach the non-ciliated airways) and the respirable fraction (i.e. inhaled par- ticles that enter the non-ciliated airways). Particles of the 2.5–10 µm size fraction are mostly deposited in the pharyngeal and tracheal region (i.e. constitute the tracheobronchial fraction), but they can be swal- lowed, reaching the gastrointestinal tract, where they come into contact with gastric juice. Smaller particles (i.e. Short‑ and long‑term exposure to trace metal(loid)s from the production of ferromanganese alloys by personal sampling and biomarkers Keywords Biomarkers · Manganese · Trace metal(loid)s · Ferromanganese alloy production · Environmental exposure B. Markiv () · A. Expósito · I. Fernández‑Olmo Departamento de Ingenierías Química y Biomolecular, Universidad de Cantabria, Santander, Spain e-mail: markivb@unican.es B. Markiv () · A. Expósito · I. Fernández‑Olmo Departamento de Ingenierías Química y Biomolecular, Universidad de Cantabria, Santander, Spain e-mail: markivb@unican.es L. Ruiz‑Azcona · M. Santibáñez Departamento de Enfermería, Universidad de Cantabria, Santander, Spain 0123456789) 3 Vol.: (011 Environ Geochem Health (2022) 44:4595–4618 4596 calculating the wind direction-weighted distance for short-term exposure, addressing Mn exposure in the vicinity of a Mn alloy plant by children living in the town of Marietta (Ohio) in the USA. 1 3 l:. (1234567890) Introduction Environ Geochem Health (2022) 44:4595–4618 4597 used to assess exposure to non-essential metals such as Pb and Cd (Henríquez-Hernández et al., 2017; Wong & Lye, 2008), highlighting the possibility of assessing acute intoxications due to the efficiency and sensitivity of the use of this biomarker. However, there are doubts about its use to assess exposure to essential trace elements such as Mn, Fe, Cu or Zn, due to their homeostatic regulation. For example, the mean-life time of Mn in blood is much shorter than in other tissues and cellular compartments, so some authors consider that it is not a good biomarker of short-term exposure to Mn (Jiang et al., 2007; Kim et al., 2015), being this more noticeable in the case of inhalation exposure, since despite finding higher levels in the blood of occupationally exposed indi- viduals, it is difficult to quantify how the pulmonary uptake of airborne Mn contributes to its increase in blood (Roth, 2006). comes from this factory (Otero-Pregigueiro et al., 2018). In addition, a source apportionment study carried out in this area also identified Cd, Fe, Pb and Zn as tracers from this plant (Hernández-Pellón & Fernández-Olmo, 2019). However, other local sources of metal(loid)s apart from the ferromanga- nese alloy exist, such as road traffic, combustion and other industrial sources, leading to moderate levels of Cu and As (Hernández-Pellón & Fernández-Olmo, 2019). comes from this factory (Otero-Pregigueiro et al., 2018). In addition, a source apportionment study carried out in this area also identified Cd, Fe, Pb and Zn as tracers from this plant (Hernández-Pellón & Fernández-Olmo, 2019). However, other local sources of metal(loid)s apart from the ferromanga- nese alloy exist, such as road traffic, combustion and other industrial sources, leading to moderate levels of Cu and As (Hernández-Pellón & Fernández-Olmo, 2019). Therefore, this area was selected to assess the exposure to airborne Mn and other trace metal(loid) s in the healthy adult population living in the vicinity of this ferromanganese alloy plant. For this purpose, the following short-term exposure markers to such metal(loid)s were taken from 130 volunteers recruited in a cross-sectional study: 24-h personal PM sam- ples of different particle sizes (PM10-2.5 and PM2.5), which accounted for the inhalation route of expo- sure, as well as whole blood samples. Introduction the respirable fraction) can travel deeper into the alveolar region of the lungs, where they interact with the lung fluid; the interstitial lung fluid has a neutral pH; however, these small particles can be phagocy- tosed by alveolar macrophages, resulting in a more acidic medium (pH 4.5). Dissolved metal(loid)s can then reach the circulatory system (Expósito et al., 2021; Mukhtar & Limbeck, 2013). l The degree of inhalation exposure can also be determined by the analysis of specific exposure bio- markers (Fernández-Olmo et al., 2021). However, the levels of metal(loid)s in such biomarkers can also account for the other routes of exposure, mainly the ingestion route; this is especially true for essential trace elements, which are included in the usual diet, but can sometimes also apply to non-essential ones, such as arsenic (As), cadmium (Cd) or lead (Pb). The usefulness of an exposure biomarker is assessed for its ability to characterise and differentiate exposed and non-exposed groups, as well as for their ability to predict health disorders, anticipating any deteriora- tion of health (Viana et al., 2014; Zheng et al., 2011). Although there is no current consensus on which bio- marker best defines the dose–effect relationship, and furthermore, the use of these biomarkers does not dif- ferentiate how much of these metal(loid)s enter the body by inhalation, biomarkers of exposure have been used in epidemiological studies designed to assess the exposure to some metal(loid)s near airborne metal(loid)s sources (Haynes et al., 2015; Rodrigues et al., 2018; Viana et al., 2014). A simple marker of the exposure to metal(loid) s by inhalation route is the distance between the known source(s) and the receptors; for example, pre- vious studies have used the distance between the par- ticipants’ home in cross-sectional epidemiological studies and the source of emission of certain air pol- lutants as a preliminary indicator of exposure (Vimer- cati et al., 2016; Zubero et al., 2010). The impor- tance of being downwind or upwind of the emission source has been considered by Haynes et al. (2012), Blood has been considered as a short-term expo- sure biomarker to some metal(loid)s (Freire et al., 2015; Stojsavljević et al., 2019); it was frequently 1 3 . (1234567890) 3 (1234567890) 1 Vol:. Introduction For long-term exposure, scalp hair and fingernails samples were col- lected, with the aim of finding associations between the amounts of metal(loid)s inhaled and those pro- cessed by the body, to determine the best biomarker of exposure to airborne Mn and other metal(loid)s. Other candidates have been considered for long- term exposure to metal(loid)s, such as hair and nails (Butler et al., 2019; Fernández-Olmo et al., 2021; Nakaona et al., 2020; Parhizkar et al., 2021), because of their slow growth: between 1 and 1.2 cm/ month of hair (Van Neste & Rushton, 2016) and about 3.47 mm/month of fingernails (Yaemsiri et al., 2010). They are easy to sample, transport, handle and store (Haynes et al., 2015; Menezes-Filho et al., 2009; Sukumar & Subramanian, 2007), although it is not possible to discriminate between the specific exposure route to them, providing us with a final concentration resulting from inhalation, oral and der- mal routes. When considering these biomarkers, it is necessary to be cautious about the use of cosmet- ics such as dyes or nail polishes. Although Directive 1223/2009 on cosmetic products forbids the use of heavy metals as additives in EU member states (Euro- pean Parliament & Council of the European Union, 2009), a recent study has pointed out that coloured nail polish still contains some metal(loid)s (Ceballos et al., 2021). Study area and population The study population was then divided into highly exposed (distance less than 1.5 km between each volunteer’s residence and the Mn alloy plant, i.e. those living in Maliaño, where the main Mn source is located) and moderately exposed (distance greater than 1.5 km, i.e. those living outside Maliaño, mainly in the city of Santander). Study area and population Study area and population The study was carried out in Santander Bay, Can- tabria, Northern Spain (about 250,000 inhabitants in 2019). Among the local industrial sources of metal(loid)s in this area, a ferromanganese alloy plant is the main emitter of metal(loid)s, mainly of Mn as reported by Otero-Pregigueiro et al. (2018), out- standing relatively high levels of Mn in ambient air that frequently exceeded the WHO guideline value (150 ng/m3, annual mean) in the town of Maliaño (about 10,000 inhabitants), where the ferromanga- nese alloys smelter is located (Hernández-Pellón & Fernández-Olmo, 2019).i Recent studies published by our research group highlighted the elevated levels of airborne Mn in Santander Bay (Cantabria region, Northern Spain), exceeding the WHO annual guideline (i.e. 150 ng/ m3 of Mn) (Hernández-Pellón & Fernández-Olmo, 2019; Hernandez-Pellón et al., 2017). These elevated levels were mainly due to the emissions from a ferro- manganese alloy production plant located in this area; approximately, 91% of air Mn emitted in this area Volunteers were recruited as specified in Ruiz- Azcona et al. (2021). All volunteers considered in this study were over 18 years old, and without pre- vious or current work in relation with the ferroman- ganese plant or any other occupational exposure to Mn. All of them resided for a minimum of one year in 1 ol.: (01 3 123456789) 3 123456789) Environ Geochem Health (2022) 44:4595–4618 4598 other lifestyles. The sampling campaign started in November 2019 and ended in November 2020 (inter- rupted from March 2020 to June 2020 due to the COVID-19 outbreak), concluding the sampling cam- paign with a total of 130 volunteers. Santander Bay at different distances from the ferroal- loy factory, which was intentionally placed in Fig. 1 in the centre of the exposure area, to account for the degree of exposure to Mn and the other studied metal(loid)s. The study population was then divided into highly exposed (distance less than 1.5 km between each volunteer’s residence and the Mn alloy plant, i.e. those living in Maliaño, where the main Mn source is located) and moderately exposed (distance greater than 1.5 km, i.e. those living outside Maliaño, mainly in the city of Santander). Santander Bay at different distances from the ferroal- loy factory, which was intentionally placed in Fig. 1 in the centre of the exposure area, to account for the degree of exposure to Mn and the other studied metal(loid)s. Sample preparation All the used reagents for treatment and sample prepa- ration were of analytical grade provided by Merck and PanReac AppliChem (Darmstadt, Germany). Whole blood samples of approximately 7.5 mL were col- lected by venipuncture after disinfection of the skin with 70% alcohol, collected in lithium heparin mon- ovettes developed for metal determination (Sarstedt, Nümbrecht, Germany). These samples were refriger- ated for up to 14 days until dilution with an alkaline solution (2% (w/v) 1-butanol, 0.05% (w/v) EDTA, 0.05% (w/v) triton X-100 and 1% (w/v) NH4OH) as described in González-Antuña et al. (2017) at a minimum whole blood/alkaline solution ratio of 1/10 (w/w), and subsequent analysis by ICP/MS. Scalp hair and fingernail samples were collected in poly- propylene flasks using clean ceramic scissors and nail clippers, respectively. A tuft of hair was cut from the occipital part of the head, using the 2 cm closest to The study was approved by the ethical committee of clinical research in Cantabria (CEIC) and by the ethical committee of research of the University of Cantabria (CEUC). Written informed consent was obtained from each subject and whole blood, scalp hair and finger- nails collection was performed only after permission was obtained. In addition to biological samples, PM samples were obtained from personal samplers that were worn for 24 h by each volunteer. Subjects were asked to complete a structured questionnaire contain- ing socio-demographic characteristics, health and medication status, occupational data, smoking habits, dietary habits including daily intake of Mn-rich foods and Mn food supplements, alcohol consumption and Fig. 1 Location of participants’ residence and the Mn alloy plant Fig. 1 Location of participants’ residence and the Mn alloy plant Fig. 1 Location of participants’ residence and the Mn alloy plant 3 (1234567890) 1 Vol:. ( Environ Geochem Health (2022) 44:4595–4618 4599 H2O2 solution in a 4/1 (v/v) ratio, up to 220 °C. The ALF extracts were stored until analysis at 4 °C for a maximum of 48 h. the scalp for analysis. By using only 2 cm from the root of the hair, alterations in hair morphology due to dyes are eliminated. This is because the hair tips may contain higher levels of metals since the hair further from the root has longer contact with the environment and additional dye applications. Metal(loid) analysis Mass spectrometry inductively coupled plasma (ICP/ MS, Agilent 7500 CE) was used for the analysis of 55Mn, 56Fe, 63Cu, 66Zn, 75As, 111Cd and 207Pb in the different samples. Internal standards (89Y, 103Rh and 185Re) were added to each vial to correct for instru- mental drifts, and a collision cell with a helium flow rate of 4.8 mL/min was used to minimise spectral interferences. Since whole blood, ALF and gastric fluid can cause spectral and non-spectral interferences (matrix effects) during ICP/MS analysis, the deter- mination of the concentration of the metals studied in these samples was performed by adding the same solution to the Multi-Element Standard Solution used to calibrate the instrument. In the case of samples from acid digestion, the calibration standards were simply prepared in dilute nitric acid (1 N). Seven cali- bration points between 0 and 25 ppb were used and samples were diluted when necessary. Fingernails of both hands were cut after asking the volunteers to wash them with the liquid soap provided and to rinse them thoroughly with tap water. Females were asked whether they dyed their hair and/or pol- ished their fingernails. When they polished them, the polish residues were removed using acetone before cutting, and the fingernails were sonicated for 10 min before proceeding to the washing protocol. Scalp hair and fingernails were washed according to the wash- ing protocol described in Eastman et al. (2013), thus removing all exogenous metals and ensuring that only endogenous metals were quantified. Once cleaned, samples were microwave digested (Milestone, Ethos One) at 200 °C in a 4/1 (v/v) HNO3/H2O2 solution, and finally analysed by ICP/MS. Limits of detection (LOD) were calculated based on the variability of 10 procedural blanks (two-tailed Student’s t-test with 95% confidence for n-1 samples (2.26) times the standard deviation of the blanks), being constant for each metal(loid) for whole blood and filters, and variable for scalp hair and finger- nails, depending on the weight of sample from each volunteer. Limit of quantification was determined as 10 times the SD of blanks (Al-Hakkani, 2019) (see Supplementary Table 1). Measurements of these pro- cedural blanks also allowed checking for possible contamination of containers and reagents. The mean values of the studied metal(loid)s measured in these blanks were subtracted from all samples except for whole blood because both calibration points and sam- ples were prepared in the same solution. Sample preparation Each additional dye application process leads to a higher level of oxida- tion of the hair, thus creating more potential binding sites, and together with the additional mechanical wear that occurs when moving from root to tip, can facilitate the diffusion of dyes into the hair (Godfrey et al., 2013). Metal(loid) analysis Results were presented for elements with a minimum of 50% of the samples above the limit of detection. i A personal two-stage modular impactor (SKC PMI coarse) was used to collect PM2.5 and PM10-2.5 sam- ples for 24 h, connected to a personal pump (SKC Aircheck XR5000) operating at a flow rate of 3 lpm. PTFE filters of 37 mm (1 µm pore size) were used for the PM2.5 fraction, and PTFE filters of 25 mm (1 µm pore size) were used for the PM10-2.5 fraction (SKC Inc., Houston, USA). An in vitro bioaccessibil- ity test was performed by extracting each filter with 10 mL of ALF (Artificial Lysosomal Fluid) as lung fluid for 24 h for PM2.5 filters, and gastric fluid for 1 h for PM10-2.5 filters, in an incubation system (MRHX- 04, LSCI) at 37 °C with end-to-end rotation (SBS) at 30 rpm. After the leaching assay, the samples were centrifuged (Mistasel-BL/Selecta) and the superna- tants were filtered using polypropylene syringe filters with a pore size of 0.45 µm. The choice of fluids and composition is described in detail in Expósito et al. (2021). Then, the insoluble fraction (non-bioacces- sible fraction) was digested based on the European standard method "EN-UNE 14,902:2006", which con- sisted of an acid digestion of each filter in a HNO3/ The analytical methods described were vali- dated using certified reference material for whole blood and human hair (Seronorm™ Trace Elements Whole Blood L-1 and ERM®-DB001, respectively), but no reference material was found for fingernails. The recoveries obtained for the different certified metal(loid)s range from 94 to 110% for whole blood 1 ol.: (01 3 123456789) 4600 Environ Geochem Health (2022) 44:4595–4618 and from 93 to 110% for hair (see Supplementary Table 2). A worse recovery was obtained for Mn in hair (144%), but its metal content was not certified in ERM®-DB001, because only one laboratory pro- vided results on hair Mn concentration. The diges- tion method was validated in the research group as described in Hernández-Pellón et al. (2018). In addi- tion to the use of certified reference materials, after calibration and at the end of each analytical run, qual- ity control standards covering the concentration range of interest were measured to check the accuracy of the measurements. Wind‑weighted distance calculation procedure for exposure metrics Then, the weighted distance (dw) was calculated for each subject by dividing the actual distance (d) by the WI, as shown in Eq. (5). To study the correlation between the levels of metal(loid)s in the short-term markers (whole blood and PM) and the distance between each volunteer’s residence and the main metal(loid) source weighted by wind, hourly wind data were taken for the personal sampling time slots of each subject. For this purpose, hourly wind direction and speed data from the San- tander—Parayas Airport weather station provided by Openair thorough the World Met package (R version 4.0.5) were used. (5) dw = d WI dw = d WI (5) With respect to the long-term markers, to calculate the weighted source/home distance, wind data were taken for the months prior to fingernails and scalp hair sampling for each subject (6 months for finger- nail samples and 2 months for scalp hair samples). For these longer periods, the procedure shown in Eqs. (1–5) sometimes failed, due to the lack of rep- resentativeness of the average wind direction over these periods. To address this, an alternative proce- dure was developed to calculate the weighted distance (d′w), using a weighting factor (wf) that considers the frequency with which the wind blows downwind (i.e. from the source to the residence) during the period considered for each subject. For this purpose, the area of interest was divided into 8 sectors, placing the emitting source in the centre of the area and assign- ing each residence a wind sector. Next, the frequency (fraction of hours) in which the wind blew from the sector opposite the volunteer’s home was calculated with respect to the total number of hours. Finally, the distance between the source and the subject’s home was weighted by dividing the raw distance by this weighting factor (Eq. 6). The procedure described by Haynes et al. (2012) was followed to calculate the wind-weighted source/ home distance. First, Wind Index (WI), a parameter between 0–1 indicating whether the subject is upwind (0) or downwind (1) of the source, was calculated, according to Eq. Metal(loid) analysis (2) Umean = −1 N ∑ ui sen(휃i ) (3) Vmean = −1 N ∑ ui cos (휃i ) (2) (3) To obtain the average direction (θm), Eq. 4 was applied (Grange, 2014), where FLOW = + 180 when ArcTan < 180 and FLOW = − 180 when ArcTan > 180: (4) 휃m = ArcTan (Umean Vmean ) + FLOW (4) Wind‑weighted distance calculation procedure for exposure metrics Description of the study population Description of the study population Data analysis was performed with IBM SPSS Sta- tistics software (version 22) and R software (version 4.0.5). Statistical analysis was only performed when at least 50% of the data above the LOD was availa- ble. For metal(loid) concentrations in biomarkers and PM samples below the LOD, a value of LOD/2 was assigned. Table 1 shows the socio-demographic character- istics of the studied population divided according to exposure (moderately exposed/highly exposed) with an age range between 20 and 71 years, with an average of 41.75 ± SD = 13.97 years. The highly exposed population lives within a radius of 0.8 km (0.25–1.5 km) from the ferromanganese factory as the main source of contamination, while the moder- ately exposed population resides within an average radius of 7.3 km (2–34 km). Significant differences were seen between exposure by years of residence, higher for the most exposed group (p < 0.001); by educational level, lower for the most exposed popu- lation (p = 0.005); and by employment status, with more people employed full time in the moderately exposed population (p = 0.045). However, no differ- ences were seen by sex and smoking habits or alcohol consumption. Regarding to diet, none participant was vegetarian and the consumption of grains, greenpeas and beans was similar. Furthermore, the study popu- lation comes from an urban/industrial area, so their diet is mainly based on products purchased in super- markets, thus ruling out the intake of potentially con- taminated food grown in local soils. In addition, the ingestion of water was not considered because of the low metal(loid) content determined in local tap water. The rest of dietary characteristics are also included in Table 1. All quantitative variables in the study were tested for normality using the Kolmogorov–Smirnov test with Lilliefors correction, and the homogeneity of variances was tested using Levene’s test. Student’s t-test (normal distribution and homogeneity of vari- ances), Welch’s t′-test (normal distribution and non- homogeneous variances) and the Mann–Whitney U test (non-normal distribution) were used to compare means/medians between the established groups.f To study the potential effect of confounders on the differences between groups as a function of source distance (i.e. highly exposed vs. moderately exposed), linear regression models were used to calculate crude and adjusted Mean Differences (MDs) of each metal(loid) concentration with their 95% confidence intervals (CI). Wind‑weighted distance calculation procedure for exposure metrics (1), where αhomesource is the direction of the home from the source (rad) and θm represents the average wind direction at the selected meteorolog- ical station in the sampling period in which the volun- teer wore the personal sampler (rad): (1) WI = 1 −cos (훼homesource −휃m ) 2 (1) WI = 1 −cos (훼homesource −휃m ) 2 (1) The average wind direction (θm) was calculated from hourly data considering the wind speed. To calculate the average direction in each period, it was necessary to first determine the Umean and Vmean com- ponents from Eqs. 2 and 3, where N is number of hours of sampling, ui is the hourly wind speed (m/s) and θi is the hourly wind direction (rad). (6) d w = d wf d w = d wf (6) 3 (1234567890) 1 Vol:. Environ Geochem Health (2022) 44:4595–4618 4601 Description of the study population Age (as a continuous variable), sex and study level (ordinal categorised) were pre-established as confounders and included in a first multivariate model. Also, a second multivariate model added as confounders employment status, tobacco smoking and dietary habits: Mn supplement intake and high Mn food consumption (nuts, tea ≥ 5/week, fish as tunas or salmon families… ≥ 3/week).fi Spearman correlation coefficients were calculated between the metal(loid)s of each matrix and between the matrices for each metal(loid). Finally, correlations were also calculated between the biomarkers stud- ied, the levels in PM and the wind-weighted distance between each volunteer’s residence and the main metal(loid) source, as well as the age of the subjects. The Chi-square test was applied to determine whether there was any relationship between exposure (catego- rised as highly/moderately exposed) and the categori- cal variables used in the description of the population (sex, education level, smoking status). All tests were bilateral, and the alpha error was set at 5%. (Bio)markers Tables 2 and 3 show the concentration of the studied metal(loid)s by sex (arithmetic mean (AM), standard deviation (SD), median and reference values (RV) with 95% confidence interval) in the short-term (bio) markers (personal filters and whole blood), as well as in the long-term biomarkers (scalp hair and finger- nails), respectively. Metal(loid)s with at least 50% values above the LOD in each matrix are shown. Ref- erence values were calculated for biological matri- ces only, as the 95th percentile with a 95% confi- dence interval, as specified by Saravanabhavan et al. (2017). With respect to whole blood as short-term biomarker, significant sex differences were observed for Fe (p = 0.039), Cu (p = 0.006) and Pb (p = 0.001), 1 ol.: (0 3 123456789) 3 ) 4602 Environ Geochem Health (2022) 44:4595–4618 1 Vol:. (Bio)markers ( 3 (1234567890) Environ Geochem Health (2022) 44:4595–4618 4603 Environ Geochem Health (2022) 44:4595–4618 Table 1 (continued) Characteristics ME (> 1.5 km) N = 65 HE (≤ 1.5 km) N = 65 Total N = 130 p-value No 57 87.7% 54 83.1% 111 85.4% Yes 8 12.3% 11 16.9% 19 14.6% Tea ≥ 5/week (n, %) 0.019* No 59 90.8% 49 75.4% 108 83.1% Yes 6 9.2% 16 24.6% 22 16.9% Fish as tunas or salmon ≥ 3/week (n, %) 0.784* No 58 89.2% 57 87.7% 115 88.5% Yes 7 10.8% 8 12.3% 15 11.5% Bold implies that the p-value is significant * Chi-square test Mann–Whitney U test with higher concentrations of Fe and Pb for males (492,650 µg/L vs. 466,302 µg/L and 11.40 µg/L vs. 8.49 µg/L, respectively), and higher levels of Cu in females (824.5 µg/L vs. 716.4 µg/L). Regarding to long-term biomarkers, significant sex differences were shown for Mn (p = 0.008), Cu (p = 0.021), Zn (p = 0.009), Cd (p = 0.020) and Pb (p = 0.007) in scalp hair, with higher concentrations for all metals except Cu in males (10,639 ng/g vs. 8450 ng/g), while no significant differences for any metal in the case of fingernails were found. The possible influence of nail varnish on the metal levels was not considered, since only four female volunteers had polished fingernails. In addition, according to the research of Ceballos et al. (2021), the internal levels of metal(loid)s meas- ured in nail technicians’ toenails were comparable to those reported in other studies in females, except for antimony, arsenic, chromium, mercury, and nickel. differences were maintained statistically significant after adjusting for the potential confounders men- tioned in Methodology, as shown in Table 4, except for Cu in scalp hair and Mn in the non-bioaccessible fine fraction, which lost significance in the multivari- ate models. (Bio)markers (1234567890) Table 1 Socio-demographic characteristics of the study population divided according to the exposure groups (highly exposed (HE) (≤ 1.5 km) versus moderately exposed (ME) (> 1.5 km) Characteristics ME (> 1.5 km) N = 65 HE (≤ 1.5 km) N = 65 Total N = 130 p-value Source distance from main point (m) Arithmetic Mean, SD 7294.68 5258.64 799.24 297.94 4046.96 4938.92 Geometric Mean 6212.08 747.87 2155.42 Median, P95 6085.55 18782.14 743.19 1447.45 1770.40 17910.06 Range: min, max 2040.80 33984.74 268.07 1500.00 268.07 33984.74 Interquartile Range (P25, P75) 4969.84 7254.11 594.43 946.25 728.56 6089.44 Age Arithmetic Mean, SD 39.77 13.45 43.66 14.31 41.72 13.97 0.092 Range: min, max 20 71 20 71 20 71 Years residing Arithmetic Mean, SD 11.62 12.42 18.85 13.96 15.23 13.65 < 0.001** Range: min, max 1 60 1 71 1 71 Sex (n, %) 0.553* Female 46 70.8% 49 75.4% 95 100.0% Male 19 29.2% 16 24.6% 35 100.0% Studies (n, %) 0.005* Primary education 3 4.6% 3 4.6% 6 4.6% Secondary Education/Vocational education and Training 7 10.8% 15 23.1% 22 16.9% High school level/Certificate of Higher Education 10 15.4% 20 30.8% 30 23.1% University studies (Bachelor’s Degree) 8 12.3% 11 16.9% 19 14.6% University studies (University Degree) 37 56.9% 16 24.6% 53 40.8% Employment status (n, %) 0.045* Employed full time 54 83.1% 43 66.2% 97 74.6% Unemployed 1 1.5% 4 6.2% 5 3.8% Housewife 0 5 7.7% 5 3.8% Retired 7 10.8% 6 9.2% 13 10.0% Full-time student 3 4.6% 7 10.8% 10 7.7% Smoking status (n, %) 0.498* Non-smoker 42 64.6% 42 64.6% 84 64.6% Former 9 13.8% 13 20.0% 22 16.9% Current 14 21.5% 10 15.4% 24 18.5% Alcohol status 0.597* Never 34 52.3% 37 56.9% 71 54.6% Ever 31 47.7% 28 43.1% 59 45.4% Average of pure ethanol (g/week) (n, %) 0.245* 0 g/week 34 52.3% 37 56.9% 71 54.6% 1–24 g/week 11 16.9% 11 16.9% 22 16.9% 25–74 g/week 16 24.6% 17 26.2% 33 25.4% ≥ 75 g/week 4 6.2% 0 0.0% 4 3.1% Mn food supplements intake (n, %) 0.154* No 63 96.9% 65 100.0% 128 98.5% Yes 2 3.1% 0 0.0% 2 1.5% Nuts ≥ 5/week (n, %) 0.456* Table 1 Socio-demographic characteristics of the study population divided according to the exposure groups (highly exposed (HE) (≤ 1.5 km) versus moderately exposed (ME) (> 1.5 km) 1 Vol:. Correlation analysis Spearman’s correlation coefficients between the concentration of metal(loid)s in each matrix (filters, whole blood, scalp hair and fingernails) and wind- weighted distance are shown in Table 5. For short-term markers, PM-bound Mn showed a significant negative correlation for all the analysed frac- tions with respect to the weighted distance from the most important source, in which those subjects who live closest to the Mn alloy factory (i.e. in the munici- pality of Maliaño) were more exposed. Table 5 also shows the lack of correlation between Mn in whole blood and weighted distance (r = 0.055, p = 0.563). On the other hand, a significant negative correlation of As in whole blood was found with distance (r = − 0.239, p = 0.011), but as we will see later, it is not possible to confirm that inhalation was the main route of exposure, since its levels in PM filters were below the LOD. Figures 2, 3 and 4 show the levels of the selected metal(loid)s in the studied short- and long-term indi- cators, according to the degree of exposure (highly vs. moderately exposed). In terms of exposure, signif- icant differences of medians were only observed for As in whole blood (p = 0.002), Mn and Cu in scalp hair (p = 0.008 and 0.001, respectively) and Mn and Pb in fingernails (p < 0.001 and 0.025, respectively), all concentrations being higher for the highly exposed group with the exception of Cu in scalp hair. For fil- ters, the most exposed population showed signifi- cantly higher concentrations for all the Mn fractions analysed (PM10-2.5 and PM2.5, both bioaccessible and non-bioaccessible), as observed in Fig. 2. These i For long-term biomarkers, Mn showed negative cor- relations with weighted distance, in agreement with that shown in Fig. 4 for both scalp hair and fingernails, respectively. However, this correlation was only statisti- cally significant for fingernails (r = − 0.607, p < 0.001). A significant negative correlation with distance to 1 ol.: (01 3 123456789) 3 123456789) Environ Geochem Health (2022) 44:4595–4618 4604 1 3 Vol:. Discussion The discussion is first focused on Mn, because the study area is characterised by the presence of a fer- romanganese alloy factory as the main source of metal(loid) emissions (Hernández-Pellón & Fernán- dez-Olmo, 2019; Hernandez-Pellón et al., 2017), and due to the lack of consensus for the choice of a suit- able biomarker for Mn, which is further difficulted by its role as a micronutrient (Aschner & Aschner, 2005; Hassani et al., 2016; Jursa et al., 2018). In addition, according to a previous study, this plant is practi- cally the only source of airborne Mn in the study area (Otero-Pregigueiro et al., 2018). i The correlations between the metal(loid) con- centrations in each matrix and the age of the sub- jects were also studied (Supplementary Table 3). In whole blood, there was a significant positive correla- tion of As and Pb with age (r = 0.367, p < 0.001 and r = 0.451, p < 0.001, respectively), and a significant negative correlation of Cu with age (r = − 0.239, p = 0.006), with younger volunteers having higher concentrations, which was also observed in scalp hair (r = − 0.337, p < 0.001). In fingernails, a significant positive correlation of Mn concentration with age (r = 0.315, p = 0.001) was observed. In general, Mn levels in the studied markers are higher in the highly exposed group, with the excep- tion of whole blood. This is corroborated when the correlation between Mn levels in these (bio)markers is analysed. Among the studied indicators of the Mn exposure, the PM-bound Mn concentration showed the highest differences between exposure groups, mainly for the bioaccessible fractions (see Figs. 2, 3, 4). Effect sizes and statistical significance were main- tained after adjusting for potential confounders except for the non-bioaccessible fine fraction (see Table 4), highlighting the importance of the inhalation route of exposure to Mn from the ferromanganese indus- try emissions, in agreement with previous modelling and stationary sampling studies (Hernández-Pellón & Fernández-Olmo, 2019; Otero-Pregigueiro et al., 2018), with the improvement of accurate 24-h expo- sure monitoring in which each volunteer carried the personal sampler. Table 5 also confirmed this hypoth- esis, since the highest correlations between Mn levels and weighted distance were obtained for PM filters, with higher correlation coefficients for the bioacces- sible fractions with respect to the non-bioaccessible fractions. Correlation analysis (r = 0.228, p = 0.018) and between Pb concentrations in whole blood and scalp hair (r = 0.210, p = 0.020). the source was also observed for Zn in scalp hair (r = − 0.207, p = 0.035), but not in fingernails. Copper and Fe show no correlation with distance in fingernails, but a positive and significant correlation in scalp hair: Fe (r = 0.269, p = 0.006), Cu (r = 0.311, p = 0.001). With respect to Pb, contradictory results were found: a positive correlation with distance in scalp hair and negative in fingernails, both significant. Negative but no significant correlations were obtained in the bioac- cessible fine fraction and whole blood. Correlation analysis (1234567890) Table 2 Summary statistics for short-term (bio)markers (filters and whole blood) by sex (Bio) marker Female Male p-value Total N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) Mn coarse fraction bioac- cessible (ng/m3) 95 68.18 (204.49) 15.17 35 41.92 (72.82) 10.57 0.527 130 61.11 (178.90) 13.61 Mn coarse fraction non- bioac- cessible (ng/m3) 95 12.73 (43.07) 3.40 35 9.36 (12.16) 2.62 0.862 130 11.82 (37.33) 3.39 Mn coarse fraction total (ng/m3) 95 80.91 (246.81) 16.51 35 51.28 (82.10) 15.46 0.723 130 79.93 (215.26) 16.47 Mn fine fraction bioac- cessible (ng/m3) 95 70.07 (147.06) 17.82 35 53.12 (100.09) 12.85 0.680 130 66.31 (135.79) 17.05 Mn fine fraction non- bioac- cessible (ng/m3) 95 14.02 (23.27) 5.73 35 8.91 (9.98) 6.61 0.858 130 12.64 (20.64) 5.80 Mn fine fraction total (ng/m3) 95 84.08 (159.31) 25.26 35 65.03 (102.57) 20.02 0.937 130 78.95 (146.07) 25.00 Mn total fraction (PM10) (ng/m3) 95 165.00 (376.19) 42.13 35 116.31 (155.90) 51.53 0.927 130 151.89 (331.66) 43.87 Environ Geochem Health (2022) 44:4595–4618 4605 1 Vol.: (012 Table 2 (continued) (Bio) marker Female Male p-value Total N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) Fe coarse fraction bioac- cessible (ng/m3) 95 62.66 (101.29) 36.30 35 32.96 (28.96) 26.10 0.112 130 54.66 (88.72) 31.65 Fe fine fraction non- bioac- cessible (ng/m3) 95 118.42 (244.32) 65.40 35 79.03 (82.99) 62.00 0.869 130 107.81 (213.59) 65.05 Pb fine fraction bioac- cessible (ng/m3) 95 13.24 (21.10) 4.50 35 11.86 (13.71) 7.40 0.634 130 12.87 (19.34) 5.25 Blood Mn (µg/L) 95 10.04 (3.14) 9.76 15.89 (15.26– 16.53) 35 9.71 (4.01) 9.01 18.51 (17.18– 19.84) 0.375 130 9.95 (3.38) 9.58 16.01 (15.43– 16.59) Blood Fe (µg/L) 95 485327 (81231) 466302 668017 (651682– 684351) 35 505143 (56523) 492650 633413 (614687– 652139) 0.039 130 490662 (75684) 478263 654062 (641052– 667072) Blood Cu (µg/L) 95 886.9 (248.1) 824.5 1425.7 (1375.8– 1475.6) 35 770.2 (167.7) 716.4 1142.9 (1087.4– 1198.5) 0.006 130 855.5 (234.4) 799.8 1368.3 (1328.0– 1408.6) Blood Zn (µg/L) 95 6071 (1897) 5799 9538 (9157– 9920) 35 6150 (1705) 5596 10187 (9622– 10752) 0.923 130 6093 (1841) 5746 9964 (9347– 9980) Blood As (µg/L) 95 4.68 (5.36) 3.06 18.38 (17.30– 19.45) 35 5.02 (5.26) 3.04 21.63 (19.89– 23.37) 0.797 130 4.77 (5.31) 3.06 18.90 (17.98– 19.81) Blood Pb (µg/L) 95 9.74 (5.52) 8.49 19.52 (18.41– 20.63) 35 14.53 (9.17) 11.40 40.24 (37.20– 43.27) 0.001 130 11.03 (6.99) 9.13 24.82 (23.62– 26.02)i 4606 Environ Geochem Health (2022) 44:4595–4618 (r = 0.228, p = 0.018) and between Pb concentrations in whole blood and scalp hair (r = 0.210, p = 0.020). 1 3 . (1234567890) Discussion Between metal(loid)s correlations in whole blood, scalp hair and fingernails are shown in Supplemen- tary Tables 4–6, respectively. In whole blood, there were significant positive correlations between Mn/ Fe (r = 0.191, p = 0.030), Fe/Zn (r = 0.283, p = 0.001), Cu/Zn (r = 0.180, p = 0.040) and As/Pb (r = 0.443, p < 0.001). Regarding to scalp hair, all of them were positive: Mn/Fe (r = 0.282, p = 0.002), Mn/Cd (r = 0.442, p < 0. 001), Mn/Pb (r = 0.348, p < 0.001), Fe/Cd (r = 0.288, p = 0.001), Cu/Cd (r = 0.189, p = 0.037), Cu/Pb (r = 0.345, p < 0.001) and Cd/Pb (r = 0.504, p < 0.001). Finally, the correlations in fin- gernails between Mn/Fe (r = 0.268, p = 0.004), Mn/ Pb (r = 0.414, p < 0.001), Fe/Zn (r = 0.340, p < 0.001), Fe/Pb (r = 0.253, p = 0.013) and Cu/Pb (r = 0.211, p = 0.039) were also significant. i Between matrices correlations for detected metal(loid)s are shown in Supplementary Tables 7 (a-e). Regarding to Mn, no significant correlations between whole blood and any of the Mn fractions analysed in PM nor other biomarkers were observed; however, scalp hair Mn showed significant positive correlations with bioaccessible and total Mn in both fractions (coarse and fine), and fingernails Mn cor- related well with all PM fractions analysed. Signifi- cant positive correlations were also observed for Fe between concentrations in scalp hair and fingernails Although the measured Mn levels in biomark- ers can result from the three routes of exposure, the results discussed above on PM-bound Mn concentra- tions and the importance of the bioaccessible frac- tion can explain the results obtained when scalp hair and fingernails are used. Thus, as shown in Fig. 4, 1 Vol:. Environ Geochem Health (2022) 44:4595–4618 4607 ( ) Vol. Discussion Table 3 Summary statistics for long-term biomarkers (scalp hair and fingernails) by sex Bold implies that the p-value is significant Biomarker Female Male p-value Total N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) N AM (SD) Median RV (95% CI) Scalp hair Mn (ng/g) 93 220.5 (205.4) 168.3 721.7 (680.0– 763.5) 29 366.3 (427.2) 295.9 1550.5 (1395.1– 1706.0) 0.008 122 255.2 (279.6) 185.1 719.0 (669.3– 768.6) Scalp hair Fe (ng/g) 93 9997 (15188) 6728 30010 (26923– 33096) 29 26519 (58257) 8615 233860 (212657– 255063) 0.094 122 13925 (31790) 6823 45887 (40246– 51528) Scalp hair Cu (ng/g) 93 14175 (14387) 10639 40706 (37782– 43630) 29 9658 (5850) 8450 26932 (24262– 28521) 0.021 122 13101 (13001) 9787 35516 (33209– 37823) Scalp hair Zn (ng/g) 93 123641 (47488) 124375 199111 (189460– 208763) 29 164080 (89082) 150307 434800 (402378– 467222) 0.009 122 133253 (62046) 130098 218907 (207897– 229917) Scalp hair Cd (ng/g) 93 12.52 (23.47) 6.74 37.21 (32.44– 41.98) 29 16.31 (15.31) 10.98 54.75 (49.18– 60.32) 0.020 122 13.42 (21.81) 7.57 48.09 (44.22– 51.96) Scalp hair Pb (ng/g) 93 210.9 (214.6) 139.0 669.5 (625.9– 713.1) 29 361.5 (368.7) 234.0 1387.2 (1253.0– 1521.4) 0.007 122 246.7 (265.7) 148.2 861.1 (813.9– 908.2) Fingernails Mn (ng/g) 87 967.5 (1097.1) 562 3778 (3547– 4008) 29 844.3 (1203.3) 532 4331 (3893– 4769) 0.337 116 936.7 (1120.5) 555 3549 (3345– 3753) Fingernails Fe (ng/g) 87 28365 (31490) 19187 93071 (86454– 99687) 29 28776 (29301) 18363 101105 (90441– 111769) 0.690 116 28468 (30832) 18746 90461 (84850– 96072) Fingernails Cu (ng/g) 73 5671 (9904) 3544 15626 (13355– 17898) 23 4730 (2887) 3840 13412 (12232– 14592) 0.328 96 5446 (8743) 3634 14470 (12721– 16219) Fingernails Zn (ng/g) 87 135378 (55913) 124375 227144 (215395– 238893) 29 117672 (34975) 150307 207368 (194639– 220097) 0.064 116 130952 (51913) 118442 220002 (210555– 229449) Fingernails Pb (ng/g) 73 127.5 (208.5) 97.3 402.0 (354.2– 449.9) 23 128.5 (119.3) 94.3 474.1 (425.3– 522.8) 0.866 96 150.5 (190.8) 96.0 424.8 (386.6– 462.9) 1 ol.: (01 Environ Geochem Health (2022) 44:4595–4618 4608 Fig. 2 Levels of Mn in filters from personal sampling accord- ing to the exposure to the main Mn source: moderately exposed (ME) versus highly exposed (HE): (a) bioaccessible, PM10-2.5; (b) non-bioaccessible, PM10-2.5; (c) total, PM10-2.5; (d) bioaccessible, PM2.5; (e) non-bioaccessible, PM2.5; (f) total, PM2.5 PM10-2.5; (b) non-bioaccessible, PM10-2.5; (c) total, PM10-2.5; (d) bioaccessible, PM2.5; (e) non-bioaccessible, PM2.5; (f) total, PM2.5 Fig. Discussion 3 Levels of metal(loid)s in whole blood as short-term biomarker according to the exposure to the main Mn source: moderately exposed (ME) versus highly exposed (HE): (a) Mn; (b) Fe; (c) Cu; (d) Zn; (e) As; (f) Pb Fig. 3 Levels of metal(loid)s in whole blood as short-term biomarker according to the exposure to the main Mn source: moderately exposed (ME) versus highly exposed (HE): (a) Mn; (b) Fe; (c) Cu; (d) Zn; (e) As; (f) Pb a reliable biomarker of exposure (Barbosa et al., 2005). However, although the levels of Pb in whole blood were higher in the highly exposed group (see Fig. 3), the difference was not statistically significant (p = 0.106). It should be noted that the steel plant is located only 3.5 km N from the ferroalloy factory, so its emissions can affect both the highly and the moderately exposed groups. Moreover, contradic- tory results were found for long-term biomarkers: a positive correlation with distance in scalp hair and negative in fingernails, both significant (see Table 5). Thus, while Fig. 4 depicts significant differences between groups in fingernails, with higher levels in the highly exposed group (103.3 vs. 82.8 ng/g, p = 0.025), but contrary to our hypothesis, Pb concen- tration in scalp hair was slightly higher in the moder- ately exposed group (p = 0.088). Although it is well known that blood and nails can be effective biomark- ers of Pb exposure (Barbosa et al., 2005; Olympio et al., 2020), the contradictory results shown here for the three biomarkers studied need further research to elucidate (i) the suitability of these biomarkers to account for the exposure to airborne Pb, and (ii) the contribution of local emission sources other than fer- roalloy smelting.i This study also confirmed that the actual levels of whole blood Pb are much lower than those of previous decades, even in industrial areas like Santander Bay, due to the strict regulations given worldwide. For example, RVs of 70 and 90 µg/L was derived by the Human Biomonitoring (HBM) Commission for women and men, respectively, in the period 1997–1999 (Schulz et al., 2011), much higher than the RV of 24.82 µg/L measured in this work. It also agrees with the report produced by the US Department of Health and Human Services (2018), with a P95 of 23.9 µg/L in 2015/2016. Discussion 2 Levels of Mn in filters from personal sampling accord- ing to the exposure to the main Mn source: moderately exposed (ME) versus highly exposed (HE): (a) bioaccessible, 9.16 µg/L vs. 9.18 µg/L, p = 0.865), even after adjust- ing for potential confounders. This is in agreement with previous studies reporting that Mn levels are tightly regulated in this matrix, with excess being quickly eliminated by the liver and excreted in bile and urine (Gurol et al., 2022). The correlation analy- sis also confirmed that whole blood cannot be used as biomarker of environmental exposure to Mn with epi- demiological purposes, as it did not show significant correlations with any of the Mn fractions analysed in PM, nor with other biomarkers, nor with distance to the source. we can consider scalp hair and mainly fingernails as potential biomarkers of long-term airborne Mn expo- sure. The most exposed population showed medians of 321.6 ng/g and 917.9 ng/g in scalp hair and fin- gernails, respectively, with these concentrations being notably lower for the less exposed population (132.7 ng/g and 331.3 ng/g, respectively), as evi- denced in other studies (Coetzee et al., 2016; Haynes et al., 2015; Levin-Schwartz et al., 2021). These dif- ferences are again maintained after adjusting for potential confounders (see Table 4). The appropriate- ness of these biomarkers is also corroborated by the results of the calculation of between matrices correla- tions for Mn. We observed positive correlations with bioaccessible and total Mn in both fractions (coarse and fine) in the case of scalp hair, and with all PM fractions analysed in the case of fingernails. With respect to Pb, Zn and Fe, their presence in the study area was also mainly attributed to the fer- roalloy factory (Hernández-Pellón & Fernández- Olmo, 2019). This was supported by the significant positive correlations between Mn, Pb, Zn and Fe found in fingernails. However, other nearby emission sources such as a steel plant for Pb, Zn and Fe and non-exhaust road traffic for Zn and Fe cannot be ruled out. Regarding to Pb, blood has been considered as i Our results also suggest that whole blood is not a good biomarker of short-term airborne Mn exposure (see Fig. 3), being unable to differentiate between highly and moderately exposed groups (medians 1 3 Vol:. (1234567890) 1 3 Vol:. (1234567890) 1 3 Vol:. (1234567890) Environ Geochem Health (2022) 44:4595–4618 4609 Fig. Discussion Other recent studies showed similar levels, such as those shown by Saravanabhavan et al. (2017) in a Canadian biomonitoring study, with a P95 of 33 µg/L, or by Ferreira et al. (2019) in an unexposed population in Brazil (P95 = 22.5 µg/L). In Spain, a former national 1 ol.: (01 3 123456789) Environ Geochem Health (2022) 44:4595–4618 4610 1 Vol:. ( 3 (1234567890) Environ Geochem Health (2022) 44:4595–4618 4611 and fingernails. The origin of As in this area needs further investigation, since although it was measured in previous studies both in PM10 collected by station- ary samplers (Hernández-Pellón & Fernández-Olmo, 2019) and in soil (Boente et al., 2020) near the ferro- manganese plant, the levels of As in the bioaccessible and non-bioaccessible fractions of the personal filters collected in the present study were below the LOD, so other sources and routes of exposure are not ruled out. Fig. 4 Levels of metals in long-term biomarkers according to the degree of exposure to the main Mn source: moderately exposed (ME) versus highly exposed (HE): (a) scalp hair Mn; (b) scalp hair Fe; (c) scalp hair Cu; (d) scalp hair Zn; (e) scalp hair Cd; (f) scalp hair Pb; (g) fingernails Mn; (h) fingernails Fe; (i) fingernails Cu; (j) fingernails Zn; k) fingernails Pb ◂ biomonitoring study carried out between 2009 and 2010 (BIOAMBIENT) reported a P95 value of 56.8 µg/L (Cañas et al., 2014), evidencing this progressive decrease of Pb levels in blood.i The differences with respect to the sex of the par- ticipants are in line with those reported in previous studies (Bocca et al., 2011; Coelho et al., 2014; Sara- vanabhavan et al., 2017; Stojsavljević et al., 2019). Coelho et al. (2014) reported higher levels of Mn in toenails in females, in agreement with our results using fingernails. The higher whole blood Pb con- centrations found in males are in accordance with those reported by Batáriová et al. (2006), Coelho et al. (2014), Schulz et al. (2011), Stojsavljević et al. (2019) and Zhang et al. (2015). Moreover, the higher whole blood Cu levels in females are in agreement with Bocca et al. (2011) and Zeng et al. (2019), and may be due to the fact that estrogen-induced cerulo- plasmin synthesis in the liver, can lead to increased blood Cu levels in females (Prasad et al., 2014). Discussion On the other hand, no significant sex differences in whole blood Mn and As levels have been found, supported by Freire et al. (2015), Haynes et al. (2010), Nisse et al. (2017), Stojsavljević et al. (2019) and Zeng et al. (2019). However, contrary to us, Zeng et al. (2019) reported higher levels of Zn, Fe and As in the whole blood of males.i With respect to Zn and Fe, a significant negative correlation with distance to the source was observed for Zn in scalp hair (r = − 0.207, p = 0.035), but not in fingernails (r = − 0.032, p = 0.754), while no cor- relation with distance in fingernails and even positive significant correlation in scalp hair was found for Fe (r = 0.269, p = 0.006). Again, the presence of other sources of these metals in the area makes it difficult to interpret these results. In addition, Zn and Fe are essential trace elements that can enter into the body by other routes.if Copper shows no significant differences as a function of exposure in whole blood and finger- nails, although it does in scalp hair, but losing sig- nificance after adjusting for the selected confounders, mainly attributed to age differences (older people in the highly exposed group, as shown in Table 1, and negative correlation between scalp hair Cu and age, as shown in Supplementary Table 3). In any case, higher scalp hair Cu levels are measured in the mod- erately exposed population, in agreement with a posi- tive significant correlation with distance (r = 0.311, p = 0.001), concluding that the origin of this metal is not due to the ferroalloys plant. According to the literature, airborne Cu originates mainly from non- exhaust emissions due to brake wear (Amato et al., 2010; Bäckström et al., 2003; Johansson et al., 2009). These results are consistent, as road traffic is similar or even higher in Santander area, where most of the moderately exposed group lives.f For males, significant higher levels of Mn in scalp hair were also observed, which is in agreement with Viana et al. (2014), although the significance they reported is limited. Nonetheless, other literature stud- ies reported no significant differences in hair Mn lev- els according to sex (Haynes et al., 2010; Menezes- Filho et al., 2009; Riojas-Rodríguez et al., 2010). The biomonitoring levels obtained in this study were also compared with the available literature. Discussion In the case of whole blood, our biomonitoring val- ues were within the range or slightly below those reported in the literature with the exception of As; for this metal(loid), some outliers were measured, corre- sponding to participants living in the vicinity of the main source of metals in Maliaño. It is unusual to find As values in whole blood higher than 15 µg/L, Finally, As exhibited important differences in whole blood (see Fig. 3), with the most exposed pop- ulation showing statistically significant higher levels (4.24 µg/L vs. 2.81 µg/L, p = 0.002). This agrees with a significant negative correlation found with the dis- tance to the factory (r = − 0.239, p = 0.011). However, it seems that it does not bioaccumulate long-term in the body, remaining below the LOD in both scalp hair 1 ol.: (01 3 123456789) 3 123456789) 4612 Environ Geochem Health (2022) 44:4595–4618 1 Vol:. Discussion (1234567890) Table 4 Crude and adjusted Mean Differences (MD)s for metal(loid)s levels between highly and moderately exposed groups Short-term exposure Highly versus moderately exposed MDcrude 95% CI p value MDa1 95% CI p value MDa2 95% CI p value PM personal samplers (ng/m3) Coarse fraction (PM10-2.5) Mn Bioaccessible 93.00 32.83 153.18 0.003 82.42 18.99 145.85 0.011 89.88 22.84 156.92 0.009 Non-Bioaccessible 15.60 2.88 28.31 0.017 14.67 1.21 28.14 0.033 16.71 2.49 30.93 0.022 Total 108.60 36.04 181.16 0.004 97.09 20.53 173.65 0.013 106.59 25.67 187.51 0.010 Fine fraction (PM2.5) Mn Bioaccessible 85.78 40.91 130.65 < 0.001 88.22 41.41 135.03 < 0.001 90.03 41.92 138.13 < 0.001 Non-Bioaccessible 8.64 1.61 15.67 0.016 6.30 − 0.95 13.54 0.088 4.38 − 2.57 11.34 0.214 Total 94.42 46.28 142.56 < 0.001 94.52 44.34 144.69 < 0.001 106.59 25.67 187.51 0.010 Total (PM10) Mn 203.02 93.05 312.99 < 0.001 191.61 75.67 307.54 0.001 201.00 78.58 323.42 0.001 Coarse fraction (PM10-2.5) Fe Bioaccessible 35.94 5.65 66.23 0.020 28.94 − 2.63 60.50 0.072 30.63 − 2.27 63.53 0.068 Fine fraction (PM2.5) Fe Non-Bioaccessible 24.15 − 50.15 98.45 0.521 18.38 − 60.02 96.78 0.643 − 8.30 − 89.28 72.68 0.840 Fine fraction (PM2.5) Pb Bioaccessible 5.19 − 1.49 11.87 0.127 3.75 − 3.27 10.77 0.293 3.68 − 3.56 10.91 0.317 Blood (µg/L) Blood Mn 0.18 − 1.00 1.36 0.759 0.52 − 0.71 1.75 0.403 0.42 − 0.85 1.70 0.513 Blood Fe − 7159.30 − 33500.51 19181.92 0.592 − 1212.46 − 28783.72 26358.79 0.931 7210.15 − 21596.27 36016.57 0.621 Blood Cu − 7.86 − 89.54 73.82 0.849 6.10 − 76.71 88.91 0.884 − 13.78 − 95.38 67.82 0.739 Blood Zn − 67.65 − 709.11 573.81 0.835 − 74.33 − 750.22 601.56 0.828 24.75 − 676.75 726.25 0.944 Blood As 2.79 1.01 4.58 0.002 2.24 0.48 4.01 0.013 2.05 0.22 3.88 0.028 Blood Pb 1.24 − 1.19 3.67 0.314 0.63 − 1.58 2.84 0.573 0.75 − 1.45 2.94 0.502 Long-term exposure Scalp hair (ng/g) Scalp hair Mn 299.76 − 207.48 806.99 0.244 311.02 − 209.23 831.27 0.239 371.24 − 177.56 920.03 0.183 Scalp hair Fe − 1574.27 − 12962.60 9814.06 0.785 1372.38 − 10245.03 12989.80 0.815 1520.24 − 10804.21 13844.70 0.807 Scalp hair Cu − 4732.05 − 9296.41 − 167.70 0.042 − 4034.22 − 8700.45 632.02 0.090 − 3422.19 − 8374.47 1530.09 0.174 Scalp hair Zn 7099.71 − 15084.49 29283.91 0.528 12442.24 − 9882.76 34767.23 0.272 15850.70 − 3814.92 35516.33 0.113 Scalp hair Cd − 1.70 − 9.48 6.08 0.666 − 1.74 − 9.91 6.43 0.674 − 0.66 − 9.27 7.94 0.879 Scalp hair Pb − 64.09 − 225.73 97.55 0.434 − 44.34 − 208.08 119.39 0.593 − 25.24 − 196.43 145.95 0.771 1 Vol:. Discussion ( Environ Geochem Health (2022) 44:4595–4618 4613 Table 5 Spearman’s correlation coefficients between concen- trations of all employed (bio)markers and wind-weighted dis- tance; markers are shown in two groups according to the dis- tance weighting methods used: short-term and long-term Bold implies that the p-value is significant MD = Mean Difference. MDa1 = MD adjusted for age, sex, and study level. MDa2 = MD adjusted for age, sex, study level, employment status, tobacco smoking and dietary habits (Mn supplement intake and food with Mn consumption (Nuts, Tea ≥ 5/week, Fish as tunas, salmon families… ≥ 3/week). Discussion A positive MD indicates higher levels on participants living in a shorter distance from the ferroalloy plant Short-term exposure Highly versus moderately exposed MDcrude 95% CI p value MDa1 95% CI p value MDa2 95% CI p value Fingernails (ng/g) Fingernails Mn 1006.68 637.21 1376.14 < 0.001 839.68 459.68 1219.68 < 0.001 861.31 467.36 1255.26 < 0.001 Fingernails Fe − 222.52 − 11613.92 11168.88 0.969 − 1030.66 − 13153.73 11092.42 0.867 774.68 − 11971.86 13521.21 0.904 Fingernails Cu 333.81 − 3240.20 3907.83 0.853 1065.19 − 2763.15 4893.53 0.582 1440.86 − 2729.58 5611.31 0.494 Fingernails Zn 878.48 − 18301.33 20058.29 0.928 − 1950.18 − 22105.12 18204.75 0.848 − 3036.20 − 24646.57 18574.16 0.781 Fingernails Pb 74.32 − 2.20 150.84 0.057 75.86 − 6.80 158.51 0.072 102.06 14.21 189.91 0.023 Bold implies that the p-value is significant r p-value Short-term (bio)markers Mn coarse fraction bioaccessible (ng/m3) − 0.609 < 0.001 Mn coarse fraction non-bioaccessible (ng/ m3) − 0.44 < 0.001 Mn coarse fraction total (ng/m3) − 0.612 < 0.001 Mn fine fraction bioaccessible (ng/m3) − 0.449 < 0.001 Mn fine fraction non-bioaccessible (ng/ m3) − 0.319 0.001 Mn fine fraction total (ng/m3) − 0.439 < 0.001 Mn total (PM10) (ng/m3) − 0.549 < 0.001 Fe coarse fraction bioaccessible (ng/m3) − 0.343 < 0.001 Fe fine fraction non-bioaccessible (ng/m3) − 0.150 0.114 Pb fine fraction bioaccessible (ng/m3) − 0.179 0.057 Whole blood Mn (µg/L) 0.055 0.563 Whole blood Fe (µg/L) 0.065 0.495 Whole blood Cu (µg/L) 0.152 0.109 Whole blood Zn (µg/L) 0.028 0.766 Whole blood As (µg/L) − 0.239 0.011 Whole blood Pb (µg/L) − 0.063 0.506 Long-term biomarkers Scalp hair Mn (ng/g) − 0.104 0.291 Scalp hair Fe (ng/g) 0.269 0.006 Scalp hair Cu (ng/g) 0.311 0.001 Scalp hair Zn (ng/g) − 0.207 0.035 Scalp hair Cd (ng/g) 0.151 0.127 Scalp hair Pb (ng/g) 0.300 0.002 Fingernails Mn (ng/g) − 0.607 < 0.001 Fingernails Fe (ng/g) 0.041 0.682 Fingernails Cu (ng/g) − 0.039 0.725 Fingernails Zn (ng/g) − 0.032 0.754 Fingernails Pb (ng/g) − 0.344 < 0.001 Bold implies that the p-value is significant although there have been previous studies report- ing As levels similar to our outliers, such as those of Tratnik et al. (2019) (maximum As in women of 28.9 µg/L and in men of 22.4 µg/L), Freire et al. (2015), (maximum As in women of 26.28 µg/L and in men of 30.75 µg/L), Henríquez-Hernández et al. Discussion (2018) (maximum As of 29.12 µg/L in population aged 20–40 years in the Canary Islands (Spain)), and Kim et al. (2017) in South Korea (maximum As of 59.8 µg/L in women and 53.1 µg/L in men). although there have been previous studies report- ing As levels similar to our outliers, such as those of Tratnik et al. (2019) (maximum As in women of 28.9 µg/L and in men of 22.4 µg/L), Freire et al. (2015), (maximum As in women of 26.28 µg/L and in men of 30.75 µg/L), Henríquez-Hernández et al. (2018) (maximum As of 29.12 µg/L in population aged 20–40 years in the Canary Islands (Spain)), and Kim et al. (2017) in South Korea (maximum As of 59.8 µg/L in women and 53.1 µg/L in men). Table 4 (continued) 1 ol.: (01 3 123456789) Environ Geochem Health (2022) 44:4595–4618 4614 In the case of Mn, the median (9.76 µg/L) was slightly higher than that found in the USA from the Fourth National Report on Human Exposure to Envi- ronmental Chemicals, 9.52 µg/L in 2015/2016 from a sample of 4987 participants (US Department of Health & Human Services, 2018). The highest values of Mn in whole blood corresponded to participants living near the manganese alloy plant, but their maxi- mum (26.76 µg/L) was clearly below some values reported near industrial sources of Mn: for example, Santos-Burgoa et al. (2001) reported a maximum of 88 µg/L near Mn ore mines in Mexico. differences between the highly exposed and mod- erately exposed, highlighting the importance of the inhalation route of exposure to Mn from emissions of the ferromanganese industry. The higher fingernails Mn levels of people living near the Mn source and the significant positive correlations between fingernails Mn and all PM fractions (bioaccessible/non-bioacces- sible coarse and fine) confirm it as the best biomarker of long-term exposure to Mn, the main pollutant in Santander Bay according to WHO guidelines. With respect to Pb, Zn and Fe, their presence in the study area was also mainly attributed to the ferroalloy factory; however, other nearby emission sources can- not be ruled out, such as a steel plant for Pb, Zn and Fe and non-exhaust road traffic for Zn and Fe; moreo- ver, Zn and Fe are essential trace elements that can enter the organism by other routes. Discussion The contradictory results shown here for the three studied biomarkers, mainly for Pb, indicate the difficulties in interpreting the results when different environmental metal(loid) sources and routes of exposure to them may occur. Finally, this study also confirmed that current whole blood Pb levels, even in an urban-industrial mixed area, are much lower than in previous decades. With respect to scalp hair, our values were in gen- eral well below those documented, except for Zn and Cu, which were within the range of other studies. This may be due to the fact that our cleaning proto- col is much more thorough than most protocols used by other authors, leading to a complete removal of exogenous contamination of scalp hair by trace met- als. The influence of pre-treatment steps on the levels of metals in hair has been discussed in the literature (Eastman et al., 2013). These differences were also observed in the case of fingernails, with the peculiar- ity that the literature studies available to compare our range of values were more limited. Acknowledgements We thank the computational assistance provided by Victoria Gallardo. We also thank Emma Campillo (Lund University) for checking the English language and Bruno Carral for his support in the calculation of the wind- weighted distance. Our ranges of concentrations in scalp hair and fingernails were in the same order of magnitude as those obtained by Butler et al. (2019), who used the same cleaning protocol previously reported by East- man et al. (2013). For example, for Mn, Cu and Pb in scalp hair, they obtained medians of 0.08, 9.57 and 0.17 µg/g, respectively, compared to the medi- ans obtained in this study of 0.19, 9.79 and 0.15 µg/g, respectively. However, studies in which a less thor- ough cleaning protocol was used showed much higher Mn levels in hair (e.g. 6.9–31.3 µg/g in Menezes- Filho et al. (2009), 12 µg/g in Mohmand et al. (2015), and 9.7 µg/g for males and 4.4 µg/g for females in Viana et al. (2014)). For nails, something similar occurred, as Butler et al. (2019) obtained medians for Mn, Cu and Pb of 0.19, 2.66 and 0.1 µg/g, respec- tively, while for the same metals our medians were 0.56, 3.63 and 0.1 µg/g, respectively. Discussion Author contribution B.M.: Investigation, Writing—Original draft preparation, Formal analysis; L.R.-A.: Investigation (sam- ples collection); A.E.: Investigation; M.S.: Supervision, Fund- ing acquisition, Formal analysis; I.F.-O.: Conceptualisation, Methodology, Reviewing and Editing, Supervision, Funding acquisition. Funding This work was supported by the Spanish Ministry of Science and Innovation (Project CTM2017-82636-R, funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”). Bohdana Markiv also thanks the MICIU for her predoctoral contract (PRE2018-085152, financed together by MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future”). Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. 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https://openalex.org/W2003385167	https://hal.science/hal-00862300/file/journal.pone.0074763.PDF	English	null	A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism	PloS one	2,013	cc-by	12,334	To cite this version: Hande Tunçay, Justin Findinier, Thierry Duchêne, Virginie Cogez, Charlotte Cousin, et al.. A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism. PLoS ONE, 2013, 8 (9), pp.e74763. 10.1371/journal.pone.0074763. hal-00862300 Distributed under a Creative Commons Attribution 4.0 International License Introduction sucrose. Transient starch turnover is crucial for normal plant growth as revealed by the reduced growth rates of mutant plants unable to synthesize or breakdown the polysaccharide [3]. Storage starch found in amyloplasts of stems, roots, seeds and tubers is remobilized to support plant development as seedling establish- ment after germination [4] or in response to high demand for carbon as for nectar secretion [5]. Starch represents the most widespread storage polysaccharide found in plants and algae. This insoluble polymer of glucose not only defines an essential source of calories for human and animal nutrition but is also used in many non-food applications including but not restricted to biofuel and hydrogen production. Starch is made of two distinct subfractions: the minor amylose fraction is synthesized by a starch granule bound isoform of starch synthase called GBSS (Granule Bound Starch Synthase). Amylopectin, the major fraction, confers to starch most of its physical properties and in particular its insoluble semi-crystalline nature. Even if amylopectin and glycogen share the same primary structure (they are both composed of linear glucose chains linked together through a-1,4 O-glycosidic bonds and branched in a-1,6), the synthesis of these two storage polysaccharides requires a completely different set of enzymes. While bacterial glycogen is synthesized and degraded through the use in most cases of no more than 5–6 activities [1] starch metabolism in green algae and land plants involves more than 30 different enzymes [2]. The high complexity of this metabolic pathway in the green lineage is conserved from the tiniest free-living green alga known to date to maize [2]. Starch is found in the plastids of both photosynthetic and nonphotosynthetic cells. In source organs as leaves it is accumulated during the light period and degraded in the dark to provide substrates for leaf respiration and to supply the plant with The starch biosynthetic process has been extensively studied thanks to both reverse and forward genetics approaches. The high number of enzymatic activities involved and the interaction found between them in the same protein complexes [6,7] reveal the high complexity of the anabolic pathway needed to achieve amylopec- tin biogenesis. It is therefore expected that mobilization of such a molecule would also require a complex catabolic pathway. Significant progress in our understanding of starch mobilisation has been made mostly in the last decade. Abstract A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding b-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae. Editor: Miguel A Blazquez, Instituto de Biologı´a Molecular y Celular de Plantas, Spain Received June 22, 2013; Accepted August 5, 2013; Published September 3, 2013 Copyright: 2013 Tunc¸ay 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 Centre National de la Recherche Scientifique (CNRS), the Universite´ des Sciences et Technologies de Lille (USTL) and the Institut Franc¸ais des Mate´riaux Agro-Source´s (IFMAS). pHD grants from the Ministe`re de la recherche et de l’enseignement supe´rieur (HT) and the Re´gion Nord Pas de Calais and the Universite´ des Sciences et technologies de Lille (JF). 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: David.Dauvillee@univ-lille1.fr * E-mail: David.Dauvillee@univ-lille1.fr . These authors contributed equally to this work. . These authors contributed equally to this work. Hande Tunc¸ay1., Justin Findinier1., Thierry Ducheˆne1, Virginie Cogez1, Charlotte Cousin1,2, Gilles Peltier3, Steven G. Ball1, David Dauville´e1* Hande Tunc¸ay1., Justin Findinier1., Thierry Ducheˆne1, Virginie Cogez1, Charlotte Cousin1,2, Gilles Peltier3, Steven G. Ball1, David Dauville´e1* 1 Unite´ de Glycobiologie Structurale et Fonctionnelle UMR 8576, CNRS- Universite´ des Sciences et Technologies de Lille, Villeneuve d’Ascq, France, 2 Institute Micalis UMR1319, INRA, Thiverval-Grignon, France, 3 UMR Biologie Ve´ge´tale et Microbiologie Environnementale, Laboratoire de Bioe´nerge´tique et Biotechnologie des Bacte´ries et Microalgues, CEA-CNRS- Aix Marseille Universite´, Saint-Paul-lez-Durance, France HAL Id: hal-00862300 https://hal.science/hal-00862300v1 Submitted on 3 Oct 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License September 2013 \| Volume 8 \| Issue 9 \| e74763 Generation of the insertional mutant libraries and phenotypic screening Two independent insertional libraries were generated by transforming our Chlamydomonas reinhardtii wild-type reference strain 137C with either the EcoRI linearized pSL18 plasmid [31] or with a 1,9 kb PCR product corresponding to the paromomycin cassette (see Methods). Prior to transformation the cell pellets were treated with home-made autolysin to allow the transformation with glass beads. The efficiency of transformation was clearly different as we obtained around 2000 paromomycin resistant clones with 1 mg of linearized plasmid while less than 100 were observed with the same quantity of PCR products. This difference could be attributed to the limited size of the non-essential region of the foreign DNA which can be used during the integration process and that most of the insertions with PCR products did not confer paromomycin resistance to the algae. With this approach, we generated 3,000 and 13,000 mutants with respectively the PCR cassette and the linearized plasmid which were thereafter screened with iodine to detect the strains defective for starch catabolism. The natural interaction between iodine and a-1,4-linked- glucans has been used extensively to study starch anabolism in Chlamydomonas [26]. However, as a Chlamydomonas strain contains enough starch to appear black while subjected to iodine vapors, the screening procedure has to be modified in order to detect strains which are potentially containing more polysaccha- ride than a wild-type. Two successive steps were then used to detect the strains in which the insertion has modified either the amount of polysaccharide or produce a defect in the rate of starch mobilization. In a first step, cells were spotted in duplicate on nitrogen free TAP plates and incubated for 5 days under continuous light to trigger massive starch deposition. For one of the plates, the starvation was removed after this incubation by applying 10 mL of 0,15 M ammonium chloride and incubated in total darkness for one more day. Both plates were then subjected to iodine vapors and immediately photographed. During the incubation in the dark, a strain competent for starch mobilization will be able to degrade nearly all its storage polysaccharide and will then loose its black stain with iodine (Figure 1). A strain which is defective for starch degradation or which contained much more polysaccharide than a wild-type before the transition to darkness will remain black revealing the presence of residual starch. Introduction In addition to offering an excellent forward genetics system with straightforward mutant screening procedure, Chlamydomonas was also shown to offer a very diverse spectrum of relevant physiological growth conditions [28] thereby mimicking source sink and storage tissues of higher plants to study the impact of diverse mutations. Introduction Moreover, even if reverse genetics was extensively used to raise this model, the most surprising steps were revealed through distinct approaches such as biochemistry for the initial dikinase discovery or forward genetics for the Mex maltose transporter. While a-amylase activities were thought not to participate to starch degradation [20] in Arabidopsis leaves, a recent study revealed that the degradation initiated by the glucan phosphorylation process would require the action of three hydrolytic activities including one particular isoform of a-amylase called AMY3 [21]. Hence there is room to better our knowledge of starch catabolism and to expand these studies to organisms which are not only amenable to very productive forward genetic approaches but which in addition would afford for a greater variety of physiological conditions not restricted to the massive carbon export displayed by Arabidopsis leaf cells. We therefore believe that the unicellular green alga Chlamydomonas reinhardtii which has been by the past intensively used to study starch synthesis [22,23,24,25,26,27] offers a very useful model to deepen our knowledge of starch mobilization. In addition to offering an excellent forward genetics system with straightforward mutant screening procedure, Chlamydomonas was also shown to offer a very diverse spectrum of relevant physiological growth conditions [28] thereby mimicking source sink and storage tissues of higher plants to study the impact of diverse mutations. In this work, we report the production and the preliminary and algae. Our data obtained so far offer valuable information for the community involved in the study of carbon metabolism in plants and reveal potential functions which may define suitable targets to control the catabolic process for bioenergy production purposes. They also not only validate the two step iodine screening procedure as a tool in identifying starch catabolic functions but also reveal the complexity of the physiological processes taking place in the cells as non directly relevant functions may have been identified too. The complete characterization of the most promising mutants including functional complementation and genetic cosegregation analyses will be described elsewhere. phosphorylation process and the ability displayed by degradation enzymes to initiate polysaccharide mobilization is not yet fully understood. It has been recently proposed that this phosphoryla- tion opens up the crystalline structure thereby facilitating subsequent hydrolysis [11]. The process is even more complex as specific phosphatases called Sex4 [12], and LSF2 [13] are necessary to achieve efficient starch degradation. Introduction Another homologous protein called LSF1 [14] is also necessary for efficient starch breakdown but this protein was not yet proved to contain a phosphatase activity and may act as a regulator. In addition to the initial discovery of GWD, the analyses performed on different T- DNA insertion mutants of Arabidopsis thaliana have yielded a working model for the recurring starch degradation occurring in leaves of this model organism [15]. Taken together, the data revealed the importance of exo-amylases (b-amylases; [16]) and a specific type of isoamylase (ISA3; [17]) as major actors of the starch degradation process in Arabidopsis leaves at night. Briefly, the starch granule is subjected to phosphorylation/dephosphory- lation thereby allowing the action of hydrolytic activities. The maltose produced by b-amylases is then exported to the cytosol via a plastidial maltose transporter MEX1 [18]. In this compartment, a transglucosylation reaction occurs allowing the release of glucose (catalysed by DPE2, [19]). Nevertheless, the catabolic model established in Arabidopsis may not represent a consensus of starch mobilization in photosynthetic tissues. It may predominantly reveal a pathway that is active in only a very specific subset of physiological conditions. These may be limited to massive and immediate export of carbon in the leaf cytosol to feed the nonphotosynthetic parts of the plant at night. Moreover, even if reverse genetics was extensively used to raise this model, the most surprising steps were revealed through distinct approaches such as biochemistry for the initial dikinase discovery or forward genetics for the Mex maltose transporter. While a-amylase activities were thought not to participate to starch degradation [20] in Arabidopsis leaves, a recent study revealed that the degradation initiated by the glucan phosphorylation process would require the action of three hydrolytic activities including one particular isoform of a-amylase called AMY3 [21]. Hence there is room to better our knowledge of starch catabolism and to expand these studies to organisms which are not only amenable to very productive forward genetic approaches but which in addition would afford for a greater variety of physiological conditions not restricted to the massive carbon export displayed by Arabidopsis leaf cells. We therefore believe that the unicellular green alga Chlamydomonas reinhardtii which has been by the past intensively used to study starch synthesis [22,23,24,25,26,27] offers a very useful model to deepen our knowledge of starch mobilization. Introduction It now defines a very active domain of research that essentially concerns diurnal Arabidopsis leaf starch mobilisation (for review, see [8]). One of the most remarkable discoveries consists of the involvement of dikinases as essential actors of starch degradation. These enzymes, glucan water dikinase (GWD; [9]) and phosphoglucan water dikinase (PWD; [10]) were first found as minor proteins bound to starch granules and were subsequently demonstrated to phos- phorylate glucose residues of amylopectin crystalline sections during starch breakdown. However, the exact link between this September 2013 \| Volume 8 \| Issue 9 \| e74763 1 September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org Genetic Dissection of Starch Catabolism phosphorylation process and the ability displayed by degradation enzymes to initiate polysaccharide mobilization is not yet fully understood. It has been recently proposed that this phosphoryla- tion opens up the crystalline structure thereby facilitating subsequent hydrolysis [11]. The process is even more complex as specific phosphatases called Sex4 [12], and LSF2 [13] are necessary to achieve efficient starch degradation. Another homologous protein called LSF1 [14] is also necessary for efficient starch breakdown but this protein was not yet proved to contain a phosphatase activity and may act as a regulator. In addition to the initial discovery of GWD, the analyses performed on different T- DNA insertion mutants of Arabidopsis thaliana have yielded a working model for the recurring starch degradation occurring in leaves of this model organism [15]. Taken together, the data revealed the importance of exo-amylases (b-amylases; [16]) and a specific type of isoamylase (ISA3; [17]) as major actors of the starch degradation process in Arabidopsis leaves at night. Briefly, the starch granule is subjected to phosphorylation/dephosphory- lation thereby allowing the action of hydrolytic activities. The maltose produced by b-amylases is then exported to the cytosol via a plastidial maltose transporter MEX1 [18]. In this compartment, a transglucosylation reaction occurs allowing the release of glucose (catalysed by DPE2, [19]). Nevertheless, the catabolic model established in Arabidopsis may not represent a consensus of starch mobilization in photosynthetic tissues. It may predominantly reveal a pathway that is active in only a very specific subset of physiological conditions. These may be limited to massive and immediate export of carbon in the leaf cytosol to feed the nonphotosynthetic parts of the plant at night. Biochemical characterizations of the CAT mutants Biochemical characterizations of the CAT mutants Starches accumulated by each of the CAT mutants were assayed after 5 days in liquid TAP-N medium under constant light. The amounts of storage polysaccharide accumulated in all these strains are displayed in figures 2a, 3a and 4a as percentages of the amount assayed in the wild-type. We were expecting that most of our selected mutants would have displayed a starch excess phenotype. This is clearly not the case as 16 of them contain between 64 to 119% of the wild-type value (the wild-type strain 137C contains 2164 mg of starch per million cells in our standard nitrogen starvation conditions). Fifteen CAT mutants contain a significant higher amount of starch under nitrogen starvation (ranging from 124% to 205%) and only 7 display a clear starch excess phenotype containing more than twice the amount of polysaccharide accumulated by the parental strain (from 233% to 468%). In parallel, we examined the kinetics of starch degradation in each of our strains in liquid cultures. Each strain was used to inoculate one liter of TAP-N liquid medium and grown for 5 days under continuous light. The cells were then transferred to TMP medium (without acetate) and aliquots of the culture were incubated in the dark for 4, 8 and 24 hours. For each time, starch was extracted and assayed in order to determine the amount of polysaccharide broken down. The amounts of residual starch after 24 hours of degradation are displayed in figures 2b, 3b and 4b while the complete kinetics can be found in figure S1. These kinetics experiments allowed us to identify three phenotypic classes in our mutant bank. The first group consisted in strains as efficient as the wild-type with respect to starch mobilization. In fact, after 24 hours darkness, the wild-type 137C strain still contained 2867% of its initial starch amount as did 12 of our mutants (figure 2b). Those strains which initially displayed a clear and confirmed phenotype during the 2 step screening procedure should be then either considered as false positives or as mutants In parallel, we tested the presence of the complete set of hydrolytic activities which are easily visualized thanks to our zymogram techniques in such mutants. In fact, some of our mutants may be defective for an enzymatic activity which is required for starch catabolism and zymograms may allow us to quickly ascertain the absence of one of them. Generation of the insertional mutant libraries and phenotypic screening The figure 1 is illustrating the phenotype of the wild-type strain 137C and of 9 mutants that have been selected as putative starch catabolic mutants after a confirmation round of screening. In total, we selected 31 strains displaying a clear black phenotype after 24 h of degradation (that we named CAT1 to CAT31) from our plasmid insertional mutagenesis procedures and 7 generated with the PCR product (called CAT32 to CAT38). The same ratio of starch catabolic mutant was then observed for both foreign DNA used in this study (1 mutant for 428 resistant clones with the PCR In this work, we report the production and the preliminary characterization of an insertional mutant bank of the green alga model Chlamydomonas reinhardtii. Our goal was to assess the suitability of the phenotypic screening procedure we set up in the green alga model [29] and that was recently used to study the link between starch degradation and hydrogen production [30]. The molecular analyses performed on our mutants allowed us to identify mutations either in enzymes involved in starch degrada- tion per se or in putative regulatory functions revealing the importance to combine both reverse and forward genetic approaches to obtain a clear view on starch catabolism in plants September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 2 Genetic Dissection of Starch Catabolism Figure 1. Two step iodine screen of putative starch catabolic mutants. Cell patches of the wild-type strain 137C, two mutant strains defective for starch biosynthesis and of the three mutant classes are displayed after staining with iodine vapors. The cells were incubated 5 days under nitrogen starvation in the light (a) or one more day in the dark after the removal of the starvation (b). The wild-type reference 137C, the BafJ4 mutant strain lacking starch and the BafR1 mutant producing only amylopectin are shown at the top while the CAT1, 6, and 9 (second lane); the CAT17, 21, and 23 (third lane); the CAT 3, 19, and 22 (fourth lane) represent respectively mutants of the three classes. doi:10.1371/journal.pone.0074763.g001 with conditional defects due to different behavior on solid and liquid medium. However, one of them called CAT37 did display a slow rate of starch mobilization during the first 8 hours of the kinetics and may represent a mutant in the signalization process (figure S1a). Figure 1. Two step iodine screen of putative starch catabolic mutants. Cell patches of the wild-type strain 137C, two mutant strains defective for starch biosynthesis and of the three mutant classes are displayed after staining with iodine vapors. The cells were incubated 5 days under nitrogen starvation in the light (a) or one more day in the dark after the removal of the starvation (b). The wild-type reference 137C, the BafJ4 mutant strain lacking starch and the BafR1 mutant producing only amylopectin are shown at the top while the CAT1, 6, and 9 (second lane); the CAT17, 21, and 23 (third lane); the CAT 3, 19, and 22 (fourth lane) represent respectively mutants of the three classes. doi:10.1371/journal.pone.0074763.g001 product and 1 for 483 with the linearized plasmid). All these mutants were then subjected to biochemical characterizations in order to determine which of the candidates identify bona fide starch catabolism mutants which are the most impaired in starch mobilization (figure 4). Despite that we did not expect mutants impaired in starch degradation to yield a structurally modified polysaccharide, we nevertheless analyzed on CL-2B gel permeation chromatography the amylopectin/amylose ratio and the amylopectin lmax which reflects the structure of this storage polysaccharide component in our CAT mutants (figure 5). The wild-type strain 137C produces a starch composed of 20% of amylose and 80% of amylopectin with a lmax of 550 nm for the latter (Figure 5a). Two of our mutants did harbor significant differences concerning either the amylopec- tin/amylose ratio or the amylopectin structure. CAT17 produced a starch containing a high lmax amylopectin at 567 nm (Figure 5b) compared to the 552 nm recorded for the wild-type. This increase in lmax is surprisingly not correlated with an amylose enrichment as is usually recorded when amylopectin synthesis is impaired (29% compared to 23% in 137C). On the other hand, the CAT31 starch displayed a high amount of amylose as this component represents 41% of the polysaccharide produced by this strain (figure 5c). In this mutant, the amylopectin structure appears unchanged (lmax 553 nm). At this stage, it seems difficult to explain these modifications and further investigations on these two strains are required to explain the observed phenotypes. September 2013 \| Volume 8 \| Issue 9 \| e74763 Generation of the insertional mutant libraries and phenotypic screening The second class of mutants displayed a confirmed unconditional defect in starch mobilization. These 13 strains (figure 3b) contain more residual starch than the wild-type after 24 h of degradation (from 35 to 49% of the initial amount while 137C contains 28%). Finally, the most defective strains (figure 4b) define the third class of mutants in which 13 strains harbor a strong starch degradation defect as they still contained between 56 to 97% of their initial amount of starch after 1 day in darkness. It is of prime importance to notice that for each class of mutants we observed strains containing completely different amounts of starch. Even for the most affected strains, some did contain less starch than the wild-type at the end of the starvation phase. This is clearly the case for example for strains CAT13, CAT34 and CAT35 which accumulate respectively 67, 86 and 73% of the wild-type starch amount but are still unable to degrade their polysaccharides as they still contain 81, 56, and 89% of this starch after 24 h in TMP in the dark while 137C was able to catabolize two thirds of its initial amount in the same time. This result and the fact that strains that seem to degrade efficiently while overaccumulating starch (CAT30 for example, figure 2a) reveal that there is not an obligate link between a starch excess phenotype and a defect in starch catabolism. However, the highest starch excess phenotypes are still found in the class 3 mutants corresponding to those strains which are the most impaired in starch mobilization (figure 4) Biochemical characterizations of the CAT mutants In one of our mutants (CAT16), an activity producing a pink band on denaturing starch containing zymograms is lacking (figure 6a, left panel). This phenotype was confirmed on starch containing zymograms performed under native (non denaturing) conditions (figure 6a, right panel). This pink/red staining of the missing activity in CAT16 is characteristic of an increase in the amount of branches in the starch contained as substrate in the gel. Two kinds of activities can be responsible of such a modification. Branching enzyme will catalyze the introduction of new a-1,6 linked chains in September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 3 Genetic Dissection of Starch Catabolism Figure 2. Starch deposition and kinetics of starch mobilization in class 1 mutants. The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g002 Figure 2. Starch deposition and kinetics of starch mobilization in class 1 mutants. The a Figure 2. Starch deposition and kinetics of starch mobilization in class 1 mutants. The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g002 mutant which is disrupted in a gene encoding the MEX transporter previously characterized in Arabidopsis [18]. We were also able to identify a disruption in a catalytic isoform of b-amylase (CAT4) which had been described as a major component of the transient starch degradation machinery in Arabidopsis [16]. Biochemical characterizations of the CAT mutants Two additional mutants can be suspected to be defective for compo- nents of the catabolic machinery even if these functions had not yet been reported as such. This is the case for the CAT33 mutant which carries a disruption for a protein of unknown function but which contains a CBM20 starch binding module. A disruption in an oligosaccharyl transferase was also detected in CAT37 and may also represent a yet undescribed enzymatic function required for efficient starch degradation. In 4 of our mutants, we were able to identify the integration sites in different kinase activities (table 1). If the link between the starch defect and the absence of these kinase activities should be confirmed, it may unravel precious informa- tion concerning the regulation of starch catabolism. However, the phenotype on starch catabolism could also reflect indirect consequences of the absence of a kinase activity since such mutants are known to yield numerous pleiotropic effects. Finally, we detected a defect in a gene described as encoding a flagellar/ the polysaccharide matrix and b-amylases by shortening the outer chains of starch will produce a so-called beta-limit dextrin which is enriched in branching points compared to the initial substrate found in the gel. To determine the nature of the missing activity in our mutant, we semi purified this activity and determined its precise nature as described below. Molecular characterizations of the CAT mutants The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g003 basal body protein which could explain the starch excess phenotype found in the CAT22 mutant as motility has been previously reported to impact starch accumulation [32]. More- over, we started backcrosses and genetic analyses of our mutants in order to check the co-segregation between the disruption and the observed phenotype. The complete genetic and biochemical characterization of some of them will be described elsewhere. containing zymograms in order to detect fractions containing the pink band forming activity free of any other hydrolytic activity. In 3 elution fractions (E1 to E3 in figure 6b which corresponds to the 3 consecutive elution with the buffer containing 0,5 mg/mL MOS) only the pink band which is missing in CAT16 could be detected on the gel while the next elutions are all contaminated with hydrolytic activities giving a white band on the gel that we already characterized as a-amylase [27]. These fractions were then used to determine the nature of the enzyme activity. To do so, 300 mL of the three fractions were incubated 2 hours in the presence of potato amylose at 30uC. The modified amylose was then stained with iodine (see methods). If the missing activity detected in the CAT16 mutant is of the beta-amylase type, then during the incubation the amylose should be entirely degraded and no interaction with iodine should be recorded. In the case of a branching enzyme activity the greenish color due to the interaction of the long amylose chains with iodine should disappear and a pink to red staining should appear due to the appearance of new branching points. In this case, the lmax of the In this work, we decided to focus on the CAT16 mutant which is the only one in our bank for which we detected a clear enzymatic defect through zymograms. Molecular characterizations of the CAT mutants In order to confirm the validity of our screening procedure, the mutants displaying a possible defect in starch mobilization (i.e. chiefly the class 2 and 3 mutants) were analyzed through molecular techniques in order to identify the genomic sequences of those regions flanking the insertions. Different strategies including plasmid rescue, inverse PCR and TAIL-PCR were attempted. Even if our efforts resulted in limited success, we were still able to determine in a few of our mutants the identity of the genes disrupted by the exogenous DNA. A summary of the genes identified and of the techniques used for the identification is listed in table 1. We were able to find in our mutant bank several insertions in genes which encoded enzymes previously reported as involved in starch catabolism. This is the case for the CAT3 September 2013 \| Volume 8 \| Issue 9 \| e74763 September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 4 Genetic Dissection of Starch Catabolism Figure 3. Starch deposition and kinetics of starch mobilization in class 2 mutants. The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g003 Genetic Dissection of Starch Catabolism Figure 3. Starch deposition and kinetics of starch mobilization in class 2 mutants. The a under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type cultivated under the same conditions. The amount of starch remaining after 1 day of degrada percentage of the initial amount measured in the corresponding strain. All data correspond t Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g003 Figure 3. Starch deposition and kinetics of starch mobilization in class 2 mutants. The CAT16 mutant is defective for starch branching enzyme 1 and displays a starch catabolism defect A crude extract from the wild-type strain 137C was used to semi-purify the activity missing in the CAT16 mutant (figure 6a). The wild-type extract was subjected to affinity chromatography on an amylose resin and the bounded proteins were eluted with purification buffer containing increasing amount of malto-oligo- saccharides (MOS). Each elution fraction was tested on starch September 2013 \| Volume 8 \| Issue 9 \| e74763 September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 5 Figure 4. Starch deposition and kinetics of starch mobilization in class 3 mutants. The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g004 Genetic Dissection of Starch Catabolism Genetic Dissection of Starch Catabolism iodine/polysaccharide complex should decrease due to the reduction in length of the glucose chains [33]. No interaction extract of the CAT (figure S2). As exp Figure 4. Starch deposition and kinetics of starch mobilization in class 3 mutants. The a under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type cultivated under the same conditions. The amount of starch remaining after 1 day of degradat percentage of the initial amount measured in the corresponding strain. All data correspond t Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g004 Figure 4. Starch deposition and kinetics of starch mobilization in class 3 mutants. The am under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type r cultivated under the same conditions. The amount of starch remaining after 1 day of degradatio percentage of the initial amount measured in the corresponding strain. All data correspond to Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g004 Figure 4. Starch deposition and kinetics of starch mobilization in class 3 mutants. The CAT16 mutant is defective for starch branching enzyme 1 and displays a starch catabolism defect The amount of starch assayed in the strains after 5 days under nitrogen starvation (a) is displayed as a percentage of the value assayed in the wild-type reference strain 137C (2164 mg per million cells) cultivated under the same conditions. The amount of starch remaining after 1 day of degradation of the same strains (b) is represented as the percentage of the initial amount measured in the corresponding strain. All data correspond to mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. doi:10.1371/journal.pone.0074763.g004 extract of the CAT16 mutant strain which is lacking this activity (figure S2). As expected, no activity was detected in the elution fractions (figure S2B) and after incubation of these fractions with amylose, no modification of the polysaccharide was detected (figure S2C) revealing the link between the ‘‘pink/red’’ producing enzyme and the branching enzyme activity. As the Chlamydo- monas genome contains three kind of branching enzymes, we tried to determine which one was missing in our mutant and if this absence was due to the insertion of the mutagenesis plasmid in the structural gene encoding the enzyme. We then designed three primers couples to amplify a part of the three branching enzyme structural genes from the CAT16 genomic DNA. We were able to amplify a fragment of BE2a and BE2b structural genes in both 137C and CAT16 while no amplification was obtained with the mutant with the primers designed to amplify a part of the BE1 gene (figure 7a). As insertional mutagenesis in Chlamydomonas is known to cause deletions, the lack of amplification is a strong argument in favor of an insertion of pSL18 into the BE1 structural iodine/polysaccharide complex should decrease due to the reduction in length of the glucose chains [33]. No interaction can be recorded between iodine and the purification buffer used alone (figure 6C, lane 1). A clear interaction of amylose and iodine prior to incubation is observed as revealed by a greenish color with a typical lmax of 643 nm due to the long chains composing the polysaccharide (figure 6C, lane 2). In the presence of 0,5 mg/mL MOS, the lmax of the amylose/iodine complex decreases but remains high at 602 nm showing a light interaction with the glucans that were used to elute the enzymatic activity (figure 6C, lane 3). The CAT16 mutant is defective for starch branching enzyme 1 and displays a starch catabolism defect Partial purification of branching enzyme 1 activity. The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native (a; left panel) and denaturing (a; right panel) starch zymograms respectively. The lack of contaminating activities in the first elution fractions (E1 to E3) of the amylose column chromatography was assessed on starch denaturing zymogram (b). (c) Interaction of polysaccharides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide modified by the enzyme contained in the 3 elution fractions are displayed in 4, 5 and 6. The values of the lmax of each complex is indicated on the figure in nanometers. doi:10.1371/journal.pone.0074763.g006 Figure 5. Separation of amylopectin and amylose by CL2B Sepharose chromatography. The optical density (N) was measured for each 0.3-mL fraction at lmax (unbroken thin line). All samples were loaded on the same column setup described by [25]. The wild-type haploid 137C strain starch extracted from nitrogen starved cultures (a) displays both amylopectin and low-molecular weight amylose. Starches from the mutant strains CAT17 and CAT31 are represented in b and c respectively. The amount of amylose (%) and the amylopectin lmax in nanometers are displayed on the corresponding graphs. doi:10.1371/journal.pone.0074763.g005 Figure 6. Partial purification of branching enzyme 1 activity. The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native (a; left panel) and denaturing (a; right panel) starch zymograms respectively. The lack of contaminating activities in the first elution fractions (E1 to E3) of the amylose column chromatography was assessed on starch denaturing zymogram (b). (c) Interaction of polysaccharides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide modified by the enzyme contained in the 3 elution fractions are displayed in 4, 5 and 6. The values of the lmax of each complex is indicated on the figure in nanometers. doi:10.1371/journal.pone.0074763.g006 Figure 6. Partial purification of branching enzyme 1 activity. The CAT16 mutant is defective for starch branching enzyme 1 and displays a starch catabolism defect After incubation with the ‘‘pink band’’ producing activity, the iodine interaction was completely changed as the color switched from green to red and the lmax drastically decreased with values around 480 nm (figure 6C, lanes 4, 5 and 6). This modification reveals that the semi purified activity which is lacking in CAT16 is a branching enzyme activity. To ensure this modification was really due to the ‘‘pink band’’ detected on zymograms, the same purification procedure was used on a crude September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 6 Genetic Dissection of Starch Catabolism Figure 6. Partial purification of branching enzyme 1 activity. The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native (a; left panel) and denaturing (a; right panel) starch zymograms respectively. The lack of contaminating activities in the first elution fractions (E1 to E3) of the amylose column chromatography was assessed on starch denaturing zymogram (b). (c) Interaction of polysaccharides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide modified by the enzyme contained in the 3 elution fractions are displayed in 4, 5 and 6. The values of the lmax of each complex is indicated on the figure in nanometers. doi:10.1371/journal.pone.0074763.g006 Genetic Dissection of Starch Catabolism gene. We then tested through RT-PCR the expression of the BE1 gene. As expected, we were not able to amplify a part of the BE1 cDNA when using the RNAs extracted from the mutant (figure 7b). These preliminary results may argue in favor of an unsuspected Figure 5. Separation of amylopectin and amylose by CL2B Sepharose chromatography. The optical density (N) was measured for each 0.3-mL fraction at lmax (unbroken thin line). All samples were loaded on the same column setup described by [25]. The wild-type haploid 137C strain starch extracted from nitrogen starved cultures (a) displays both amylopectin and low-molecular weight amylose. Starches from the mutant strains CAT17 and CAT31 are represented in b and c respectively. The amount of amylose (%) and the amylopectin lmax in nanometers are displayed on the corresponding graphs. doi:10.1371/journal.pone.0074763.g005 Figure 6. The CAT16 mutant is defective for starch branching enzyme 1 and displays a starch catabolism defect The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native (a; left panel) and denaturing (a; right panel) starch zymograms respectively. The lack of contaminating activities in the first elution fractions (E1 to E3) of the amylose column chromatography was assessed on starch denaturing zymogram (b). (c) Interaction of polysaccharides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide modified by the enzyme contained in the 3 elution fractions are displayed in 4, 5 and 6. The values of the lmax of each complex is indicated on the figure in nanometers. doi:10.1371/journal.pone.0074763.g006 gene. We then tested through RT-PCR the expression of the BE1 gene. As expected, we were not able to amplify a part of the BE1 cDNA when using the RNAs extracted from the mutant (figure 7b). These preliminary results may argue in favor of an unsuspected role of branching enzyme 1 in starch catabolism in Chlamydo- monas. to produce mutants and study the link between polysaccharide degradation and photosystem II-independent hydrogen biopro- duction [30]. However, in this study, the mutations were not characterized and it appeared important to ascertain that true starch catabolic mutants could be obtained through this screening procedure. Several physiological processes suspected to be highly regulated impact the amount of polysaccharide remaining after incubation in darkness during the phenotypic screening procedure. First, the cells are incubated 5 days on solid medium without nitrogen which triggers starch deposition. Depending of the strain genotype, the amount of storage polysaccharide accumulated can be different from strain to strain and thereby affect the intensity of Discussion While the iodine vapors staining of cell patches has been proved to be an efficient way to selectively detect algal mutants affected in starch biosynthesis and (or) starch structure [26]; the use of this natural interaction had to be tested with respect to starch catabolism. A two-step procedure consisting of the staining of Chlamydomonas cell patches with iodine after massive starch accumulation under the light and a duplicate which is incubated one more day in darkness to trigger degradation was recently used September 2013 \| Volume 8 \| Issue 9 \| e74763 September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 7 Genetic Dissection of Starch Catabolism Table 1. List of mutants whose molecular nature was identified through molecular or biochemical techniques. Table 1. List of mutants whose molecular nature was identified through molecular or biochemical techniques. Strain Origin Identification Technique Insertion (Phytozome v9.0) CAT3 Plasmid Inverse (T1&T2) BssHII Cre12.g486600, Maltose exporter-like protein CAT4 Plasmid Inverse (P1&P2) BssHII Cre06.g307150, Beta-amylase CAT16 Plasmid Zymograms Cre06.g289850, Branching enzyme 1 CAT33 PCR TAIL Cre02.g091750, CBM20 Starch Binding Domain containing protein CAT37 PCR TAIL Cre07.g330100, Oligosaccharyl transferase STT3 subunit CAT15 Plasmid Plasmid rescue ApaI Cre16.g689550, Putative tyrosine kinase CAT24 Plasmid Inverse (P1&P2) EaeI Cre06.g307100, ABC1/COQ8 Ser/Thr kinase CAT26 Plasmid Inverse (P1&P2) BsrBI Cre02.g107000, Putative protein Kinase CAT14 Plasmid Inverse (P1&P2) BssHII Cre06.g266150, Putative kinase CAT22 Plasmid Inverse (P1&P2) BssHII Cre07.g338300, Putative flagellar/basal body protein doi:10.1371/journal.pone.0074763.t001 the stains. New signals have to be integrated by the cells when starvation is removed and cell patches transferred to darkness. At this stage again, a high amount of residual starch which will impact the stain intensity can reflect not only the presence of a defect for a starch catabolic function but also any mutation influencing the complex machinery responsible for the detection of either the switch to darkness or the starvation status and the equally complex signaling cascades. We then decided to conduct an insertional mutagenesis assay in Chlamydomonas in order to ensure that true starch catabolic mutants could indeed be recovered. Our major goal in this work was then to ascertain that we could recover mutations in genes previously reported or suspected to be selectively involved in starch mobilization. In this case, the effectiveness and at least partial selectivity of the screening procedure would be confirmed thereby allowing a certain level of confidence for the discovery of unsuspected functions. Figure 7. Discussion Molecular characterization of the CAT 16 mutant. (a) Amplification of a part of each of the three branching enzyme structural genes was performed on genomic DNA extracted from the wild-type reference 137C (WT) and from the mutant (16). The amplification products sizes for the BE1, BE2a and BE2b structural genes were 650, 516 and 615 bp respectively. (b) RT-PCR analysis performed on total RNAs extracted from the wild-type and the CAT16 mutant allowing the amplification of a 736 bp fragment of the PHOB structural gene and a 357 bp fragment of the BE1 structural gene (indicated by an arrow). Smart ladder (Eurogentec) was used as molecular weight marker (MW), the standard fragments corresponding to 0.4, 0.6 and 0.8 kbp are indicated. doi:10.1371/journal.pone.0074763.g007 From 16,000 paromomycin resistant colonies obtained after insertional mutagenesis (13,000 with the linearized pSL18 plasmid and 3,000 with a PCR product corresponding to the resistance cassette) we were able to isolate 38 independent strains which harbor a clear defect for starch mobilization with the two-step iodine screening procedure. Through kinetics of starch mobiliza- tion, we were able to show that 12 of these 38 mutants do degrade starch with the same efficiency as the wild-type. Those strains may be either false positive or strains in which the efficiency of starch degradation is not the same on solid and liquid medium. The 26 other mutants were all less effective than the wild-type with respect to starch mobilization and define two different phenotypic classes. The so-called class 2 mutants display a mild phenotype but with a significant starch degradation impairment as these 13 mutants still contain between 35 to 49% of their initial polysaccharide amounts after 1 day of degradation compared to the 28% recorded for the isogenic wild-type. The most defective strains were found in the class 3 for which 13 mutants still contain from 56 to 97% of the initial polysaccharide amounts assayed before degradation. It is worth noting that starch over accumulation and impairment in starch degradation were not linearly linked as overproducers are found in the mutants of class 1 (the one degrading starch with good efficiency) while even in class 3 mutants containing the mutants the most dramatically impaired for starch degradation, some of the strains accumulated nearly wild-type amounts of polysaccharide. These results may reflect the high complexity of the process involved in the regulation of starch deposition and catabolism in Chlamydomonas. Figure 7. September 2013 \| Volume 8 \| Issue 9 \| e74763 Discussion Molecular characterization of the CAT 16 mutant. (a) Amplification of a part of each of the three branching enzyme structural genes was performed on genomic DNA extracted from the wild-type reference 137C (WT) and from the mutant (16). The amplification products sizes for the BE1, BE2a and BE2b structural genes were 650, 516 and 615 bp respectively. (b) RT-PCR analysis performed on total RNAs extracted from the wild-type and the CAT16 mutant allowing the amplification of a 736 bp fragment of the PHOB structural gene and a 357 bp fragment of the BE1 structural gene (indicated by an arrow). Smart ladder (Eurogentec) was used as molecular weight marker (MW), the standard fragments corresponding to 0.4, 0.6 and 0.8 kbp are indicated. September 2013 \| Volume 8 \| Issue 9 \| e74763 September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 8 Genetic Dissection of Starch Catabolism screening procedure in Chlamydomonas is quite sensitive and effective in this respect. For the 26 strains for which a real defect for starch degradation was confirmed thanks to the kinetics of polysaccharide mobiliza- tion in liquid cultures, we used several strategies including the starch structure determination, visualization of the starch hydro- lytic activities through the use of zymograms and molecular techniques in order to identify the missing function responsible for the catabolic phenotype. The discovery of mutants for Mex or b-amylase which are already known as important starch degradation enzymes reveals the two step iodine phenotypic screen as an efficient tool to isolate novel starch catabolic mutants. We believe that the sensitivity of this mutant screening procedure and the physiological plasticity of the Chlamydomonas model will enable this system to bring further light into the complex machinery responsible for starch mobiliza- tion in green plants and algae. Even if a defect in starch catabolism is not expected to lead to a modification of starch structure, two of our mutants displayed modification either in the amylopectin/amylose ratio or in the amylopectin structure. The CAT31 mutant contains a high amount of amylose as this starch subfraction represents 41% of the total polysaccharide for only 23% in the wild-type. This increase in amylose is not accompanied by a strong modification of amylopectin which displays the classical lmax value around 550 nm. Methods Chlamydomonas reinhardtii Strains, Growth Conditions, and Media Discussion At the opposite, the CAT17 mutant produces a modified amylopectin with a high lmax at 567 nm but no amylose increase in this polysaccharide can be recorded. As for now, it is difficult to explain these modifications and the link between the modified structure and the defect in starch degradation cannot be easily explained. The identification of the disrupted functions in these strains will possibly yield some understanding on the reasons for these observations. Chlamydomonas reinhardtii Strains, Growth Conditions, and Media The wild-type reference C. reinhardtii strains used in this study were 137C (mt- nit1 nit2) and 37 (mt+ ac14 pab2). All experiments were carried out in continuous light (40 mE m22 s21) in the presence of acetate at 24uC in liquid cultures that were shaken without air or CO2 bubbling. Late-log phase cultures were inoculated at 105 cells mL21 and harvested at 2–3.106 cells mL21. Nitrogen-starved cultures were inoculated at 5.105 cells mL21 and were harvested after 5 days at a final density of 1 to 2.106 cells mL21. Recipes for media can be found in [37]. For starch degradation kinetics experiments, the cells harvested after 5 days under nitrogen starvation were collected in sterile conditions and transferred in TMP (TAP medium without acetate; [37]) liquid medium and incubated in the dark for 4, 8 and 24 hours. Starch was purified from these samples and the amount of remaining polysaccharide was assayed and compared to the initial starch amount assayed before the switch to darkness. The molecular experiments including plasmid rescue, inverse PCR and TAIL PCR allowed us to identify several candidates which still need to be validated as responsible of the starch degradation defects. Nevertheless, the detection of a copy of the mutagenesis plasmid in the structural genes encoding the maltose transporter Mex (CAT3) or an isoform of b-amylase (CAT4), both functions already demonstrated to impact efficient starch mobili- zation, are encouraging with respect to the specificity of our mutant screen. Several disrupted functions which may be related to polysaccharide metabolism were also identified as a starch binding domain containing protein in CAT 33 or an oligosac- charyl transferase in CAT 37. Such oligosaccharyl transferase are known to be involved in protein N-glycosylation in the ER lumen [34]. The effect on starch catabolism if really linked to this insertion may reflect a consequence of the malfunction of important proteins involved in starch catabolism or also be related to the building in a direct or indirect manner of the heteroglycan which plays a central role in maltose breakdown in the cytosol [35]. Four independent insertions in structural genes encoding kinase activities were also recorded in the CAT14, 15, 24 and 26 mutants. However, at this stage we are not sure these mutations are directly responsible for the slower starch degradation observed in these strains. Determination of Starch Levels, Starch Purification, and Spectral Properties of the Iodine-Starch Complex A full account of amyloglucosidase assays, starch purification on Percoll gradient, separation of amylose and amylopectin on CL-2B gel permeation chromatography and lmax (maximal absorbance wavelength of the iodine polysaccharide complex) measures can be found in [25]. Generation of the Mutant Library The wild-type Chlamydomonas reference strain 137C was transformed with either EcoRI linearized pSL18 vector [31] which carries the AphVIII gene (aminoglycoside 39-phosphotransferase from Streptomyces rimosus) conferring resistance to paramomycin, or with a 1,9 kb PCR product of this plasmid corresponding to the resistance cassette in order to produce transformants according to the protocol described in [38]. The paromomycin cassette was amplified from the pSL18 plasmid with the primers ParoF (59- ACCATGATTACGCCAAGCGCGCAA-39) and ParoR (59- CTCGACATGCGTTCACTTCCTGTC-39) using Dynazyme Ext (Finnzymes, Espoo, FI) following the manufacturer recom- mendation with an annealing temperature at 55uC and an extension time of 2 minutes for 30 cycles. One single mutation was identified through the use of zymogram techniques allowing visualization of hydrolytic activi- ties. The missing activity in the CAT 16 mutant was identified as a branching enzyme activity. The molecular characterizations allowed us to prove that this strain was defective for branching enzyme 1, an enzyme for which no function in starch metabolism has been reported so far. A recent study performed in maize revealed a slight modification of starch structure in this homol- ogous mutant [36]. The endosperm starch produced by the sbe1a mutant was more resistant to a-amylase digestion. During kernel germination, shorter coleoptile lengths and higher residual starch were recorded leading to the hypothesis that less efficient starch utilization was taking place in this mutant. However, at this stage a role of branching enzyme 1 in starch catabolism in Chlamydo- monas can only be suggested as this and the other mutants reported in this work have to go through a more detailed and comprehensive characterization. Nevertheless, the recovery of such a ‘‘mild’’ defect in starch catabolism suggests that the iodine After transformation, cells were plated on paromomycin supplemented TAP (10 mg/l) plates for the selection of transfor- mant lines. Nucleic acid Techniques Total RNA was extracted from Chlamydomonas according to Merendino et al., 2003[40]. RT-PCR analysis was performed on 1 mg of total RNA using the One Step RT-PCR Kit (Qiagen) following the manufacturer recommendation. Amplification was achieved with an annealing temperature of 60uC in the presence of Q-solution with the 4 following primers. The BE1RT5 (59- GAGAGTCACGACCAGGCTCTG-39) and BE1RT3 (59- GAAGCCGTAGTGGTTGTCGAG-39) allow the amplification of a 357 bp fragment of BE1 mRNA while PhoBRT5 (59- GCATGTTCCGCCAGACCA-39) and PhoBRT3 (59-TGCAG- GAAGCGCCAGTTGA-39) primers are used to amplify a 736 bp fragment of the PhoB mRNA used as an internal control. PCR products [23] were resolved in 2% (w/v) agarose gels. Iodine screening of putative starch catabolism mutants TAIL PCR Reaction Step Thermal settings N6 of cycles Primary 1 93uC, 2 min; 95uC, 1 min 1 2 94uC, 1 min; 65uC, 1 min; 72uC, 3 min 7 3 94uC, 1 min; 25uC, 1 min; ramping to 72uC over 3 min; 72uC, 2.5 min 1 4 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 48uC, 1 min; 72uC, 3 min 15 5 72uC, 5 min 1 Secondary 1 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 44uC, 1 min; 72uC, 3 min 15 2 72uC, 5 min 1 Tertiary 1 94uC, 1 min; 50uC, 1 min; 72uC, 3 min 30 2 72uC, 5 min INVERSE PCR Step Thermal Settings N6 of cycles 1 95uC, 1 min 1 2 95uC, 1 min; 55uC, 1 min; 72uC, 3 min 30 3 72uC, 10 min 1 doi:10.1371/journal.pone.0074763.t002 y g p g TAIL PCR Reaction Step Thermal settings N6 of cycles Primary 1 93uC, 2 min; 95uC, 1 min 1 2 94uC, 1 min; 65uC, 1 min; 72uC, 3 min 7 3 94uC, 1 min; 25uC, 1 min; ramping to 72uC over 3 min; 72uC, 2.5 min 1 4 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 48uC, 1 min; 72uC, 3 min 15 5 72uC, 5 min 1 Secondary 1 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 65uC, 1 min; 72uC, 3 min; 94uC, 30 s; 44uC, 1 min; 72uC, 3 min 15 2 72uC, 5 min 1 Tertiary 1 94uC, 1 min; 50uC, 1 min; 72uC, 3 min 30 2 72uC, 5 min INVERSE PCR Step Thermal Settings N6 of cycles 1 95uC, 1 min 1 2 95uC, 1 min; 55uC, 1 min; 72uC, 3 min 30 3 72uC, 10 min 1 doi:10.1371/journal.pone.0074763.t002 doi:10.1371/journal.pone.0074763.t002 reagent (0.01% I2; 0.1% KI) and 700 mL of distilled water. The absorbance was measured every nanometer between 400 and 700 nm to determine the wavelength of the maximal absorbance of the iodine-polysaccharide complex (lmax) which reflects the polysaccharide structure. Iodine screening of putative starch catabolism mutants The putative catabolic mutants were screened on the basis of their iodine/starch interaction which reflects the amount of polysaccharide in the cells. Cellular patches (20 mL) of the wild- type reference strain 137C, the already characterized mutants September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org 9 Genetic Dissection of Starch Catabolism Table 2. TAIL-PCR and inverse PCR cycling parameters used to isolate flanking DNA from insertional mutants. Table 2. TAIL-PCR and inverse PCR cycling parameters used to isolate flanking DNA from insertional mutants. Table 2. TAIL-PCR and inverse PCR cycling parameters used to isolate flanking DNA from insertional mutants. Iodine screening of putative starch catabolism mutants BafJ4 [27] and BafR1 [25] which are respectively defective for isoamylase and granule-bound starch synthase and of putative mutants were incubated on nitrogen free solid medium (TAP-N) for 5 days under continuous light in duplicate. Ten microliters of 0.15 M ammonium chloride were added to each spot of one of the two plates and these Petri dishes were incubated 24 hours in complete darkness in order to trigger starch degradation. Both control and test dishes were exposed to iodine vapor and photographed. References 15. Weise SE, Schrader SM, Kleinbeck KR, Sharkey TD (2006) Carbon balance and circadian regulation of hydrolytic and phosphorolytic breakdown of transitory starch. Plant Physiol 141(3): 879–886. 1. Wilson WA, Roach PJ, Montero M, Baroja-Ferna´ndez E, Mun˜oz FJ, et al. (2010) Regulation of glycogen metabolism in yeast and bacteria FEMS Microbiol Rev 34(6): 952–985. 2. Deschamps P, Moreau H, Worden AZ, Dauville´e D, Ball SG (2008) Early gene duplication within Chloroplastida and its correspondence with relocation of starch metabolism to chloroplasts. Genetics 178: 2373–2387. 16. Fulton DC, Stettler M, Mettler T, Vaughan CK, Li J, et al. (2008) Beta- AMYLASE4 a noncatalytic protein required for starch breakdown acts upstream of three active beta-amylases in Arabidopsis chloroplasts. Plant Cell 20(4): 1040–1058. 3. Smith AM, Stitt M (2007) Coordination of carbon supply and plant growth. Plant Cell Environ 30: 1126–1149. 17. Wattebled F, Dong Y, Dumez S, Delvalle´ D, Planchot V, et al. (2005) Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin. Plant Physiol 138(1): 184–195. 4. Fincher GB (1989) Molecular and cellular biology associated with endosperm mobilization in germinating grain cereal grains. Annu Rev Plant Physiol Plant Mol Biol 40: 305–346. 18. Niittyla¨ T, Messerli G, Trevisan M, Chen J, Smith AM, et al. (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303(5654): 87–89. 5. Razem FA, Davis AR (1999) Anatomical and ultrastructural changes of the floral nectary of Pisum sativum L during flower development. Protoplasma 206: 57–72. 19. Chia T, Thorneycroft D, Chapple A, Messerli G, Chen J, et al. (2004) A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night. Plant J 37(6): 853–863. 6. Hennen-Bierwagen TA, Liu F, Marsh RS, Kim S, Gan Q, et al. (2008) Starch biosynthetic enzymes from developing maize endosperm associate in multi- subunit complexes. Plant Physiol 146(4): 1892–1908 20. Yu TS, Zeeman SC, Thorneycroft D, Fulton DC, Dunstan H, et al. (2005) Alpha-Amylase is not required for breakdown of transitory starch in Arabidopsis leaves. J Biol Chem 280(11): 9773–9779. 7. Tetlow IJ, Beisel KG, Cameron S, Makhmoudova A, Liu F, et al. (2008) Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes. Plant Physiol 146(4): 1878– 1891. 21. Streb S, Eicke S, Zeeman SC (2012) The simultaneous abolition of three starch hydrolases blocks transient starch breakdown in Arabidopsis. References J Biol Chem 287(50): 41745–41756. 8. Smith AM, Zeeman SC, Smith SM (2005) Starch degradation. Annu Rev Plant Biol 56: 73–98. 22. Colleoni C, Dauville´e D, Mouille G, Bule´on A, Gallant D, et al. (1999) Genetic and biochemical evidence for the involvment of a-14 glucanotransferase in amylopectin synthesis. Plant Physiol 120: 993–1004. 9. Ritte G, Lloyd JR, Eckermann N, Rottmann A, Kossmann J, et al. (2002) The starch-related R1 protein is an alpha-glucan water dikinase. Proc Natl Acad Sci USA 99(10): 7166–7171. y p y y 23. Dauville´e D, Chochois V, Steup M, Haebel S, Eckermann N, et al. (2006) Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii. Plant J 48: 274–285. 10. Ko¨tting O, Pusch K, Tiessen A, Geigenberger P, Steup M, et al. (2005) Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves: The phosphoglucan water dikinase. Plant Physiol 137(1): 242–252. 24. Dauville´e D, Colleoni C, Mouille G, Morell M, D’Hulst C, et al. (2001) Biochemical characterization of wild-type and mutant isoamylases of Chlamydo- monas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation. Plant Physiol 125: 1723–1731. 11. Hejazi M, Fettke J, Haebel S, Edner C, Paris O, et al. (2008) Glucan water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubili- zation. Plant J 55(2): 323–334. 12. Hejazi M, Fettke J, Ko¨tting O, Zeeman SC, Steup M (2010) The Laforin-like dual-specificity phosphatase SEX4 from Arabidopsis hydrolyzes both C6- and C3-phosphate esters introduced by starch-related dikinases and thereby affects phase transition of alpha-glucans. Plant Physiol 152(2): 711–722. 25. Delrue B, Fontaine T, Routier F, Decq A, Wieruszeski JM, et al. (1992) Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structur- ally modified amylopectin. J Bacteriol 174(11): 3612–3620. phase transition of alpha-glucans. Plant Physiol 152(2): 711–722. 13. Santelia D, Ko¨tting O, Seung D, Schubert M, Thalmann M, et al. (2011) The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3- position in Arabidopsis. Plant Cell 23(11): 4096–4111. 26. Hicks GR, Hironaka CM, Dauville´e D, Funke RP, D’Hulst C, et al. (2001) When simpler is better: Unicellular green algae for discovering new genes and functions in carbohydrate metabolism. Plant Physiol 127(4): 1334–1338. 14. Comparot-Moss S, Ko¨tting O, Stettler M, Edner C, Graf A, et al. (2010) A putative phosphatase LSF1 is required for normal starch turnover in Arabidopsis leaves. Zymograms, Partial Purification and characterization of branching enzyme activity Zymograms in starch containing gels allowing the detection of most starch hydrolases and branching enzymes have been described for both undenatured samples [39] and for denatured enzymes [27]. The semi-purification of the activity generating the pink/red staining band on starch-containing zymograms was performed from 1 liter of exponential phase TAP culture of the reference strain 137C. Algae were disrupted by sonication in purification buffer (10 mM Tris/HCl pH 7.5; 10 mM EDTA; 8 mM DTT). Twenty five mg of total proteins were incubated with an amylose affinity resin (New England Biolabs, Ipswich, MA) for 10 minutes at 4uC. The affinity resin was then washed 3 times with 5 mL of purification buffer and bound proteins were eluted with 3 times one mL of buffer containing increasing concentra- tions of malto-oligosaccharides (0.5, 1, 2, 5 and 10 mg/mL MOS in purification buffer). Fifty microliters of each elution fraction were subjected to zymogram analysis to detect the elution fractions free of interfering activities (i.e. containing only the pink band). These fractions (300 mL) were incubated for 2 hours at 30uC in the presence of 1 mg of potato amylose (Sigma, St. Louis, MO). The polysaccharides were then collected by ethanol precipitation (1.2 mL 100% ethanol; 13000 g 10 min., 4uC). The dried pellets were then resuspended in 100 mL of 10 mM NaOH and the interaction with iodine was assessed by adding 200 mL of Lugol Total DNA from wild-type and mutant strains was extracted as previously described [41]. The integrity of branching enzymes genes was checked by amplifying parts of the corresponding DNA with Dynazyme Ext (Finnzymes, Espoo, FI) following the manufacturer recommenda- tion with an annealing temperature of 60uC and an extension time of 1 minute for 30 cycles. A 650 bp genomic DNA fragment corresponding to BE1 (XM_001695339) was amplified in the reference wild-type using the BE1 FOR5 (59-ATGGCTGC- GAGGCCGCTTCAG-39) and BE1 REV3 (59-ACGACCGCC- TACACGCCCTG-39) primers, while BE2aFor5 (59-CAATGG- CACACCTCCTCCAC-39) and BE2aRev3 (59-GTTGAACTG GATCTCGTTCCAG-39) primers were used to amplify a 516 bp DNA fragment and BE2bFor5 (59-CTTACTCGCACCAGCA AGCTG-39) and BE2bRev3 (59-CTTGAAGGTGTACTGCTT September 2013 \| Volume 8 \| Issue 9 \| e74763 10 PLOS ONE \| www.plosone.org Genetic Dissection of Starch Catabolism GTC-39) a 615 bp fragment corresponding respectively to the BE2a (XM_001696177) and BE2b (XM_001690270) genes. GTC-39) a 615 bp fragment corresponding respectively to the BE2a (XM_001696177) and BE2b (XM_001690270) genes. Author Contributions Figure S1 Kinetics of starch mobilization in the inser- tional mutants. The amount of starch measured in the CAT mutant strains is displayed as percentages of the initial content after 4, 8 and 24 h of degradation. The class 1 mutants are Conceived and designed the experiments: GP SGB DD. Performed the experiments: HT JF TD VC CC. Analyzed the data: GP SGB DD. Wrote the paper: SGB DD. Conceived and designed the experiments: GP SGB DD. Performed the experiments: HT JF TD VC CC. Analyzed the data: GP SGB DD. Wrote the paper: SGB DD. Zymograms, Partial Purification and characterization of branching enzyme activity presented in a while the class 2 and 3 are shown in b and c respectively. Each bar is mean 6SE of three independent experiments. Significant differences with the wild-type 137C (p,0.05) are indicated with a star. (TIF) Thermal asymmetric interlaced PCR and inverse-PCR were used to obtain the DNA flanking the paramomycin cassette or pSL18 insertion sites [42, 43,.44] Homology searches were performed using the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST; [45]), and at Phytozome site (http://www.phytozome.net/search. php?show = blast&method = Org_Creinhardtii). For TAIL-PCR the primers used were the degenerated primer AD2 (59-NGT CGASWGANAWGAA-39) and 3 specific primers, TAIL1 (59- GTGCTCGTTTGTCGCTGAAAGT-39), TAIL2 (59-CAAAT- CAGTCCTGTAGCTTCA-39), and TAIL3 (59-ACATACGCA CCAATCATGTCA-39) used respectively for the primary, secondary and tertiary reactions. For inverse PCR, the P1 (59- CATGTTTGCCCGAACTCGGAG-39) and P2 (59-CATTTG CCTGCCTTCACGCATC-39) or the T1 (59-TGCATGTA ATGGCCAGGCCATG -39) and T2 (59-ACTGGCTCACGCA CACGCTAAC-39) primers allowed the amplification of the genomic DNA flanking each side of the unique EcoRI site used to linearize the pSL18 plasmid. The cycling parameters for all reactions of TAIL-PCR and inverse PCR are described in Table 2. Figure S2 Partial purification of branching enzyme 1 activity from the CAT16 mutant crude extract. The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native (a; left panel) and denaturing (a; right panel) starch zymograms respectively. This activity cannot be detected in the first elution fractions (E1 to E3) of the amylose column chromatography as it was the case with the wild-type crude extract as reveled by analysis on starch denaturing zymogram (b). (c) Interaction of polysaccha- rides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide incubated with the 3 elution fractions (E1 to E3) are displayed in 4, 5 and 6. The values of the lmax of each complex is indicated on the figure in nanometers. (TIF) Genetic Dissection of Starch Catabolism decreased digestibility and reduced coleoptile growth during germination. BMC Plant Biol 11: 95. decreased digestibility and reduced coleoptile growth during germination. BMC Plant Biol 11: 95. 28. Libessart N, Maddelein ML, Koornhuyse N, Decq A, Delrue B, et al. (1995) Storage Photosynthesis and Growth: The Conditional Nature of Mutations Affecting Starch Synthesis and Structure in Chlamydomonas. Plant Cell 7(8): 1117–1127. 37. Harris EH (1989) The Chlamydomonas sourcebook: A comprehensive guide to biology and laboratory use. Academic Press.San Diego. 37. Harris EH (1989) The Chlamydomonas sourcebook: A com biology and laboratory use. Academic Press.San Diego. gy y g 38. Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87: 1228–1232. 29. Bellanger F (1994) «La mutagene`se par insertion d’ADN chez Chlamydomonas reinhardtii: une nouvelle voie pour l’e´lucidation de la biosynthe`se de l’amidon» PhD thesis Universite´ de Technologie de Compie`gne France. 39. Kakefuda G, Duke SH (1984) Electrophoretic transfer as a technique for the detection and identification of the plant amylolytic enzymes in polyacrylamide gels. Plant Physiol 75: 278–280. 30. Chochois V, Constans L, Dauville´e D, Beyly A, Soliveres M, et al. (2010) Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii. Int J Hydrogen Energy 35: 10731–10740. g y 40. Merendino L, Falciatore A, Rochaix JD (2003) Expression and RNA binding properties of the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii. Plant Mol Biol 53(3): 371–382. 31. Fischer N, Rochaix JD (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genom 265: 888–894. 41. Rochaix JD, Mayfield S, Goldschmidt-Clermont M, Erickson J (1991) Molecular biology of Chlamydomonas. In C. Shaw ed Plant Molecular Biology: A Practical Approach IRL Press Oxford pp 253–275. JD, Mayfield S, Goldschmidt-Clermont M, Erickson J (1991) Molecular 32. Hamilton BS, Nakamura K, Roncari DA (1992) Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants. Biochem Cell Biol 70(3–4): 255–258. 42. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463. 33. Banks W, Greenwood C, Khan K (1971) The interaction of linear amylose oligomers with iodine. Carbohyd Res 17: 25–33. J 43. Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120(3): 621–623. 34. References Plant Physiol 152(2): 685–697. 27. Mouille G, Maddelein ML, Libessart N, Talaga P, Decq A, et al. (1996) Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants. Plant Cell 8(8): 1353–1366. September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e74763 11 Genetic Dissection of Starch Catabolism Kelleher DJ, Gilmore R (2006) An evolving view of the eukaryotic oligo- saccharyltransferase. Glycobiology 16: 47R–62R. 44. Colombo SL, Pollock SV, Eger KA, Godfrey AC, Adams JE, et al. (2002) Use of the bleomycin resistance gene to generate tagged insertional mutants of Chlamydomonas reinhardtii that require elevated CO2 for optimal growth. Funct Plant Biol 29: 231–241. 35. Fettke J, Eckermann N, Tiessen A, Geigenberger P, Steup M (2005) Identification subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L: distinct SHG reside in the cytosol and in the apoplast. Plant J 43(4): 568–585. SHG reside in the cytosol and in the apoplast. Plant J 43(4): 568 45. Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. 36. Xia H, Yandeau-Nelson M, Thompson DB, Guiltinan MJ (2011) Deficiency of maize starch-branching enzyme I results in altered starch fine structure September 2013 \| Volume 8 \| Issue 9 \| e74763 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 12
https://openalex.org/W4293171637	http://ejournal.uin-suska.ac.id/index.php/gurindam/article/download/12786/7058	Indonesian	null	PARADIGMA PEMBELAJARAN EFEKTIF BAHASA DAN SASTRA INDONESIA	Gurindam	2,021	cc-by	6,482	PARADIGMA PEMBELAJARAN EFEKTIF BAHASA DAN SASTRA INDONESIA Febri Wahyuni1, Herlinda2 Universitas Islam Negeri Sultan Syarif Kasim Riau febriwahyuni531@gmail.com Informasi Artikel: DOI: 10.24014/gjbs.v1i2.12786 http://ejournal.uin-suska.ac.id/index.php/gurindam/index ISSN: 2798-6675 Abstrak: Paradigma pembelajaran efektif bahasa dan sastra Indonesia. Pembelajaran bahasa dan sastra Indonesia secara formal dimulai ketika memasuki sekolah dasar hingga sekolah menengah atas. Tujuan pembelajaran bahasa dan sastra Indonesia adalah agar peserta didik memiliki kemampuan menggunakan bahasa Indonesia yang baik dan benar untuk meningkatkan kemampuan intelektual, dan dapat menghargai serta membanggakan bahasa Indonesia sebagai bahasa persatuan dan bahasa negara. Pembelajaran bahasa dan sastra Indonesia dapat efektif apabila mencapai tujuan pembelajaran yang diinginkan sesuai dengan indikator pencapaian. Kata kunci: paradigma, pembelajaran efektif, bahasa dan sastra Indonesia Abstract: The paradigm of effective learning of Indonesian language and literature. Learning Indonesian language and literature formally begins when entering elementary school to high scool. The purpose of learning Indonesian language and literature is so that students have the ability to use good and correct Indonesian to improve intellectual abilities, and be able to appreciate and pride themselves on Indonesian as the language of unity and the language of the country. Indonesian language and literature learning can be effective if it achieves the desired learning objectives according to the achievement indicators. Open Journal System UIN Sultan Syarif Kasim Riau http://ejournal.uin-suska.ac.id/index.php/index/index Email: jurnalgurindam@uin-suska.ac.id Open Journal System UIN Sultan Syarif Kasim Riau http://ejournal.uin-suska.ac.id/index.php/index/index Email: jurnalgurindam@uin-suska.ac.id Open Journal System UIN Sultan Syarif Kasim Riau http://ejournal.uin-suska.ac.id/index.php/index/index Email: jurnalgurindam@uin-suska.ac.id Gurindam: Jurnal Bahasa dan Sastra Volume 1 Nomor 2 2021 Hlm. 40—51 Keywords: paradigm, effective learning, Indonesian language and literature Pembelajaran bahasa dan sastra Indonesia pada hakikatnya adalah membelajarkan peserta didik tentang keterampilan berbahasa Indonesia yang baik dan benar sesuai tujuan dan fungsinya. Menurut Atmazaki dalam Ali (2020: 41) mata pelajaran bahasa Indonesia bertujuan agar peserta didik memiliki kemampuan berkomunikasi secara efektif dan efisien sesuai dengan etika yang berlaku, baik secara lisan, maupun tulis, menghargai dan bangga menggunakan bahasa Indonesia sebagai bahasa persatuan dan bahasa negara, memahami bahasa Indonesia dan menggunakannya dengan tepat dan kreatif untuk berbagai tujuan. Kedudukan dan fungsi serta peranan bahasa Indonesia dalam kehidupan bermasyarakat tidak hanya digunakan dalam satu bidang saja, namun dalam berbagai bidang, baik dalam bidang sosial, ekonomi, politik, budaya, maupun pendidikan. Khusus dalam bidang pendidikan, bahasa Indonesia memiliki peranan yang sangat penting sebagai bahasa pengantar pendidikan. Selain itu, dalam bidang pendidikan bahasa Indonesia juga masuk dalam kurikulum pengajaran khusus untuk mata pelajaran bahasa 40 Wahyuni, Paradigma Pembelajaran Efektif … 41 mengkomunikasikan dengan tetap memperhatikan karakteristik siswa, b) menggunakan ilmu pengetahuan sebagai penggerak pembelajaran untuk semua mata pelajaran, c) menuntun siswa mencari tahu, bukan diberi tahu (discovery learning), d) menekankan kemampuan berbahasa sebagai alat komunikasi, pembawa pengetahuan dan berpikir logis, sistematis, dan kreatif (Kemendikbud, 2014). Indonesia yang diajarkan pada siswa mulai dari sekolah dasar hingga menengah atas bahkan sampai di perguruan tinggi (Gesrianto, 2017: 65). Hadirnya Kurikulum 2013 sebagai pengganti KTSP diyakini akan membawa perubahan yang lebih baik. Berbagai pertimbangan telah dilakukan. Sementara itu, dalam UU Sisdiknas dinyatakan bahwa tujuan pendidikan yang harus dicapai salah satunya melalui penerapan kurikulum berbasis kompetensi. Kompetensi lulusan program pendidikan harus mencakup tiga kompetensi, yakni sikap, pengetahuan, dan keterampilan, sehingga yang dihasilkan adalah manusia seutuhnya. Kurikulum 2006 (KTSP) dikembangkan menjadi Kurikulum 2013 dengan dilandasi pemikiran tantangan masa depan yaitu tantangan abad ke-21 yang ditandai dengan abad ilmu pengetahuan, knowledge-based society dan kompetensi masa depan (Yusnawarni, 2014: 172). ) Pengesahan kurikulum 2013 merupakan penyempurnaan dari kurikulum sebelumnya, yaitu kurikulum 2006 atau KTSP (Kurikulum Tingkat Satuan Pendidikan) telah membawa perubahan mendasar dalam pembelajaran bahasa Indonesia. Pada kurikulum 2006, mata pelajaran bahasa Indonesia lebih mengedepankan pada keterampilan berbahasa, sedangkan dalam kurikulum 2013, pembelajaran bahasa Indonesia digunakan sebagai sarana untuk mengembangkan kemampuan dan keterampilan menalar. Perubahan ini terjadi dilatarbelakangi oleh kenyataan bahwa kemmapuan menalar peserta didik Indonesia masih sangat rendah. Pembelajaran Efektif Menurut Sagala (2005: 61) pembelajaran merupakan membelajarkan siswa menggunakan asas pendidikan maupun teori belajar yang merupakan penentu utama keberhasilan pendidikan. Pembelajaran merupakan proses komunikasi dua arah, mengajar dilakukan oleh pihak guru sebagai pendidik, sedangkan belajar dilakukan oleh peserta didik. Sementara menurut Hamalik (2003: 61) pembelajaran adalah suatu kombinasi yang tersusun meliputi unsur-unsur manusiawi (siswa dan guru), material (buku, papan tulis, kapur, dan alat belajar), fasilitas dan proses yang saling mempengaruhi mencapai tujuan pembelajaran. Dari beberapa pendapat ahli di atas, dapat penulis simpulkan bahwa secara umum pembelajaran adalah suatu kegiatan yang dilakukan oleh pengajar dalam kondisi tertentu, sehingga kognitif, afektif, dan psikomotorik peserta didik berubah ke arah yang lebih baik. GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 suasana pembelajaran yang menyenangkan sehingga memberikan kreatifitas siswa untuk mampu belajar dengan potensi yang sudah mereka miliki yaitu dengan memberikan kebebasan dalam melaksanakan pembelajaran dengan cara belajarnya sendiri. Di dalam menempuh dan mewujudkan tujuan pembelajaran yang efektif maka perlu dilakukan sebuah cara agar proses pembelajaran yang diinginkan tercapai yaitu dengan cara belajar efektif. Untuk meningkatkan cara belajar yang efektif perlu adanya bimbingan dari guru. Selain itu, menurut Mulyono (2012: 7) pembelajaran yang efektif apabila kegiatan mengajar dapat mencapai tujuan sesuai pada perencanaan awal. Pembelajaran dikatakan efektif ketika peserta didik dapat menyerap materi pelajaran dan efisien. Dalam setiap pembelajaran guru ataupun pendidik seharusnya memiliki perencanaan awal secara tertulis dalam bentuk RPP (Rencana Pelaksanaan Perencanaan) maupun sejenisnya. Dapat disimpulkan bahwa pembelajaran efektif merupakan sebuah proses perubahan seseorang dalam kognitif, tingkah laku, dan psikomotorik dari hasil pembelajaran yang ia dapatkan dari pengalaman dirinya dan dari lingkungannya yang membawa pengaruh, makna dan manfaat tertentu. Untuk mengimplementasikan tujuan mata pelajaran bahasa Indonesia tersebut, maka pembelajaran bahasa Indonesia dalam kurikulum 2013 disajikan dengan menggunakan pendekatan berbasis teks. Teks dapat berwujud teks tertulis maupun teks lisan. Teks merupakan ungkapan pikiran manusia yang lengkap di dalamnya memiliki situasi dan konteks. Dengan kata lain, belajar bahasa Indonesia tidak sekadar memakai bahasa Indonesia sebagai alat komunikasi, tetapi perlu juga mengetahui makna atau bagaimana memilih kata yang tepat yang sesuai tatanan budaya dan masyarakat pemakainya (Nasution, 1999: 23). Keywords: paradigm, effective learning, Indonesian language and literature Hal ini diketahui dari studi Trends in Internatioal Mathematics and Science Study (TIMSS) tahun 2011, hanya 5% peserta didik Indonesia yang mampu memecahkan persoalan yang membutuhkan pemikiran, sedangkan sisanya 95% hanya sampai pada level menengah, yaitu memecahkan persoalan yang bersifat hapalan. Ini membuktikan, bahwa pendidikan Indonesia baru berada pada tatanan konseptual. Untuk itu, pembelajaran bahasa Indonesia merupakan salat satu solusi yaitu dengan menjadi bahasa sebagai penghela ilmu pengetahuan dan pembelajaran berbasis teks (Jurnal Ummul Khair, 2018: 88). Paradigma penempatan bahasa Indonesia sebagai penghela ilmu pengetahuan merupakan langkah lebih maju dan konkret dalam memaksimalkan pembelajaran bahasa dan sastra Indonesia. Dikatakan demikian karena kedudukan bahasa Indonesia sebagai bahasa nasional, bahasa berfungsi mempersatukan berbagai etnis yang berbeda serta kedudukannya sebagai bahasa resmi. Melalui penguasaan bahasa Indonesia, peserta didik dapat mempelajari ilmu pengetahuan yang lain. Bahasa Indonesia menjadi sarana untuk mengembangkan dan mengomunikasikan berbagai pengetahuan. Pada Kurikulum 2013, kedudukan Bahasa Indonesia adalah suatu hal yang istimewa, menurut Nuh (2013) Kurikulum 2013 adalah menempatkan bahasa sebagai penghela ilmu pengetahuan (Mahsun, 2014). Bahasa Indonesia sebagai penghela berarti bahasa Indonesa sebagai bahasa pengantar di semua jenjang pendidikan sekaligus sebagai bahasa disemua bidang ilmu. Hal ini menjadi paradigma baru dalam pembelajaran bahasa dan sastra Indonesia, karena dengan pergantian kurikulum akan selalu ada perubahan disemua mata pelajaran. Pada proses pembelajaran bahasa dan sastra Indonesia perlunya penguatan karakteristik yang mencakup: a) menggunakan pendekatan saintifik melalui mengamati, menanya, mencoba, mengolah, menyajikan, menalar, mencipta dan Adanya perubahan dalam pembelajaran bahasa Indonesia tersebut diiringi dengan kompetensi guru dalam mengimplementasikan pembelajaran dengan paradigma baru yaitu pembelajaran berbasis teks. Mata pelajaran bahasa Indonesia ini disuguhkan pada peserta didik bertujuan untuk melatih peserta didik terampil berbahasa dengan menuangkan ide dan gagasan secara kreatif dan kritis. Namun kenyataannya banyak guru terjebak dalam tatanan konsep sehingga pembelajaran cenderung membahas teori-teori bahasa. Teori- teori bahasa hanya sebagai pendukung atau penjelas dalam konteks yaitu yang berkaitan dengan keterampilan tertentu yang tengah diajarkan. GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 42 GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 Indikator Pembelajaran Efektif Menurut Wotruba and Wright dalam Anwar (2017: 471), ada tujuh indikator yang menunjukan pembelajaran efektif yaitu Pertama, pengorganisasian materi yang baik. Pengorganisasian adalah bagaimana cara mengurutkan materi yang akan disampaikan secara logis dan teratur, sehingga dapat terlihat kaitan yang jelas antara topik satu dengan topik lainnya selama pertemuan berlangsung. Pengorganisasian materi terdiri atas; perincian materi, urutan materi dari yang mudah ke yang sukar, dan kaitannya dengan tujuan. Faktor lain yang perlu dipertimbangkan dalam penyajian materi adalah bagaimana kemampuan daya serap peserta didik. Daya serap tersebut berkaitan erat dengan motivasi dan kesiapan belajar mereka. Motivasi peserta didik dipengaruhi oleh minat dan perhatian, yaitu hubungan materi pelajaran dengan harapan dan kesiapan belajar sebelumnya. Kesiapan belajar individu ditentukan oleh penguasaan pengetahuan yang telah dipelajari sebelumnya, keterampilan membaca dan mendengar, tingkat pendidikan Menurut Mulyasa (2003: 149) efektif adalah perubahan yang membawa makna dan manfaat tertentu. Pembelajaran yang efektif ditandai dengan sifatnya yang menekankan pada pemberdayaan siswa secara aktif. Pembelajaran menekankan pada penguasaan pengetahuan tentang apa yang dikerjakan, tetapi lebih menekankan pada internalisasi, tentang apa yang dikerjakan sehingga tertanam dan berfungsi sebagai muatan murni dan hayati serta dipraktekkan dalam kehidupan oleh siswa. p p Slameto (1995: 75-76) menjelaskan bahwa pembelajaran efektif juga akan melatih dan menanamkan sikap demokratis bagi siswa. Pembelajaran efektif juga dapat menciptakan Wahyuni, Paradigma Pembelajaran Efektif … 43 yang telah dicapai, dan tingkat kesulitan materi. Penguasaan materi juga mencakup faktor penunjang lainnya yang digunakan selama proses penyajian. Faktor penunjang tersebut antara lain yaitu penggunaan media, sikap, gerak-gerik mengajar, dan cepat lambat penyajian. sendiri untuk memecahkan masalah yang dihadapinya karena hal ini sesuai dengan prinsip belajar mandiri. Bantuan kepada peserta didik sebaiknya diberikan setelah usaha mereka sendiri kurang berhasil. Bantuan itu tidak berarti memecahkan masalah yang dihadapi peserta didik, melainkan memberikan saran jalan keluar, memberikan dorongan, dan membangkitkan motivasi. Kedua, komunikasi efektif. Komunikasi yang efektif dalam pembelajaran mencakup penyajian yang jelas, kelancaran berbicara, interpretasi gagasan abstrak dengan contoh- contoh, kemampuan wicara yang baik (nada, intonasi, ekspresi) dan kemampuan mendengar. Kemampuan komunikasi tidak hanya diwujudkan melalui penjelasan verbal, tetapi dapat juga berupa makalah yang ditulis, serta silabus yang jelas dan mudah dimengerti. Kelima, pemberian nilai yang adil. Keadilan dalam pemberian nilai tercermin dari adanya seperti kesesuaian soal tes dengan materi yang diajarkan, sikap konsisten terhadap pencapaian tujuan pelajaran, kejujuran peserta didik dalam memperoleh nilai, dan pemberian umpan balik terhadap hasil pekerjaan peserta didik. Indikator Pembelajaran Efektif Keadilan pemberian nilai tidak berarti peserta didik nilai A kalau mereka seharusnya tidak mendapatkan nilai itu. Sesuai tidaknya ujian dan penilai dengan tujuan materi pelajaran dapat diketahui oleh teman sejawat atau pimpinan langsung. Demikian pula penilaian terhadap prestasi peserta didik, adakalanya nilai yang diberikan kepada seseorang pendidik dipengaruhi oleh rasa senang tidak senang dengan peserta didik tertentu. Peserta didik dapat pula diminta pendapatnya tentang tingkat keadilan pendidik. Tetapi kita harus berhati-hati karena peserta didik juga tidak selalu dapat bersikap objektif. y g j g Ketiga, penguasaan dan antusiasme dalam materi pelajaran. Penguasaan dan antusiasme terhadap materi pelajaran yaitu seorang pendidik dituntut untuk menguasai materi jika telah menguasainya maka dapat diorganisasikan secara logis dan sistematis. Penguasaan materi harus pula diiringi dengan kemauan dan semangat untuk memberikan pengetahuan dan keterampilan kepada peserta didik. Pemilihan buku wajib dan bacaan, penentuan topik pembahasan, pembuatan ikhtisar dan pembuatan bahan sajian merupakan indikator penguasaan atas bahan materi pelajaran. Penguasaan bahan materi saja tidak cukup. Penguasaan itu harus diiringi dengan kemampuan dan semangat untuk memberikan penguasaan itu kepada para peserta didik. Tidak jarang pendidik yang ahli dalam suatu bidang kajian ingin memiliki keahlian itu sendiri, karena khawatir mendapat persaingan. Inilah yang dimaksudkan antusiasme yang tinggi. Penguasaan atas bahan materi ini dapat diketahui dengan baik melalui teman sejawat dalam bidang disiplin yang sama. Keenam, keluwesan dalam pendekatan pembelajaran. Pendekatan pembelajaran bervariasi, merupakan salah satu petunjuk adanya semangat dalam mengajar. Berbagai pendekatan mungkin dapat bermanfaat dalam mencapai berbagai tujuan atau dalam menanggapi latar belakang dan kemampuan peserta didik. Umpamanya simulasi dan teknik permainan dapat bermanfaat dalam mengajar analisa, sintesa dan kemampuan berfikir kritis. Media dapat dipakai dalam menambah daya cerna pembelajaran jadi memberikan keuntungan bagi peserta didik. Dengan memberikan kesempatan waktu yang berbeda kepada peserta didik yang kemampuannya berbeda sudah berarti adanya pendekatan yang luwes. Kegiatan pendidik seharusnya ditentukan berdasarkan karakteristik peserta didik, karakteristik mata pelajaran, dan hambatan. Karakteristik yang berbeda dan hambatan yang berbeda menghendaki pendekatan yang berbeda pula. Usaha yang pertama untuk pendekatan yang luwes mungkin belum dapat menunjukkan hasil Keempat, sikap positif terhadap peserta didik. Sikap positif terhadap peserta didik dapat ditunjukkan baik pada kelas kecil maupun kelas besar, tentu saja dengan cara yang berbeda. Dalam kelas yang kecilg, sikap ini dapat ditunjukkan dengan memberikan perhatian pada orang per orang, sedangkan dalam kelas besar dapat diberikan pada kelompok yang menghadapi masalah yang sama. Beberapa pendidik menganggap bahwa bersikap positif terhadap peserta didik artinya dengan memanjakan mereka. Indikator Pembelajaran Efektif Pendidik seperti ini berpendapat bahwa peserta didik harus berusaha GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 44 yang baik. Kesediaan untuk melakukan eksperimen atau memberikan umpan balik merupakan usaha untuk menghasilkan pembelajaran yang lebih baik. atau mengaplikasikan hasil suatu pembelajaran. Dengan kecepatan unjuk kerja di atas standar yang ditetapkan maka peserta didik mencapai tujuan pembelajaran secara maksimal. Terkait dengan tingkat alih belajar pada masing-masing peserta didik berbeda-beda tergantung kematangan penguasaan materi masing- masing. Bagi peserta didik dengan penguasaan pembelajaran cepat tentunya memiliki tingkat alih belajar yang cepat, karena untuk beralih pada pembelajaran berikutnya tak jarang dipengaruhi oleh pemahaman pembelajaran sebelumnya. Keluwesan pendekatan mengajar mungkin hanya dapat diketahui oleh pendidik yang bersangkutan dan peserta didik yang mengikuti pembelajaran. Adakalanya pendekatan yang digunakan pendidik ditentukan secara situasional yaitu disesuaikan dengan suasana dan peristiwa yang ada pada waktu pembelajaran diberikan. Dalam keadaan seperti ini sebaiknya pendidik mencatat suasana dan pendekatan yang digunakan, karakteristik dari perubahan serta hasil yang diperolehnya. Berdasarkan beberapa pendapat ahli di atas, dapat disimpulkan bahwa indikator pembelajaran efektif terdiri atas pengelolaan pembelajaran, respon peserta didik, aktivitas belajar, dan hasil belajar. Dengan demikian, pembelajaran dinyatakan efektif bila semua indikator tersebut dalam kategori minimal baik. Jika salah satu dari indikator yang dimaksud belum terolong baik maka belum dinyatakan efektif. Ketujuh, hasil peserta didik yang baik. Seberapa banyak yang dipelajari oleh peserta didik dalam suatu pembelajaran adalah hasil dari berbagai faktor, yang tidak semuanya berhubungan dengan kegiatan pendidik. Kemampuan dan motivasi peserta didik umpamanya sangat berhubungan dengan apa yang dicapai peserta didik. Beberapa peserta didik dapat belajar sendiri, tanpa harus mendapat pelajaran terlebih dahulu. Oleh karena itu, memisahkan hasil pembelajaran dan proses belajar merupakan sesuatu yang sangat sukar. Meskipun ada kesukaran adalah hal penting untuk mempertimbangkan usaha belajar peserta didik pada waktu menilai efektivitas pembelajaran. Hasil belajar dapat dibedakan atas tiga ranah atau kawasan, yaitu kognitif, afektif, dan psikomotorik. Proses untuk menentukan jenjang dan tujuan merupakan tugas yang tidak mudah. Pedoman yang perlu dipegang adalah hasil belajar peserta didik itu harus sesuai dengan tujuan pembelajaran. Karakteristik Pembelajaran Efektif memberikan kebebasan untuk menari sendiri, sehingga menumbuhkan rasa tanggumg jawab yang besar pada pekerjaannya dan lebih percaya diri sehingga anak tidak menggantungkan pada diri orang lain. (7) Pemberian remedial dan diagnosa pada kesulitan belajar yang muncul, mencari faktor penyebab dan memberikan pengajaran remedial sebagai perbaikan. j Menurut Mac Gregor (2007) dalam Setyosari (2014: 23) pembelajaran yang efektif, sesungguhnya bukan sesuatu yang mudah dan sederhana. Pembelajaran yang efektif, bukan hanya masalah tercapainya seluruh tujuan khusus pembelajaran. Banyak aspek yang terlibat di dalamnya. Kita nampaknya sepaham bahwa sebagian besar kajian atau literature menyatakan pembelajaran yang efektif itu merupakan suatu proses yang benar-benar kompleks. Sementara, Kyriacou (2009) dalam Setyosari (2014: 24) mengatakan pembelajaran efektif sesungguhnya terkait dengan aspek-aspek pembelajaran dan seberapa kemampuan guru menentukan suatu pengalaman belajar yang mengarah pada pencapaian hasil belajar yang diharapkan. Agar supaya hal ini bisa terwujud, maka setiap peserta didik harus dilibatkan dalam aktivitas pembelajaran. Sedikitnya ada dua unsur pokok dalam pembelajaran yang efektif, yaitu (1) guru harus memiliki suatu gagasan jelas tentang tujuan belajar yang diharapkan dan (2) pengalaman belajar yang direncanakan dan disampaikan dapat tercapai. Tujuan Pembelajaran Efektif Tujuan pembelajaran efektif adalah untuk mencapai hasil belajar yang memuaskan. Apabila peserta didik belajarnya tidak tuntas, tidak teratur, tidak berkesinambungan dan juga tidak bersungguh-sungguh baik itu di sekolah ataupun di rumah maka akan menyebabkan tidak tercapai tujuan belajar yang di harapkan. Menurut Hamalik (2003: 65) tujuan pembelajaran adalah sejumlah hasil belajar yang menunjukkan bahwa siswa setelah melakukan perbuatan belajar yang umunya meliputi pengetahuan, keterampilan dan sikap- sikap yang baru yang diharapkan tercapai oleh siswa. Hal ini juga senada dengan Yusuf yang menjelaskan bahwa tujuan pembelajaran pada prinsipnya mengandung arti pernyataan atau gambaran perubahan pada pengetahuan, pemahaman, sikap, tingkah laku, penampilan atau kondisi psikologi dan lainnya pada peserta didik, baik yang dapat dilihat langsung atau tidak, tetapi tidak diukur atau dinilai. Dari beberapa pendapat yang dikemukakan dapat penulis simpulkan bahwa secara umum tujuan pembelajaran adalah sebagai upaya membekali diri siswa dengan kemampuan-kemampuan yang bersifat pengalaman, pemahaman moral dan keterampilan sehingga mengalami perkembangan yang positif. g j p j Reigeluth (1983: 234) dalam Magdalena, dkk (2020: 368) mengemukakan bahwa indikator pembelajaran efektif yaitu: (1) Kecermatan penguasaan, (2) Kecepatan unjuk kerja, (3) Tingkat alih belajar, dan (4) Tingkat retensi. Untuk kecermatan penguasaan difokuskan pada peserta didik maupun pengajar. Peserta didik dengan penguasaan yang baik setelah pembelajaran itu berarti tujuan pembelajaran sudah tercapai. Sedangkan bagi pengajar yang memiliki kecermatan penguasaan tergolong baik, hal ini merupakan modal utama dalam proses belajar mengajar untuk sampai pada tujuan pembelajaran yang ditetapkan. Kecepatan unjuk kerja dapat diartikan sebagai kemampuan secara cepat untuk memperlihatkan Wahyuni, Paradigma Pembelajaran Efektif … 45 Kondisi Pembelajaran Efektif Nawawi (1989: 117) menjelaskan bahwa guru sebagai pembimbing diharapkan mampu menciptakan kondisi yang strategi yang dapat membuat peserta didik nyaman dalam mengikuti proses pembelajaran tersebut. dalam menciptakan kondisi yang baik, hendaknya guru memperhatikan dua hal: pertama, kondisi internal merupakan kondisi yang ada pada diri siswa itu sendiri, misalnya kesehatan, keamanannya, ketentramannya, dan sebagainya. Kedua, kondisi eksternal yaitu kondisi yang ada di luar pribadi manusia, umpamanya kebersihan rumah, penerangan serta keadaan lingkungan fisik yang lain. Untuk dapat belajar yang efektif diperlukan lingkungan fisik yang baik dan teratur, misalnya ruang belajar harus bersih, ruangan cukup terang, tidak gelap dan tidak menganggu mata, sarana yang diperlukan dalam belajar yang cukup atau lengkap. Keberhasilan dalam proses pembelajaran di kelas memang tidak semata tergantung guru, tetapi melibatkan banyak faktor, diantaranya keaktifan siswa, tersedianya fasilitas belajar, kenyamanan dan keamanan ruangan kelas dan beberapa faktor lainnya, kendati memang keberadaan guru merupakan faktor penentu dalam mencipttakan kondisi pembelajaran yang efektif. Hal ini senada dengan Slameto (1995: 94) yang mengatakan bahwa pembelajaran dapat efektif apabila mencapai tujuan pembelajaran yang diinginkan sesuai dengan indikator pencapaian. Untuk mengetahui bagaimana memperoleh hasil yang efektif dalam proses pembelajaran, maka sangat penting untuk mengetahui ciri-cirinya. Adapun pembelajaran yang efektif dapat diketahui dengan ciri-ciri sebagai berikut: (1) Belajar secara aktif baik mental maupun fisik. Aktif secara mental ditunjukkan dengan mengembangkan kemampuan intelektualnya, kemampuan berfikir kritis. Dan secara fisik, misalnya menyusun intisari pelajaran, membuat peta dan lain-lain. (2) Metode yang bervariasi, sehingga mudah menarik perhatian siswa dan kelas menjadi hidup. (3) Motivasi guru terhadap pembelajaran di kelas. Semakin tinggi motivasi seorang guru akan mendorong siswa untuk giat dalam belajar. (4) Suasana demokratis di sekolah, yakni dengan menciptakan lingkungan yang saling menghormati, dapat mengerti kebutuhan siswa, tenggang rasa, memberi kesempatan kepada siswa untuk belajar mandiri, menghargai pendapat orang lain. (5) Pelajaran di sekolah perlu dihubungkan dengan kehidupan nyata. (6) Interaksi belajar yang kondusif, dengan p j y g Dalam mewujudkan kondisi pembelajaran yang efektif, maka perlu dilakukan langkah- langkah yaitu pertama, melibatkan siswa secara aktif. Mengajar adalah membimbing kegiatan belajar siswa sehingga ia mau belajar. Dengan demikian aktivitas siswa sangat diperlukan dalam kegiatan pembelajaran. Aktivitas belajar siswa dapat digolongkan ke dalam beberapa hal sebagai berikut: (a) Aktivitas visual, seperti membaca, menulis, dan melakukan eksperimen. (b) Aktivitas lisan, seperti bercerita, dan tanya jawab. (c) Aktivitas mendengarkan, seperti mendengarkan penjelasan guru, dan mendengarkan pengarahan guru. (d) Aktivitas gerak, seperti melakukan praktek di tempat praktek. Kondisi Pembelajaran Efektif (e) Aktivitas menulis, seperti GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 46 mengarang, membuat surat, membuat karya tulis, dan sebagainya. guru di dalam memberikan variasi pembelajaran agar dapat diserap oleh semua ssiwa dalam berbagai tingkatan kemampuan, dan disini pulalah perlu adanya pelayanan individu siswa. Kedua, menarik minat dan perhatian siswa. Rosyada (2004: 56) mengatakan bahwa kondisi pembelajaran yang efektif adalah adanya minat dan perhatian siswa dalam belajar. Minat merupakan suatu sifat yang relatif menetap pada diri seseorang. Minat ini besar sekali pengaruhnya terhadap belajar, sebab dengan minat seseorang akan melakukan sesuatu yang diminatinya. Sebaliknya tanpa minat seseorang tidak mungkin melakukan sesuatu. Keterlibatan siswa dalam pembelajaran erat kaitannya dengan sifat, bakat, dan kecerdasan siswa. Pembelajaran yang dapat menyesuaikan sifat, bakat dan kecerdasan siswa merupakan pembelajaran yang diminati. Kelima, menyiapkan dan menggunakan berbagai media dalam pembelajaran. Alat peraga/media pembelajaran adalah alat-alat yang digunakan guru ketika mengajar untuk membantu memperjelas materi pelajaran yang disampaikan kepada ssiwa dan mencegah terjadinya verbalisme pada diri siswa. Sebab, pembelajaran yang menggunakan banyak verbalisme tentu saja akan membosankan. Sebaliknya pembelajaran akan lebih menarik, bila siswa merasa senang dan gembira setiap menerima pelajaran dari gurunya. Prinsip Dasar Pembelajaran Efektif Adapun prinsip-prinsip pembelajaran yang relatif berlaku umum yaitu: prinsip perhatian dan motivasi, keaktifan, keterlibatan langsung/ berpengalaman, pengulangan, tantangan, balikan dan penguatan, serta perbedaan individual (Ali, 2013: 33-38). Pertama, perhatian dan motivasi. Perhatian mempunyai peranan penting dalam kegiatan belajar. Tanpa adanya perhatian, proses belajar tidak mungkin terjadi. Perhatian akan timbul pada peserta didik apabila bahan pembelajaran dirasakan sebagai: sesuatu yang dibutuhkan; diperlukan untuk belajar lebih lanjut; atau diperlukan dalam kehidupan sehari- hari. Thorndike sebagaimana dikutip Muhammad Thobrani dan Arif Mustofa menjelaskan jika anak tertarik dan merasa senang pada suatu kegiatan, maka akan menghasilkan prestasi memuaskan. Ketiga, keterlibatan langsung/ pengalaman. Pembelajaran akan lebih bermakna jika peserta didik “mengalami sendiri apa yang dipelajarinya” bukan “mengetahui” dari informasi yang disampaikan guru. Pentingnya keterlibatan langsung dalam belajar dikemukakan oleh John Dewey dengan “learning by doing” nya. Belajar sebaiknya dialami melalui perbutan langsung dan harus dilakukan oleh peserta didik secara aktif. Prinsip ini didasarkan pada asumsi bahwa para didik dapat memperoleh lebih banyak pengalaman dengan cara keterlibatan secara aktif dan proporsional, dibandingkan dengan bila mereka hanya melihat materi/konsep. Adapun motivasi dalam konteks pembelajaran adalah usaha sadar oleh guru untuk menimbulkan motif-motif pada peserta didik yang menunjang pencapaian tujuan pembelajaran. Motivasi erat kaitannya dengan minat. Peserta didik yang memiliki minat terhadap suatu bidang studi tertentu cenderung tertarik perhatiannya dan dengan demikian timbul motivasinya untuk mempelajari bidang studi tersebut. Motivasi juga dipengaruhi oleh nilai-nilai yang dianggap penting dalam kehidupan. Nilai-nilai tersebut mengubah tingkah laku dan motivasinya. Modus Pengalaman belajar adalah sebagai berikut: seseorang belajar 10% dari apa yang dibaca, 20% dari apa yang didengar, 30% dari apa yang dilihat, 50% dari apa yang dilihat dan didengar, 70% dari apa yang dikatakan, dan 90% dari apa yang dikatakan dan dilakukan. Hal ini menunjukkan bahwa jika guru mengajar dengan banyak ceramah, maka peserta didik akan mengingat hanya 20% karena mereka hanya mendengarkan. Sebaliknya, jika guru meminta peserta didik untuk melakukan sesuatu dan melaporkannya, maka mereka akan mengingat sebanyak 90%. Kedua, keaktifan. Menurut pandangan psikologi, anak adalah makhluk yang aktif. Anak mempunyai dorongan untuk berbuat sesuatu, mempunyai kemauan dan aspirasi sendiri. Belajar tidak bisadipaksakan oleh orang lain dan jugatidak bisa dilimpahkan pada orang lain. Belajar hanya mungkin terjadi apabila anak aktif mengalaminya sendiri. John Dewey sebagaimana dikutip Abuddin Nata mengemukakan, belajar adalah menyangkut apa yang harus dikerjakan peserta didik untuk dirinya sendiri, maka inisiatif harus datang sendiri. Guru sekadar pembimbing dan pengarah. Suasana Pembelajaran Efektif Disinilah sebenarnya perlunya keterampilan Wahyuni, Paradigma Pembelajaran Efektif … 47 sehingga tujuan pembelajaran prestasi dapat dicapai dengan maksimal. berpikir. Agar ia berpikir sendiri (aktif) ia harus diberi kesempatan untuk berbuat sendiri. Lebih lanjut Piaget menjelaskan, bahwa belajar menunjukkan adanya jiwa yang sangat aktif, jiwa yang mengolah informasi, jiwa yang tidak sekadar menyimpan informasi, tetapi mengadakan transformasi. Keaktivan dapat berupa kegiatan fisik dan kegiatan psikis. Kegiatan fisik bisa berupa membaca, mendengar, menulis, berlatih keterampilan, dan sebagainya. Sedangkan kegiatan psikis, misalnya, menggunakan khasanah pengetahuan yang dimiliki dalam memecahkan masalah yang dihadapi, membandingkan satu konsep dengan yang lain, menyimpulkan hasil percobaan, dan sebagainya. Suasana Pembelajaran Efektif Ketiga, membangkitkan motivasi siswa. Menurut Santrock (2008: 9) motivasi adalah semacam daya yang terdapat dalam diri seseorang yang dapat mendorongnya untuk melakukan sesuatu, dan suatu proses untuk menggiatkan motif- motif menjadi perbuatan atau tingkah laku untuk memenuhi kebutuhan dan mencaapai tujuan. Tugas guru adalah bagaimana membangkitkan motivasi siswa sehingga ia mau belajar. Berikut ini beberapa cara bagaimana membangkitkan motivasi siswa: (a) Guru berusaha menciptakan persaingan diantara siswanya untuk meningkatkan prestasi belajarnya. (b) Pada awal kegiatan pembelajaran, guru hendaknya terlebih dahulu menyampaikan kepada siswa tentang tujuan yang akan dicapai dalam pembelajaran tersebut, sehingga siswa terpancing untuk ikut serta di dalam mencapai tujuan tersebut. (c) Guru berusaha mendorong siswa dalam belajar untuk mencapai tujuan pembelajaran. (d) Guru hendaknya banyak memberikan kesempatan kepada siswa untuk meraih sukses dengan usahanya sendiri. (e) Guru selalu berusaha menarik minat belajar siswa. (f) Sering-seringlah memberikan tugas dan memberikan nilai seobjektif mungkin. Keberhasilan pengajaran dalam arti tercapainya tujuan-tujuan pengajaran sangat tergantung pada kemampuan mengatur kelas yang dapat menciptakan situasi yang Keberhasilan pengajaran dalam arti tercapainya tujuan-tujuan pengajaran sangat tergantung pada kemampuan mengatur kelas yang dapat menciptakan situasi yang memungkinkan anak didik dapat belajar, sehingga merupakan titik awal keberhasilan pengajaran. Siswa dapat belajar dengan baik dalam suasana yang wajar, tanpa tekanan dalam kondisi yang merangsang untuk belajar. Dalam kaitan dengan proses belajar mengajar hendaknya guru dapat mengarahkan dan membimbing siswa untuk aktif dalam kegiatan belajar mengajar sehingga tercipta suatu interaksi yang baik antara guru dengan siswa maupun siswa dengan siswa. Hal ini senada seperti yang ditulis Madri M. dan Rosmawati (2004: 274) terjadinya proses pembelajaran itu ditandai dengan dua hal yaitu: (1) siswa menunjukkan keaktifan, seperti tampak dalam jumlah curahan waktunya untuk melaksanakan tugas ajar, (2) terjadi perubahan perilaku yang selaras dengan tujuan pengajaran yang diharapkan. Untuk menciptakan suasana yang dapat menumbuhkan gairah belajar, meningkatkan prestasi belajar siswa, dan lebih memungkinkan guru memberikan bimbingan dan bantuan terhadap siswa dalam belajar, maka diperlukan pengorganisasian kelas yang memadai. Upaya- upaya yang dilakukan ini merupakan usaha dalam menciptakan sekaligus memelihara kondisi dan suasana belajar yang kondusif, optimal, dan menyenangkan agar proses pembelajaran dapat berjalan secara efektif Keempat, memberikan pelayanan individu siswa. Madri dan Rosyada (2004: 57) salah satu masalah utama dalam pendekatan pembelajaran adalah kurangnya pemahaman guru tentang perbedaan individu antar siswa. Guru sering kurang menyadari bahwa tidak semua siswa dalam suatu kelas dapat menyerap pelajaran dengan baik. Kemampuan individual mereka dalam menerima pelajaran berbeda- beda. Prinsip Dasar Pembelajaran Efektif Hal ini ada kaitannya dengan pendapat yang dikemukakan oleh seorang filosof Cina yaitu Confocius, bahwa apa yang saya dengar, saya lupa; apa yang saya lihat, saya ingat; dan apa yang saya lakukan saya paham. Dari kata- kata bijak ini seseorang dapat mengetahui betapa pentingnya keterlibatan langsung dalam pembelajaran. Jean Piaget yang dikutip Ahmad Rohani mengemukakan, seorang anak akan berpikir sepanjang ia berbuat, tanpa berbuat anak tak 48 GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 Keempat, pengulangan. Hamalik (2003: 8) mengatakan bahwa pengulangan dalam kaitannya dengan pembelajaran adalah suatu tindakan atau perbuatan berupa latihan berulangkali yang dilakukan peserta didik yang bertujuan untuk lebih memantapkan hasil pembelajarannya. Pemantapan diartikan sebagai suatu perbaikan dan sebagai usaha perluasan yang dilakukan melalui pengulangan- pengulangan. Kelima, tantangan. Teori medan (field theory) dari Kurt Lewin mengemukakan bahwa peserta didik dalam situasi belajar berada dalam suatu medan atau lapangan psikologis. Dalam belajar, peserta didik menghadapi suatu tujuan yang ingin dicapai, tetapi selalu terdapat hambatan yaitu menguasai bahan belajar, maka timbullah motif untuk mengatasi hambatan itu, yaitu dengan mempelajari bahan belajar tersebut. Tantangan yang dihadapi dalam bahan belajar membuat peserta didik bergairah untuk mengatasinya. Bahan belajar yang baru, yang banyak mengandung masalah yang perlu dipecahkan, membuat peserta didik tertantang untuk mempelajarinya. Penggunaan metode eksperimen, inkuiri, discovery juga memberikan tantangan bagi peserta didik untuk belajar secara lebih giat dan sungguh-sungguh. Penguatan positif maupun negatif juga akan menantang peserta didik dan menimbulkan motif untuk memperoleh ganjaran atau terhindar dari hukum yang tidak menyenangkan. Pembelajaran yang efektif dilakukan dengan berulang kali sehingga peserta didik menjadi mengerti. Bahan ajar bagaimanapun sulitnya yang diberikan oleh pendidik kepada peserta didik, jika mereka sering mengulangi bahan tersebut niscaya akan mudah dikuasai dan dihafalnya. Zayadi dan Majid (2005: 74) mengatakan bahwa penguatan dorongan serta bimbingaan pada beberapa peristiwa pembelajaran peserta didik dapat meningkatkan kemampuan yang telah ada pada perilaku belajarnya. Hal ini mendorong kemudahan bagi peserta didik untuk melakukan pengulangan atau mempelajari materi pelajaran secara berulang kali. Hal ini senada dengan Sagala (2005: 54) yang mengatakan adanya pengulangan terhadap materi pelajaran yang diberikan mempermudah penguasan dan dapat meningkatkan kemampuannya. Keenam, balikan dan penguatan. Prinsip belajar yang berkaitan dengan balikan dan penguatan terutama ditekankan oleh teori belajar operant conditioning dari B.F. Skinner. Kalau pada teori conditioning yang diberi kondisi adalah stimulusnya, maka pada operant conditioning yang diperkuat adalah responnya. Kunci dari teori belajar ini adalah law of effect versi Thorndike. Prinsip Dasar Pembelajaran Efektif p y Salah satu teori pembelajaran yang menekankan perlunya pengulangan adalah teori psikologi asosiasi atau koneksionisme dengan tokohnya yang terkenal Thorndike mengemukakan ada tiga prinsip atau hukum dalam belajar yaitu: (a) Law of readines, belajar akan berhasil apabila individu memiliki kesiapan untuk melakukan perbuatan tersebut. (b) Law of exercise, belajar akan berhasil apabila banyak latihan dan ulangan. (c) Law of effect, belajar akan bersemangat apabila mengetahui dan mendapatkan hasil yang baik. Belajar akan berhasil apabila peserta didik itu memiliki kesiapan untuk belajar, pelajaran itu selalu dilatihkan/diulangi serta peserta didik lebih bersemangat apabila mendapatkan hasil yang memuaskan. Peserta didik belajar sungguh- sungguh dan mendapatkan nilai yang baik dalam ulangan. Nilai yang baik itu mendorong anak untuk belajar lebih giat lagi. Nilai yang baik dapat merupakan operant conditioning atau penguatan positif. Sebaliknya, anak yang mendapat nilai yang jelek pada waktu ulangan akan merasa takut tidak naik kelas. Hal ini juga bisa mendorong anak untuk belajar lebih giat. Inilah yang disebut penguatan negatif atau escape conditioning. Format sajian berupa tanya jawab, diskusi, eksperimen, metode penemuan dan sebagainya merupakan cara pembelajaran yang memungkinkan terjadinya balikan dan penguatan. Ketujuh, perbedaan individu. Pada dasarnya tiap individu merupakan satu kesatuan, yang berbeda antara satu dengan yang lainnya. Tidak ada yang sama baik dari aspek fisik maupun psikis. Dimiyati dan Mudjiono (2009: 92) berpendapat bahwa peserta didik merupakan Fungsi utama pengulangan adalah untuk memastikan peserta didik memahami persyaratan-persyaratan kemampuan untuk suatu mata pelajar, peserta didik akan belajar dengan mudah dan mengingat lebih lama jika mereka mengulangi apa yang mereka pahami. Wahyuni, Paradigma Pembelajaran Efektif … 49 komponen dari suatu set materi termasuk aktivitas sebelum pembelajaran, dan partisipasi peserta didik yang merupakan prosedur pembelajaran yang digunakan kegiatan selanjutnya. (3) Suparman (1997), strategi pembelajaran merupakan perpaduan dari urutan kegiatan, cara mengorganisasikan materi pelajaran peserta didik, peralatan dan bahan, dan waktu yang digunakan dalam proses pembelajaran untuk mencapai tujuan pembelajaran yang telah ditentukan. (4) Hilda Taba, strategi pembelajaran adalah pola atau urutan tingkah laku pendidik untuk menampung semua variabel-variabel pembelajaran secara sadar dan sistematis. (5) Gerlach dan Ely (1990), Strategi pembelajaran merupakan cara-cara yang dipilih untuk menyampaikan metode pembelajaran dalam lingkungan pembelajaran tertentu. (6) Kemp (1995), Stategi pembelajaran adalah suatu kegiatan pembelajaran yang harus dikerjakan pendidik dan peserta didik agar tujuan pembelajaran dapat dicapai secara efektif dan efisien. individu yang unik, artinya tidak ada dua orang peserta didik yang sama percis, tiap peserta didik memiliki perbedaan satu sama lain. Prinsip Dasar Pembelajaran Efektif Perbedaan itu terdapat pula pada karakteristik psikis, kepribadian dan sifat-sifatnya. Hamalik (2003: 92) mengemukakan bahwa perbedaan individu manusia, dapat dilihat dari dua sisi yakni horizontal dan vertikal. Perbedaan horizontal adalah perbedaan individu dalam aspek mental, seperti tingkat kecerdasan, bakat, minat, ingatan, emosi, dan sebagainya. Sedangkan perbedaan vertikal adalah perbedaan individu dalam aspek jasmaniah seperti bentuk badan, tinggi, dan besarnya badan, tenaga, dan sebagainya. Masing-masing aspek tersebut besar pengaruhnya terhadap kegiatan dan keberhasilan pembelajaran yang dilakukan. Ramayulis (1990: 61) mengatakan perbedaan individual ini berpengaruh pada cara dan hasil belajar peserta didik. Oleh karena itu perbedaan individu ini perlu menjadi perhatian dalam aktivitas pembelajaran dengan memperhatikan tipe-tipe belajar setiap individu. Para ahli didik mengklasifikasi tipe belajar peserta didik atas empat macam yaitu: (a) Tipe auditif, yaitu peserta didik yang mudah menerima pelajaran melalui pendengaran. (b) Tipe visual, yaitu peserta didik yang mudah menerima pelajaran melalui penglihatan. (c) Tipe motorik, yaitu peserta didik yang mudah menerima pelajaran melalui gerakan. (d) Tipe campuran, yaitu peserta didik yang mudah menerima pelajaran melalui penglihatan dan pendengaran. Strategi dalam kegiatan pembelajaran dapat diartikan dalam pengertian secara sempit dan pengertian secara luas. Dalam pengertian sempit bahwa istilah strategi itu dapat sama dengan pengertian metode yaitu sama- sama merupakan cara dalam rangka pencapaian tujuan. Dalam pengertian luas sebagaimana dikemukakan Newman dan Logan (Abin Syamsuddin Makmun, 2003) mengemukakan empat unsur strategi dari setiap kegiatan, yaitu: (1) Mengidentifikasi dan menetapkan spesifikasi dan kualifikasi hasil (output) dan sasaran (target) yang harus dicapai, dengan mempertimbangkan aspirasi dan selera masyarakat yang memerlukannya. (2) Mempertimbangkan dan memilih jalan pendekatan utama (basic way) yang paling efektif untuk mencapai sasaran. (3) Mempertimbangkan dan menetapkan langkah- langkah yang akan ditempuh sejak titik awal sampai dengan sasaran. (4) Mempertimbangkan dan menetapkan tolok ukur (criteria) dan patokan ukuran (standard) untuk mengukur dan menilai taraf keberhasilan (achievement) usaha. Mengetahui perbedaan individu dalam belajar, memudahkan bagi pendidik dalam menentukan media yang akan digunakan, hal tersebut sangat urgen dalam pencapaian hasil pembelajaran yang optimal. Strategi Pembelajaran Efektif Strategi pembelajaran merupakan suatu serangkaian rencana kegiatan yang termasuk di dalamnya penggunaan metode dan pemanfaatan berbagai sumber daya atau kekuatan dalam suatu pembelajaran dan disusun untuk mencapai suatu tujuan tertentu. Beberapa pengertian tentang strategi pembelajaran menurut para ahli adalah sebagai berikut: (1) Hamzah B. Uno (2008), strategi pembelajaran merupakan hal yang perlu diperhatikan pendidik dalam proses pembelajaran. (2) Dick dan Carey (2005), strategi pembelajaran adalah komponen- Untuk diterapkan dalam konteks pembelajaran keempat unsur tersebut dilakukan dengan cara berikut: (1) Menetapkan spesifikasi dan kualifikasi tujuan pembelajaran yakni perubahan profil perilaku dan pribadi peserta didik. (2) Mempertimbangkan dan memilih sistem pendekatan pembelajaran yang dipandang GURINDAM: JURNAL BAHASA DAN SASTRA, Volume 1, Nomor 2, 2021, hlm. 40—51 50 kelompok, diskusi dan sebagainya. Mereka bertanggung jawab bersama dalam proses pemecahan masalah. paling efektif. (3) Mempertimbangkan dan menetapkan langkah-langkah atau prosedur, metode dan teknik pembelajaran. (4) Menetapkan norma-norma dan batas minimum ukuran keberhasilan atau kriteria dan ukuran baku keberhasilan. Keempat, individualisasi. Dalam mengorganisasi belajar mengajar guru memperhatikan taraf kesanggupan siswa dan merangsangnya untuk menentukan bagi dirinya sendiri apa yang dapat dilakukan sebaik-baiknya. Efektivitas merupakan unsur pokok untuk mencapai tujuan atau sasaran yang telah ditentukan. Efektivitas disebut juga efektif, apabila tercapainya tujuan atau sasaran yang telah ditentukan sebelumnya. Hal tersebut sesuai dengan pengertian efektivitas menurut Hidayat (1986) bahwa efektivitas adalah suatu ukuran yang menyatakan seberapa jauh target (kuantitas, kualitas dan waktu) yang telah tercapai. Dengan demikian efektif lebih mengarah kepada pencapaian sasaran/tujuan. Kelima, urutan. Belajar sebagai gejala tersendiri dan pada mengorganisasikannya dengan tetap berdasarkan prinsip konteks, fokalisasi, sosialisasi, dan individualisasi. Namun demikian, guru juga harus mempertimbangkan efektivitas dari serangkaian pelajaran yang disusun secara tepat menurut waktu atau urutannya. Keenam, evaluasi. Evaluasi dilaksanakan untuk meneliti hasil dan proses belajar siswa, untuk mengetahui kesulitan- kesulitan yang melekat pada proses belajar itu. g p p p j Cara belajar yang efektif dapat membantu siswa dalam meningkatkan kemampuan yang diharapkan sesuai dengan tujuan instruksional yang ingin dicapai. Untuk meningkatkan cara belajar yang efektif diperlukan strategi yang tepat agar pembelajaran dapat berjalan dengan optimal dan seefektif mungkin. Oleh karena itu, dalam belajar para siswa menghendaki hasil belajar yang efektif. Demi tuntutan tersebut guru harus membantu dengan cara mengajar yang efektif pula. Mengajar efektif adalah mengajar yang dapat membawa belajar yang efektif. Untuk dapat mengajar secara efektif guru harus mampu menciptakan iklim belajar yang menunjang terciptanya kondisi yang optimal bagi terjadinya proses belajar. Strategi Pembelajaran Efektif Kondisi yang dimaksudkan hanya dapat terjadi apabila guru mengaajr menggunakan prinsip-prinsip mengajar, diantaranya yaitu Pertama, konteks. Belajar sebagian besar tergantung pada konteks belajar itu sendiri. Situasi problematis yang mencakup tugas untuk belajar hendaknya dinyatakan dalam kerangka konteks yang dianggap penting dan memaksa bagi pelajar dan melibatkan siswa menjadi peserta yang aktif, justru karena tujuan itu sendiri. PENUTUP Pembelajaran efektif adalah proses belajar mengajar yang bukan saja terfokus kepada hasil yang dicapai peserta didik, namun bagaimana proses pembelajaran yang efektif mampu memberikan pemahaman yang baik, kecerdasan, ketekunan, kesempatan dan mutu serta dapat memberikan perubahan perilaku dan mengaplikasikannya dalam kehidupan. Indikator pembelajaran efektif terdiri atas pengorganisasian materi yang baik, komunikasi efektif, penguasaan dan antusiasme dalam materi pelajaran, sikap positif terhadap peserta didik, pemberian nilai yang adil, keluwesan dalam pendekatan pembelajaran, dan hasil peserta didik yang baik. Tujuan pembelajaran efektif ini adalah untuk mencapai hasil belajar yang memuaskan, apabila peserta didik belajarnya tidak tuntas, tidak teratur, tidak berkesinambungan dan juga tidak bersungguh-sungguh baik itu di sekolah ataupun di rumah maka akan menyebabkan tidak tercapai tujuan belajar yang diharapkan. Kedua, fokus. Proses pembelajaran perlu diorganisasikan dengan bahan belajar. Di samping itu pembelajaran yang penuh makna dan dektit harus diorganisasikan di sekitar suatu fokus. Pengajaran akan berhasil dengan menggunakan fokalisasi, sehingga mutu pembelajaran lebih meningkat. Dalam pembelajaran efektif ini terdiri atas karakteristik pembelajaran efektif, prinsip- prinsip pembelajaran efektif, kondisi pembelajaran efektif, suasana pembelajaran efektif, dan strategi pembelajaran efektif. Ketiga, sosialisasi. Dalam proses belajar siswa melatih bekerja sama dalam kerja 51 Wahyuni, Paradigma Pembelajaran Efektif … DAFTAR PUSTAKA Gesrianto, A. Jamaluddin. 2017. Analisis Sikap Bahasa dan Motivasi Belajar Bahasa Indonesia Siswa Kelas XI SMA Bosowa International School. Jurnal Nalar Pendidikan, Vol. 1 No.1. Nasution, S. 1999. Kurikulum dan Pengajaran. Jakarta: Bumi Aksara. Nawawi, Hadari. 1989. Organisasi Sekolah dan Pengelolaan Kelas. Jakarta: PT. Haji Mas Agung. Abdurrahman, Mulyono. 2012. Pendidikan Bagi Anak Berkesulitan Belajar. Jakarta: Rineka Cipta. Ramayulis. 1990. Metodologi Pengajaran Islam. Jakarta: Kalam Media. Ali, Muhammad. 2020. Pembelajaran Bahasa Indonesia dan Sastra (BASASTRA) di Sekolah Dasar. PERNIK Jurnal PAUD. Vol. 3 No. 1. Rosyada, Dede. 2004. Paradigma Pendidikan Demokratis: Sebuah Model Pelibatan Masyarakat Penyenggaraan Pendidikan. Jakarta: Prenada Media. Ali, St. Hansniyati Gani. 2013. Prinsip- prinsip Pembelajaran dan Implikasinya Terhadap Pendidik dan Peserta Didik. Jurnal Al- Ta’did, Vol. 6 No. 1. Sagala, Syaiful. 2005. Konsep dan Makna Pembelajaran. Bandung: Alfa Beta. Anwar, Muhammad. 2017. Menciptakan Pembelajaran Efektif Melalui Hypnoteaching. Jurnal Ekspose, Vol. 16 No. 2. Santrock, Jhon W. 2008. Educational Psychology, Terjemahan Tri Wibowo B.S. Psikologi Pendidikan. Jakarta: Prenada Media. Dimiyati dan Mudjiono. 2009. Belajar dan Pembelajaran. Jakarta: Rineka Cipta. Setyowati, Punaji. 2014. Menciptakan Pembelajaran Yang Efektif dan Berkualitas. Jurnal Inovasi dan Teknologi Pembelajaran, Vol. 1 No. 1. Hamalik, Oemar. 2003. Kurikulum dan Pembelajaran. Jakarta: Bumi Aksara. Slameto. 1995. Belajar dan Faktor- faktor Yang Mempengaruhi Belajar. Jakarta: PT. Rineka Cipta. Khair, Ummul. 2018. Pembelajaran Bahasa Indonesia dan Sastra (BASASTRA) di SD dan MI. AR-RIAYAH: Jurnal Pendidikan Dasar. Vol. 2 No. 1. Yusnawarni. 2014. Peran Bahasa Indonesia dalam Pembelajaran Tematik Terpadu Melalui Pendekatan Saintifik. Jurnal Madah, Vol. 5 No. 2. Kemendikbud. 2014. Materi Pelatihan Guru Implementasi Kurikulum 2013. Zayadi, Ahmad dan Abdul Majid. 2005. Tadzkiyah: Pembelajaran Pendidikan Agama Islam Berdasarkan Pendekatan Kontekstual. Jakarta: Raja Grafindo Persada. Mahsun, M.S. 2014. Teks dalam Pembelajaran Bahasa Indonesia Kurikulum 2013. Jakarta: Raja Grafindo. Magdalena, Ina, dkk. 2020. Pengelolaan Pembelajaran Daring Yang Efektif Selama Pandemi di SDN 1 Tanah Tinggi. EDISI: Jurnal Edukasi dan Sains. Vol. 2 No. 2. Mulyasa, E. 2003. Menjadi Kepala Sekolah Profesional dalam Konteks Menyukseskan MBS dan KBK. Bandung: Remaja Rosdakarya.
https://openalex.org/W4361861780	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/16171EAAB2193717F880D9C6F8FF6D5A/S0001972023000116a.pdf/div-class-title-a-share-in-the-sands-trips-pits-and-potholes-in-accra-ghana-div.pdf	English	null	A share in the sands: trips, pits and potholes in Accra, Ghana	Africa	2,023	cc-by	12,324	© The Author(s), 2023. Published by Cambridge University Press on behalf of the International African Institute. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract This article deploys sand as a potential way of engaging with contemporary livelihoods in the Ghanaian city of Accra – one of many metropolitan nodes in an urbanizing region of West Africa. Both as a very real material at the heart of concrete urbanization and as metaphorically indica- tive of the shifting landscapes of opportunity and income on which lives and livelihoods are marked out, sand is offered as a way of seeing and writing about the city. The article brings these two facets of urban sand together in more concrete ways, considering how the material produc- tion of the city becomes the uneven, uncertain ground of urban life-making. Drawing from four- teen months of ethnographic fieldwork in Accra, the article engages with the movements of sand across the city, as it travels from extraction zones (or pits) to lorries and then to places of consumption. By honing in on the material behaviours and temporal junctures of sand as it shifts its shape, form and directions, the article draws out the ways in which sand emerges as a plat- form for exchange, negotiation and ultimately income for different people across the city region. In turn, it offers a share in the sands as a tentative holding space for the kinds of claims made on and through sand, positioning them as indicative of a dweller-led, emergent politics that claims a share of an income, livelihood and urban future. A share in the sands: trips, pits and potholes in Accra, Ghana Katherine Dawson University College London, London, UK Email: katherine.dawson@ucl.ac.uk Katherine Dawson University College London, London, UK Email: katherine.dawson@ucl.ac.uk University College London, London, UK Email: katherine.dawson@ucl.ac.uk Africa (2023), 93, 40–59 doi:10.1017/S0001972023000116 https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Résumé Cet article déploie le sable comme un moyen potentiel d’aborder les moyens de substance contem- porains dans la ville ghanéenne d’Accra, l’un des nombreux pôles métropolitains dans une région d’Afrique de l’Ouest en voie d’urbanisation. À la fois comme matériau très réel au cœur d’une urbanisation de béton et comme révélateur métaphoriquement des paysages changeants d’opportunité et de revenu sur lesquels se dessinent les existences et les moyens de subsistance, le sable est proposé comme un moyen de voir et d’écrire la ville. L’article rassemble ces deux face- ttes du sable urbain de manière plus concrète, en considérant comment la production matérielle de la ville devient le terrain inégal et incertain de vie urbaine. S’appuyant sur quatorze mois de travaux ethnographiques menés sur le terrain à Accra, l’article traite des mouvements de sable à travers la ville, depuis les zones d’extraction (ou sablières) jusqu’aux camions, puis jusqu’aux lieux de consommation. En se focalisant sur les comportements matériels et sur les tournants temporels du sable alors qu’il change de forme et de direction, l’article fait ressortir les manières dont le sable émerge comme une plateforme d’échange, de négociation et en 41 Africa Africa définitive de revenu pour différentes personnes de cette région urbaine. Enfin, il se propose comme une tentative d’espace d’ouverture pour les types de revendications faites sur le sable et à travers lui, en les positionnant comme un révélateur de politique émergente menée par les citadins qui revendique une part de futur de revenu et de moyens de subsistance, et de futur urbain. Resumo Este artigo utiliza a areia como uma forma potencial de envolvimento com os meios de subsistência contemporâneos na cidade ganesa de Accra - um dos muitos n´os metropolitanos numa região urbanizadora da África Ocidental. Tanto como um material muito real no coração da urbanização de betão como, metaforicamente indicativo das paisagens mutáveis de oportunidades e rendimentos sobre os quais as vidas e meios de subsistência são marcados, a areia é apresentada como uma forma de ver e escrever sobre a cidade. O artigo reúne estas duas facetas da areia urbana de formas mais concretas, considerando como a produção material da cidade se torna o terreno irregular e incerto da produção da vida urbana. Baseado em catorze meses de trabalho de campo etnográfico em Acra, este artigo analisa os movimentos de areia através da cidade, à medida que esta se desloca das zonas de extracção (ou fossos) para camiões e depois para locais de consumo. Destacando os comportamentos materiais e as conjunções temporais da areia à medida que esta muda de contorno, forma e direcção, o artigo examina as formas como a areia emerge como uma plataforma de troca, negociação e, em última análise, rendimento para diferentes pessoas em toda a região da cidade. Por sua vez, este oferece uma parte nas areias como um espaço de detenção provis´orio para os tipos de reivindicações feitas sobre e através da areia, posicionando-as como indicativo de uma política emergente, liderada por um habi- tante que reivindica uma parte de um rendimento, subsistência e futuro urbano. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A city built on sand Sand is a material deployed with the intention of solidifying urban form. Indeed, mixed with water and cement, sand becomes the concrete backbone of cities across the planet, and it is doing this at an accelerating rate – a process of ‘cementification’, in the words of Choplin (2020). For homes, roads, skyscrapers, shopping centres and so on, sand is enlisted on a huge scale, as residents, investors and states attempt to produce a materially stable city in which they can live and through which they might profit and govern. A hard city. Situating sand as central to urban materiality is an argument that I and others have made elsewhere (Dawson 2021a; Mendelsohn 2018; Zalasiewicz et al. 2017; Jamieson 2021) and that chimes with the growing public interest in sand as an environmental issue of global significance (UNEP 2019). Yet, thinking through the city as a sandy landscape also evokes the kinds of evolving movements, choreographies and engagements that majorities perform in the ‘urban now’ (De Boeck and Baloji 2016). Simone’s (2004) ‘people as infrastructure’ drew attention to such relations, recalling the rumours, calculations and forms of knowledge that structure life and living in cities from ‘Jakarta to Dakar’ (Simone 2009). More broadly, a literature on African urbanism has played an important role in this space, moving the focus from a view of some cities as failing sets of infrastruc- ture to a view that sees cities made in spite of, through and beyond such scaffolding (Pieterse 2008; Pieterse and Simone 2013; Diouf and Fredericks 2014; Ernstson et al. Katherine Dawson 42 Figure 1. Concrete blocks, made up of sand, water and cement, in plots of land, Greater Accra region. Figure 1. Concrete blocks, made up of sand, water and cement, in plots of land, Greater Accra region. Figure 1. Concrete blocks, made up of sand, water and cement, in plots of land, Greater Accra region. 2014; Myers 2011; 2016; Quayson 2014). Such concepts and ways of thinking appeal to notions of shifting urban landscapes that, when not romanticized, depict the deep struggles that urban majoritities face to secure homes, livelihoods and futures in many of the world’s cities (Simone and Pieterse 2017). In this rendition, the city is a landscape in constant formation: a fluid platform, remaking itself over time and space. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A city built on sand In tension with the hard concrete city of sand, the urban landscape might also be understood as a set of shifting sands – the uncertain ground on which lives and livelihoods must be marked out. In this article, I bring these two facets of urban sand together, to consider the ways in which the material production of the city becomes the uncertain ground of life and livelihood-making. More concretely, the article engages with the material spheres of sand in Accra, Ghana, where, like elsewhere, sand is unearthed from the city’s outer edges and surrounding regions to support the expanding conglomer- ation of the Greater Accra region (Figure 1). Situated in the space between a hard city and a fluid city, sand becomes the material entry point into the ways in which urban livelihoods in particular are elaborated. Drawing from fourteen months of ethno- graphic research in Accra in 2017 and 2021, I engage with the material movements of sand across the city, as it travels from extraction zones (or pits) to lorries and then to places of consumption. The article reveals the diverse labours, livelihoods and income-seeking practices that coalesce around sand’s material economies and their multiple temporalities (Kothari and Arnall 2020) – aligning with a literature that recognizes the complex and differentiated livelihood implications of sand’s extraction and consumption (Anokye et al. 2022; Marschke and Rousseau 2022; Marschke et al. 2021; Lamb and Fung 2022; Lamb et al. 2019). https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Africa 43 By honing in on the material behaviours and temporal junctures of sand as it shifts its shape, form and direction, the article draws out the ways in which sand emerges as a platform for exchange, negotiation, different forms of labour, diverse livelihoods – and ultimately income. In this sense, sand is revealed to be as much a social infra- structure as a concrete infrastructure in the city. Critically, set in the context of ‘wageless life’ (Denning 2010), precarious urban incomes and unequal revenues flowing from sand to various communities and individuals, I recall events that expose a politics of claim-making to the values embedded in sand. In this vein, I offer a share in the sands as a tentative holding space for the kinds of claims made on and through sand, positioning them as indicative of a dweller-led, emergent politics that claims a share to an income, livelihood and urban future. A city built on sand The article is structured as follows. I first introduce a literature that deals with the future of (urban) work globally, in sub-Saharan Africa, and specifically in Accra. This offers an important context for thinking through the significance of labour, liveli- hoods and claims to an urban income that become apparent as the analysis progresses. I then situate sand in Accra and outline my research methods. From here, I turn to an analysis of the ethnographic narratives and encounters that emerged in the research, examining the various ways in which the material economies of sand coalesce as a platform for value extraction and income demands. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Beyond the ‘proper job’ Yet, as United Nations Special Rapporteur on Extreme Poverty and Human Rights Philip Alston commented: ‘[I]nequality is higher than it has ever been in Ghana, while almost one-quarter of the population lives in poverty, and one person in every twelve lives in extreme poverty. As a result, a large number of Ghanaians do not enjoy their basic economic and social human rights’ (Yirenkyi 2020). Significantly, in 2018, the Ibrahim Index of African Governance concluded that ‘even though the patterns of growth and job-creation in Africa are complex and change from region to region, “the continental trend is one of resilient but jobless growth”’ (Mo Ibrahim Foundation 2018: 52). In December 2018, the IMF published a report that shared a projected analysis of the future of work on the continent in the face of dramatic uncertainties surrounding technological innovation, climate change and global economic integration. The IMF concluded that an average of 20 million jobs per year across the continent will be required to keep pace with forecast increases in population growth and given projected expansions of urban populations; this requirement will likely be marked by the need for ‘urban jobs’ (Abdychev et al. 2018: 1). For Ferguson (2015), this ‘jobless growth’ has given rise to urban economies characterized by stark struggle and forced flexibility. With these realities in mind, studies that engage with work, livelihoods and labour in Accra are not difficult to locate. The term ‘informal economy’ in fact arose through insights derived from the neighbourhood of Nima in Accra (Hart 1973). Since then, a proliferation of work has explored the contours of informal work throughout the city. In more recent decades, this has been shaped by the consequences of neoliberal reform from the 1980s onwards, which saw the loss of urban jobs as well as subsidy reductions for housing and basic infrastructural provision (Obeng-Odoom 2012; Grant 2009; Murray and Myers 2006). Where population increases in urban areas ‘have not been matched by opportunities for wage-paid employment, expectant work seekers in the cities and towns have been forced to find other means to generate income’ (Murray and Myers 2006: 120). Beyond the ‘proper job’ In the introduction to Critique of Black Reason, Mbembe sketches out grand moments in a ‘biography of the vertiginous assemblage that is Blackness and race’ (2017: 2). In this analysis, Mbembe extends the condition of Blackness, arguing that, ‘for the first time in human history, the term “Black” has been generalized’ – indeed, ‘institutionalized as a new form of existence and expanded to the entire planet’ (ibid.: 5). Painting a picture of a present marked by the triumph of finance capital, digital technologies and a ‘postimperial military complex’, Mbembe writes: ‘If yesterday’s drama of the subject was exploitation by capital, the tragedy of the multitude today is that they are unable to be exploited at all. They are abandoned subjects, relegated to the role of a “superfluous humanity”’ (ibid.: 3). This reading captures a broader sentiment expressed across academic disciplines, within governments and among policymakers, marked by an anxiety of the prospect of the ‘wageless life’ (Denning 2010). Such anxiety prompted the International Labour Organization (ILO) to assign to the newly formed Commission on the Future of Work the task of producing a series of independent reports on ‘how to achieve a future of work that provides decent and sustainable work opportunities for all’ (ILO 2016). In a landmark report, the ILO (ibid.) sets out a future of work in the context of the expanding economies of digitization, automation and low-carbon transitions. According to Ferguson and Li, in order to engage with this contemporary conjuncture, questions must be asked that depart from a fixation ‘on the old story-line of ever- expanding wage employment’ (2018: 4). They pose a series of questions related to distributive claims, gender, generation, identity and land that together ‘offer points of entry for understanding lives and livelihoods, membership and meaning minus the telos (though not spectre) of the “proper job”’ (ibid.: 5). In attempting to denaturalize the idea of a ‘proper job’ while simultaneously re-politicizing forms of labour that Katherine Dawson 44 sustain vast numbers of people around the world, Ferguson and Li conclude that making sense of what lies beyond the ‘proper job’ demands ‘a focus on the empir- ical contours of the present – what is there, and what is emergent’ (ibid.: 20). In Ghana, economic growth at a national level has been significant. Ghana was enshrined as the ‘the world’s fastest growing economy’ by the International Monetary Fund (IMF) in 2019 (Edmond 2019). https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Beyond the ‘proper job’ This rough sketching has formed the basis of diverse empirical engagements with livelihoods in Accra, including, among many others, e-waste economies, the gendering of different forms of entrepreneurship, the economy of free time and struggles over space in the city (Amankwaa 2013; Grant and Oteng-Ababio 2012; Oteng-Ababio 2012; Langevang and Gough 2012; Quayson 2014; Gillespie 2016). In recent years, important work has appeared that examines the impact of the Covid-19 pandemic on informal livelihoods and household poverty in Accra and elsewhere in Ghana (Amoah-Nuamah et al. 2020; Adom et al. 2020; Asante and Mills 2020). These studies point to the exacerbated struggles facing inhab- itants who rely on markets and busy streets to make a living, with residents suffering from reduced sales, forceful relocations, supply shortages and business closures, with observers pointing to an increased number of households entering conditions of poverty (Bukari et al. 2021). Africa Africa 45 The analysis in this article, however, moves away from engagements defined strictly by the ‘informal economy’. Invoking both Kate Meagher and Keith Hart, Ferguson argues that ‘the sorts of economic activities the term was originally intended to capture have become so pervasive as to call into question the very concept of an “informal economy”’ (2015: 11). Indeed, as Hart has argued, ‘when most of the economy is “informal”, the usefulness of the category becomes questionable’ (2006: 27). This is indeed a reality in sub-Saharan Africa, where a significant percentage of workers are in vulnerable employment – or in labour marked by ‘limited access to social protection schemes’ and ‘confronted by low and highly vola- tile earnings’ (ILO 2016: 3). The material production of the city is perhaps no different; its realization is supported by a host of labours and practices that could be considered informal. However, moving beyond a need to identify the informal and often exploited forms of labour and nature that underpin the production of the concrete city, this article looks more closely at the lives, livelihoods, narratives and encounters that are central to ‘the story of the economy of sand’ in Accra (Lamb et al. 2019: 1525). In doing so, and while keeping in view the uneven distribution of incomes that char- acterize the sand economy, the article reveals the ways in which sand – and its multiple materialities and temporalities – becomes a source of income for a host of different people, in different ways. 1 ‘Populations by regions, Greater Accra’, Statsghana.gov.gh, 2021 <https://www.statsghana.gov.gh/ regionalpopulation.php?population=MTM0NTk2MjQzOS4yMDE1&&Greater%20Accra&regid=3>, accessed August 2022. Beyond the ‘proper job’ This, I suggest, is indicative of the ways in which urban incomes and claims to them co-emerge with the making of the material city. Situating sand in Accra The empirical data deployed in this article draws from my doctoral research in Accra, Ghana. Accra sits on the southern coast of Ghana, bordering the Atlantic Ocean. As the headquarters of the Ga people and the former seat of British colonial power, the city possesses a hybrid history of contested rules, governance and identities (Parker 2000). The city region of Greater Accra reached a population of 5.4 million people in 2021,1 and the boundaries of built-up Accra continue to stretch into surrounding villages and farming areas, where individuals and companies buy up land for residential use (Akubia and Bruns 2019). The extraction of sand often – though not exclusively – takes place in peri-urban Accra and beyond, extending into the rural landscapes of Accra, depending on the availability of and access to land. The extraction of sand – otherwise known as ‘sand winning’ – can be classified as legal or illegal; however, as described to me, the boundary is somewhat blurred. At the legal end of the spectrum, sand contractors were responsible for acquiring the land from various landowners, bringing machinery to the site, extracting the sand and loading it onto tipper trucks, taking a fee from each truck. Contractors were responsible for obtaining licences and permissions from the Minerals Commission and paid a fee to the landowner, depending on the quantity of sand extracted from the particular parcel of land. 1 ‘Populations by regions, Greater Accra’, Statsghana.gov.gh, 2021 <https://www.statsghana.gov.gh/ regionalpopulation.php?population=MTM0NTk2MjQzOS4yMDE1&&Greater%20Accra&regid=3>, accessed August 2022. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press 46 Katherine Dawson In many ways, sand conjures up a similar story to other extraction processes. Sand is extracted from pits and revenues flow both to those with the capital and to the owners of the land in which the resource is embedded. Mirroring the sale of land leases across the Greater Accra region, revenues from sand tend to flow to more powerful individuals and families who already own land. I was told that the majority of landowners in peri-urban Accra are Ga who had long occupied strips of land stretching from family quarters in downtown Accra out into the peri-urban and rural landscapes of the wider city region (Parker 2000). Other groups do occupy land in these areas, including Ewe communities, who migrated at various points in history. Situating sand in Accra However, I was told that, in many cases, these non-Ga communities rent the land from indigenous groups and have little power or say over the future of that land. This was discussed during interviews with communities in Greater Accra, with residents complaining that their farmland was being handed over to sand contractors and estate developers, leaving them with limited access to food or income and feeling powerless to resist. Oduro and Adamtey’s (2017) research suggests that revenues from the sale of sand are rarely distributed widely into the communities where sand is found, with money largely retained by landowning groups. This was also confirmed in conversations I had with those working in the sand industry. g y The pits from which sand is unearthed in Greater Accra and the surrounding area varied depending on the quality and quantity of sand as well as on the amount of land available for extraction. Once the sand has been extracted from the land, contractors move to a new stretch of land and new sources of sand. In this way, the pits were incredibly mobile and demanded that their location be sought on a daily basis. As a researcher, locating these pits was achieved via word of mouth – that is, asking local inhabitants and shop owners where sand was being extracted – as well as by following the flags that were planted in the earth on the roadside to indicate the pits’ locations. The afterlives of the land from which sand was extracted varied. In some cases, land had already been sold to estate developers, who would proceed to prepare the ground for residential building. In other cases, if the contractors abided by the regulations, the land would be reclaimed, a process that involved restoring the topsoil on the surface of the land in order to allow previous occupants – often farming communities – to continue farming the land. In various conversations, there were debates about whether it was possible for land to operate at its previous yield; people argued, for instance, that machines compacted the topsoil, which rendered it less fertile. The bulk of my research took place between 2017 and 2019, and I have since returned to Accra for several shorter visits. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Situating sand in Accra The research was multi-sited and involved engaging with various facets of the sand economy and the residents who lived and worked in a small portion of Awoshie Junction in western Accra. I spent many months visiting this community, learning from residents who shared experiences of their precarious occupation of government land, which sat under an electricity transmis- sion wire (Dawson 2021b). On the opposite side of the multi-lane road was a busy sand station, hosting trucks and piles of sand. It was one of many spaces in the city that acted as a depot for fresh sand, gathered from the rural edges of Accra and the surrounding regions and brought here for sale. As my interest in this material grew, and as its centrality to urban life as built form became more apparent to me, I began to talk to those working in this space. In particular, I spoke with Francis, a young Africa 47 Figure 2. Performing the duties of the mate, Greater Accra region. Figure 2. Performing the duties of the mate, Greater Accra region. Ghanaian man from Accra who had been working in the sand industry for around five years. At the time, he was working as a ‘mate’; this involved assuming the labour- intensive role of directing sand on and off the sand truck and, crucially, securing it in place to limit sand spills from the truck’s bucket (Figure 2). Due to the fact that they moved around in tipper trucks all day, the ‘mates’ and truck drivers in the sand industry were known as ‘tippers’. Francis and the truck’s driver, Kojo, kindly allowed me to join them on truck journeys throughout Accra as they collected sand from pits and delivered it to various destinations. I went on many trips that encompassed the outer edges of rural Greater Accra and extended into the neighbouring Central and Eastern regions. Most often, we collected sand from pits near the growing town of Ashalaja in Greater Accra and distributed it between block factories, construction sites and the tipper station, from where it would later be sold. Our conversations ranged from their thoughts on the sand economy, the growing spaces of Greater Accra and the difficulty of securing incomes and building homes to the UK economy and Premier League football. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Situating sand in Accra On the truck, I was able to ask more detailed questions about the sand economy, how it functioned and who benefited, as well as observe practices, engagements and encounters as they unfolded on the road. Ghanaian man from Accra who had been working in the sand industry for around five years. At the time, he was working as a ‘mate’; this involved assuming the labour- intensive role of directing sand on and off the sand truck and, crucially, securing it in place to limit sand spills from the truck’s bucket (Figure 2). Due to the fact that they moved around in tipper trucks all day, the ‘mates’ and truck drivers in the sand industry were known as ‘tippers’. Francis and the truck’s driver, Kojo, kindly allowed me to join them on truck journeys throughout Accra as they collected sand from pits and delivered it to various destinations. I went on many trips that encompassed the outer edges of rural Greater Accra and extended into the neighbouring Central and Eastern regions. Most often, we collected sand from pits near the growing town of Ashalaja in Greater Accra and distributed it between block factories, construction sites and the tipper station, from where it would later be sold. Our conversations ranged from their thoughts on the sand economy, the growing spaces of Greater Accra and the difficulty of securing incomes and building homes to the UK economy and Premier League football. On the truck, I was able to ask more detailed questions about the sand economy, how it functioned and who benefited, as well as observe practices, engagements and encounters as they unfolded on the road. As the research progressed, I also approached authorized sand contractor compa- nies in the Greater Accra region and the immediate surroundings. With the guidance of an interlocutor, permission was sought to spend time at the pits of one particular contractor, Piam. I followed the pits as they shifted across the region, largely in the vicinity of Ashalaja and Hobor. Mornings, afternoons and evenings were spent at the Katherine Dawson 48 pit, capturing the shifting economies of sand throughout the day; by visiting in different months of the year, I was also able to observe changes in the market. A trip of sand Francis lived near a row of shops at the centre of the community where I spent a significant amount of time during my research. He regularly passed this line of shops on his way home from the sand station, which was slowly disappearing to make way for an extension to the headquarters of a large charismatic church. We would often greet one another and, if he had time, he would sit next to me to discuss the day’s events. It was almost a year after we first met and as we sat watching the daily burning of local waste, Francis expressed his anxiety about securing work. During the time that I had known Francis, work was precarious. On this particular evening, for reasons I am not entirely sure about, Francis was no longer employed irregularly as a mate on Kojo’s truck. He would later make headway in finding another truck, with himself as a spare driver and another friend as the main driver, but this never came to fruition. Like many others, he regularly sought irregular work as a mate. Viewing the remnants of the sand station, Francis gestured to the other side of the road, pointing out a Sinotruk Howo truck2 laden with fresh sand. He said, ‘Some guy bought this truck and two more. US$85,000. If you buy a truck, you can make money.’ I asked how this man could have earned the money to buy three, to which he replied: ‘In Africa, you can’t ask someone how they get their money.’ Compared with the people who owned trucks, those working on them received much smaller incomes that varied according to their roles. As it was explained to me, there is an owner of the truck, who assigns a caretaker, most commonly the driver. Kojo was the main driver. He would make 800 cedis a month (approximately US$170 at the time of research), based on the premise that he would make 800 cedis a day in profit, six days a week, all of which would be remitted to the truck owner. ‘If you make 600 a day, you get 600 a month. If you bring 200 a day, you make 200 a month.’ The driver therefore had to ensure that they made profits of 800 cedis a day in order to receive 800 cedis a month as payment from the owner. 2 Howo is a model of truck produced by Sinotruk, whose parent company is the China National Heavy Duty Truck Group Co. Ltd. Situating sand in Accra Having sought permission from the operating site manager, Mr Osei, I was able to spend time speaking with people working at the pits, either those directly employed by Piam as bulldozer drivers, payloader drivers, land analysts, researchers or admin- istrators, or those who worked in the pits as food providers or selling clothes or shoes. With the assistance of an interlocutor, who introduced me to people and explained my presence, translating from Ga and Twi to English where appropriate, I was able to speak to many of those working in the pits. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A trip of sand There was no guaranteed minimum income, but higher profits would result in increased income. The spare driver was not paid anything by the truck owner, nor was the mate. Kojo paid the spare drivers and mates, but sometimes they weren’t paid at all: it depended on how much money was made per day, which in turn depended on the number of ‘trips’ of sand. A ‘trip’ referred to the journey to and from the pit to collect sand 2 Howo is a model of truck produced by Sinotruk, whose parent company is the China National Heavy Duty Truck Group Co. Ltd. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Africa 49 Figure 3. A truck laden with sand leaves the sandpit, Greater Accra region. Figure 3. A truck laden with sand leaves the sandpit, Greater Accra region. (Figure 3). For the tipper drivers, this meant driving out to the pit, collecting sand and delivering it to a site or the sand station. Talking in terms of ‘trips’ was widespread among tipper drivers, but also in the construction industry more broadly. A customer might request ‘three trips of sand’, meaning three truckloads of sand to be delivered to the site. Plots of land for sale may also include ‘a first trip of sand’ as an added bonus to attract prospective buyers. For the tipper drivers, trips of sand were used as a way to calculate their costs and profits. To explain this, Francis listed the exchanges in a regular trip of sand: ‘The sand is 200, the fuel for one trip is 250, council ticket is 15, police stop is 10. You have to bring 800 a day. Sometimes you go twice and you’re at a loss, so you have to use some of your own money to make it up.’ As Francis explained: ‘You will go for three trips in a day before you make 800.’ This was in a context where the sale of sand stabilized at approximately 800 cedis per trip. (Figure 3). For the tipper drivers, this meant driving out to the pit, collecting sand and delivering it to a site or the sand station. Talking in terms of ‘trips’ was widespread among tipper drivers, but also in the construction industry more broadly. A customer might request ‘three trips of sand’, meaning three truckloads of sand to be delivered to the site. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A trip of sand and were now on what would be their fifth trip of sand: ‘If you have a baby, they will not know you; if you have a girlfriend, they will not see you.’ When we discussed in greater detail the cost of a tipper truck (US$85,000), I asked who had this kind of money. Francis replied that plenty of people in Ghana are financially capable of inves- ting, usually politicians and businesspeople. He said that the owner of this truck had a managerial position in a bank and owned as many as eleven trucks. The truck owners could thus be considered the most significant beneficiaries of the sand economy. Their incomes were stable, guaranteed and ongoing. While, for the tippers, daily costs were subject to change (fuel, food, police stops), the money handed over to the truck owner was consistent, meaning that any increases in short-term costs were absorbed by the tippers. And while landowners also received significant sums of money, their land was finite and thus their ability to cash in from sand was somewhat limited by the boundaries of their plot and by their ability to obtain uncontested access to it. In comparison, truck owners could benefit from the repeated extraction of sand from whatever land the tippers travelled to, cashing in on the shifting geography of the sand economy. As I thought more about this huge sum of money required to purchase a truck, I also considered the need to import it; through some light-touch research, it became apparent that this was even more expensive due to import duties and the possibility of other unforeseen and unpredictable costs at the border. Buying a truck was beyond the reach of most. y The shifting of sands was thus structured by a distinct hierarchy of truck owners, managers, drivers and mates. While it presented job opportunities for some, the proceeds flowing from this work were unevenly distributed. Those with access to capital could acquire trucks and make significant money, while mates, whose access to trucks remained insecure, experienced ongoing income volatility and periodic unemployment. The laborious, yet meagrely paid, work of shifting sands often prompted a critique of both the sand economy and the social structure of Ghanaian society more broadly. Complaining about the level of inequality in the country, Francis said that he felt cheated by the broader social system in Ghana; his peers agreed. A trip of sand Plots of land for sale may also include ‘a first trip of sand’ as an added bonus to attract prospective buyers. For the tipper drivers, trips of sand were used as a way to calculate their costs and profits. To explain this, Francis listed the exchanges in a regular trip of sand: ‘The sand is 200, the fuel for one trip is 250, council ticket is 15, police stop is 10. You have to bring 800 a day. Sometimes you go twice and you’re at a loss, so you have to use some of your own money to make it up.’ As Francis explained: ‘You will go for three trips in a day before you make 800.’ This was in a context where the sale of sand stabilized at approximately 800 cedis per trip. Francis had worked as a mate and his income was irregular. Unlike the main driver, who made a stable income every month, work and wages for mates were volatile. Income depended on the number of trips of sand in a day and the number of sales, as well as the number of mates or spare drivers working on the truck that day. On some days, the mate might make nothing at all. Despite this, even as a mate, shifting sand around the city offered a way to make money in an increasingly satu- rated urban economy of selling goods and performing services. However, demand for jobs on trucks was extremely high and it was difficult to affiliate oneself to a truck. Francis explained that work could be hard to come by, so acting faithfully towards your manager was crucial. Within this context, I would later ask Francis who was responsible for inscribing the front panel of the truck with the Twi words Di Nokore – ‘Be Truthful’ – to which he replied that Kojo had pasted the phrase on Katherine Dawson 50 the truck to let the owner know that he was faithful, reliable and could be trusted: it was a matter of retaining employment. Even when work was secured on a truck, it was difficult and laborious. On an early trip, Francis and his colleague Kwaku noted that they had been awake since 2 a.m. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A trip of sand Thus, as Francis situated himself in the economy of shifting sands, he positioned himself in the city and the nation more broadly, generating a space in which he could critique the structure of Ghanaian society. In many ways, Francis’s and Kwaku’s descriptions of working on tipper trucks expressed a broader senti- ment that securing a livelihood in Accra was tough and uneven and that the work secured was often laborious. Yet the tippers were only one element in a more expansive sand economy. Indeed, the labours of sand were far wider, encapsulating a variety of spaces and individuals. Together, these broader labours reflect the ways in which sand was materialized as a platform for value extraction, speaking not only to the challenges of improvising incomes in Greater Accra, but also hinting at an emergent set of claims to the values embedded in sand. It is these labours to which I now turn. Africa Africa 51 Figure 4. Preparing food for sale at the sandpit, Greater Accra region. Figure 4. Preparing food for sale at the sandpit, Greater Accra region. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Labours of sand As I moved through the city with the tipper drivers, it was clear that their labours were integral to the material construction of the city. Through their bringing of sand from pit to city, in many senses, they were city makers. Labours at the sandpit were also paramount in the production of the city. Operating machinery, performing administrative roles, driving motorbikes and loading sand were indeed significant elements in the sand economy. Of equal importance were the economies of consump- tion that shifted with the sands – the reproductive labour that kept the sandpits moving. This labour was performed almost exclusively by women, who prepared food and sold items of clothing, shifting with the sandpits as they moved across the region. Notable exceptions to this largely female economy included an elderly gentleman selling medicine for truck drivers’ aches and pains as well as one or two men who frequently sold work boots and strong-soled sandals at the pits. During the time I spent with Piam, some of these women would become familiar. Many followed the company from pit to pit, preparing food such as eggs and bread in the morning and rice and stew in the afternoon. Other women would move between sandpits during the course of the day, trying to find the best market. Other women would come from nearby villages, depending on the location of the sandpit that day, with petrol, fish, clothes and shoes, hoping to make sales in these temporary spaces of consumption (Figure 4). For some women, selling in the sandpits was a better option than selling in a village or Accra, where rents were higher. Moreover, the sandpits offered an inflated economy; as a woman selling bread and drinks explained, ‘If something is selling for two and a half cedis, you can sell it here for three cedis. If it’s three cedis, you can sell it for four.’ 52 Katherine Dawson This kind of work, however, was hard. Over time, as I listened to women share their experiences of working in the sandpits, I gained a sense of how these mobile sand frontiers were experienced. After preparing eggs, bread and tea, Ama shared her history of moving with the sands. She recalled how she had been working in the sand- pits for thirty years, having moved with them from places such as Ablekuma and Amasaman. Labours of sand ‘Back then,’ she explained, ‘it was hand loading, everyone was using their hands. The trucks were smaller too. Ten years ago, the trucks changed and the machines came too.’ She explained that people move with the sandpits, picking up their belongings as they go. Each working day she would bring what was required to prepare and sell hot food, including a wooden table, plastic stools, gas, water and food ingredients. It’s tiring work. Sometimes you can come and not sell too much. The market is on and off. Sometimes we have to move to another pit. And then we have to walk with our things : : : Your waist becomes sick. Many of the other women have stopped because they’re tired, they want to find another job. It’s tiring work. Sometimes you can come and not sell too much. The market is on and off. Sometimes we have to move to another pit. And then we have to walk with our things : : : Your waist becomes sick. Many of the other women have stopped because they’re tired, they want to find another job. Ama’s accounts of the labour involved in providing those working in the sandpits with sustenance seemed to stress the embodiment of these frontiers. On the ground, these feminized reproductive labours were vital to producing mobile economies that enabled companies such as Piam to shift the sandpit. Other facets of the sand economy kept the sandpit shifting and exposed the embeddedness of sand extraction in spaces beyond the limits of the pit. For example, if machines were out of fuel, young men would bring supplies from small petrol stations nearby, where fuel was stored in yellow containers and distributed to vessels via a hand pump. At the city’s limits, formal fuel supply was limited and thus small- scale informal petrol stations were important institutions in the sand economy. The fuel was carried in what had long been politicized as ‘Kufuor gallons’. These ‘gallons’, which would have once carried Frytol oil, became synonymous with John Kufuor’s ruling years (2001–09), which were marked by a depleted water supply in Accra. During this time, they functioned as containers for collecting, transporting and storing water sourced from various supply points in the city. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Potholes The transportation of sand from the pit to the city had to respond to the shifting location of the sandpit. Once sand from a relatively small pit has been exhausted, the contractors move to a new plot of land to unearth the sand there. The tippers reacted to the shifting geography of the sandpit, following the flags that were regu- larly repositioned. Thus, while tippers generally moved in similar directions, each day, as they both collected and delivered sand, their route was subject to change. In this way, a ‘trip of sand’ was an unfolding field of interactions that was repeatedly carved out in different ways across the Greater Accra region. The more trips I took with the tipper drivers, the more this unfolding field of interactions emerged to me as a significant site for the extraction of incomes, beyond the landowner, contractor, tipper driver, consumer and even the pit itself. These relations, I suggest, are integral to the way in which sand became part of the life of the city and an alternative to its implication in the production of the hard concrete city. y During one of my first trips of sand with Francis and Kwaku, we drove through the town of Ashalaja and the north-west of Greater Accra towards the boundary that separated the eastern part of the Central region and the southern part of the Eastern region. With little space to swerve around its edges, a deep depression drew the truck into it. As we rose, the truck rattled and we regained our balance. Francis said that Howo trucks – the type of truck we were in – were good for the roads out here since they have excellent suspension, giving them the capacity to dip and dive across the uneven roads. He explained that these trucks, imported from China, had begun appearing on the roads around seven years earlier. Prior to this, the most widely used trucks were European or American; these were expensive, and, while their engines lasted a long time, their springs weren’t as good – they were vulnerable to snapping due to the frequent stress. The Howo trucks would most likely suffer engine failure within four or five years, he suggested, but within this timeframe, they required little maintenance and could get the job done. Labours of sand In one of their newest lives, the containers had become an artistic medium in the work of Serge Clottey, where they morphed into high-end sculptures that hung from the walls of the new Kempinski hotel in downtown Accra, among other spaces in the city. Now, as I watched the yellow vessels transport fuel to bulldozers, they seemed to embody yet another life in these sandy landscapes. They were the vessels that kept things in motion, kept the sandpits shifting and kept the city supplied with sand. Meanwhile, men occupied the very edges of the sandpit, collecting sand with a shovel and loading it into small carts attached to a moped. This, I was told by someone at Piam, was ‘allowed’. They were probably living close by and building something for themselves. Indeed, an employee explained that, as long as they did not bring large trucks, it was generally allowed, proclaiming that it was Piam’s ‘social responsibility’. This kind of social responsibility was also enabled by the very materiality of the pits and sand’s liquidity once it came to the surface. Indeed, once the topsoil was removed, sand was a material that was accessible to all those who came to the pit, requiring only a spade and a cart to be collected. Whether through the perceived difficulty, a Africa Africa 53 lack of desire, or indeed a sense of citizenship, little effort was made to police the boundaries of the pit – in this instance, its edges were open and available to all those who came to make a collection. Thus, the sandpit was more than just a space; it was a material event that leaked into its surroundings, and into which its surroundings leaked in turn. The unearthing of sand was a moment when the material properties of its extraction – whether the shifting nature of the sandpit or the susceptibility of sand to being collected by nearby residents – presented opportunities where value could be extracted from the other- wise exclusive transactions of sand. This process of extraction was also apparent once sand left the pit and moved towards the city on tipper trucks. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Potholes The potholed roads he was describing were those at the edges of Accra; funds may be less likely to be directed there and timeframes for repair and general maintenance were understood to be longer. Moreover, it was widely recognized that sand trucks were in part to blame for the poor condition of the roads, burdening the concrete with tonnes of sand, year after year. While limited in their long-term performance, the ability of Howo trucks to respond to a network of potholed roads – a situation in which they themselves might be implicated – was considered paramount. In this way, the material landscape in Katherine Dawson 54 which the sandpits were located was marked by the uneven roads of peri-urban and rural Accra, and this in turn shaped the kind of technology required to keep trucks mobile and the sand shifting. Potholes were significant in shaping engagements as sand moved from pit to city. We regularly passed people moving sand and gravel to fill both deep and shallow depressions in the road. This, Francis explained, was a regular practice, performed by those looking to make two or three cedis from passing truck drivers who were sometimes grateful for their service. ‘Sometimes we dash [give] them small,’ he said. ‘But not always.’ This practice was visible on roads close to sites of sand consumption and also on stretches of road at the rural edges of the region. Men, women and chil- dren evening out the road with sand thus became a familiar sight as we moved from pit to city, city to pit. There was no guarantee that they would receive an income for their service; it therefore represented a speculative form of work that sought to extract potential value from the movement of sand. At times frustrated by this uncer- tainty, young men might occupy the roads and be more demanding, explicitly requesting money from drivers. On one occasion, a small group of young men, clearly frustrated by the ability of drivers to navigate around the potholes and pass without payment, had moved a large branch from a tree and placed it across the road, preventing any vehicle from passing without stopping and engaging in some form of (monetary) exchange. While unspoken, the threat of a potential confrontation was one that drivers recognized and responded to. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press Potholes g Much like De Boeck and Baloji’s (2016) interpretation of the productivity of potholes in Kinshasa, the shifting of sand in, around and through the pothole produced negotiated spaces of exchange. In their analysis, De Boeck and Baloji describe the ways in which potholes worked to slow people down, redirecting pedes- trian flow and traffic, offering new opportunities for commerce, while also presenting an occasion for ‘refilling’ services. They write that it is these ‘vulnerable infrastruc- tures [that] impose their own spatial and temporal logic on the city. They close off many possibilities, but they also create new social infrastructures, alternative spheres of social interaction, and different coping strategies’ (ibid.: 11). Situating their analysis within a conversation surrounding infrastructure more broadly, they write that these ‘syncopated rhythms : : : also generate new possibilities and opportunities, as well as different kinds of spaces’ (ibid.: 108). This urban ‘syncopation’ is read as presenting both limits and opportunities, yet it is significant in an understanding of how the city is lived. Indeed, in this reading of a politics of syncopation, they argue that ‘small scale modes of action that punctuate such urban living provide residents with an urban politics of the possible. Often these unsteady, provisional and continually shifting possibilities and action schemes are all that is available to urban dwellers; it is, there- fore, impossible to underestimate their importance’ (ibid.: 108). In the material lives of shifting sand, perhaps the most extreme form of syncopa- tion centred on a collapsed bridge near Ashalaja – a growing town on the western periphery of the Greater Accra region. The story of this syncopation unfolded on a trip of sand. Following an uneasy journey over rough roads, we passed onto smooth terrain; in front of us a steel bridge lay stretched out ahead. Francis pointed to a dislo- cated steel structure that hung precariously over the Densu River, running parallel to us. It was the bridge that had formerly served as the passage from Ayikai Doblo to Ashalaja, but it had collapsed in a fatal accident in 2015. Gradually tested by the Africa Africa 55 weight of trucks loaded with sand, the structure had succumbed to a tipper truck, sinking quickly into the water, taking with it the life of the driver. This new bridge had been open for only a few months. Potholes In later conversations, Francis recalled the temporary bridge that was constructed during the time when the new bridge was being built. I was told that a man had seized the opportunity to construct a makeshift bridge by lining up four concrete tunnels across the river, allowing the water to flow through them when the river was high. He used sand to bring the top of the tunnels level with the road either side of the river, allowing one lorry to pass at a time. When the sand was worn or washed away, Francis continued, they brought more sand to level it out. Francis explained: You would pay twenty cedis when you come back from the bush with your sand. When you go four times, it was eighty cedis. There are plenty of tipper trucks, so the guy was making a lot of money. You can’t say you won’t pay because big men are standing there. If you say no, they will throw a stone at the glass. I asked how long it was like that for, and he replied: ‘It was that way for six months. It made it more expensive, and you made less money.’ Indeed, as Francis lamented, this extra cost could mean that, in a week, you might be required to make several extra trips to balance this new expense in the equation of a sand trip. In this narra- tive, the shifting of sand made, unmade and remade the city in its image. In doing so, it gave rise to moments when the transactions embedded in sand could be rewritten, even if only temporarily. I asked how long it was like that for, and he replied: ‘It was that way for six months. It made it more expensive, and you made less money.’ Indeed, as Francis lamented, this extra cost could mean that, in a week, you might be required to make several extra trips to balance this new expense in the equation of a sand trip. In this narra- tive, the shifting of sand made, unmade and remade the city in its image. In doing so, it gave rise to moments when the transactions embedded in sand could be rewritten, even if only temporarily. Tracing the empirical details of shifting sand as it moved from pit to city thus exposes the livelihood practices that extend beyond the sandpit. Potholes These details also reveal the ways in which livelihood practices become possible through the multiple materialities of sand and their differing temporalities. As a substance that can break down existing infrastructure, provide temporary repairs, and then be blown away again, sand was never a constant; rather, it was a material in suspension that offered up moments for potential engagements, exchanges and, essentially, income. Indeed, transforming the socio-material landscape of the region as it moved, generating and engaging with a series of syncopations, sand in motion presented ongoing moments when value could be extracted, the terms of exchange could be remade, and new possibilities could come to the fore. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A share in the sands As the world continues apace with urbanization, it seems important to ask what kinds of livelihoods will be made possible for the ‘urban majority’ (Simone 2018). As agri- cultural land is swallowed up and new real estate crystallizes unequal power relations (Simone and Pieterse 2017), what forms of living and methods of livelihood can – and, indeed, should – be sustained? This article has deployed sand as a potential way in which to examine contemporary livelihoods and living in Accra – an expanding metropolitan region in West Africa. Situated in the space between a hard city of concrete and a fluid city of negotiation, sand has been presented as a material entry Katherine Dawson 56 point into the ways in which urban livelihoods are elaborated. Moving from trips to pits to potholes, the article has attended to the materialities of sand and its extrac- tion, looking at the substantial behaviours and temporal junctures of sand as its shifts its shape, form and direction. In doing so, it brings to the fore the ways in which sand emerges as a platform for exchange, negotiation and ultimately income, making it possible to see sand as a social infrastructure as much as an infrastructure of the concrete city. Yet, I also suggest that it might be possible to think of sand as a political infrastructure. The many labours embedded in the expanded material economies of sand speak to the difficulties of securing work in Accra and its peripheries. Indeed, this economy is a testament to the sheer energy, improvisation and ingenuity required to make a living in an economy where, in the words of an interlocutor, the ‘market is not easy’ – nor is it fair. In this way, the broader extractive economy of sand exposed yet another perspective on a difficult urban life, in which inhabitants are made to think deeply and relentlessly about potential values hidden in the shifting sands of the city. However, beyond improvisation, perhaps the most revealing facets of this extractive economy were the practices that were not so much determined by the creation of a service but rather were something closer to a demand. Indeed, watching young men simply take sand from the sandpit or demand money from a tipper on a potholed road hinted at a different kind of practice: a distributive demand directed to the values flowing from sand, from which they were otherwise excluded. https://doi.org/10.1017/S0001972023000116 Published online by Cambridge University Press A share in the sands I do not suggest that these actions should be romanticized as a form of resistance to extractive economies, and I recognize the need to consider how these claims might be better directed towards truck owners and landowning groups rather than drivers or mates on the road. Yet, I think it might be possible to discern a set of questions emerging from such demands. If cities continue to expand with limited opportunities for reasonable working lives, perhaps the city needs to be rethought as a place where value can be claimed, distributions called for and incomes demanded. Thus, if a right to space and housing has been widely politicized, could a right to an urban income be too? What would a de-linking of labour and income in the city actually look like? And how could this extend beyond the city itself? What would a demand not just for a job but for a flow of resources look like? What kinds of claims could be made, and on which flows? And what would these necessaraily multi-scalar (global) geographies look like? y (g ) g g p ? In an analysis of new welfare states in Southern Africa, Ferguson locates an emer- gent politics in the ideas and practices of cash transfers and universal basic income. Here, ‘such a politics is based on a kind of claim-making that involves neither a compensation for work nor an appeal for “help” but rather a sense of a rightful enti- tlement to an income that is tied neither to labor nor to any sort of disability or capacity’ (Ferguson 2015: 183). In this reading, the vast mineral wealth that under- girds the economies of Southern Africa are reconceptualized as awaiting distribution among the region’s inhabitants. Ferguson argues that, instead of positioning this distribution as a kind of market exchange or gift, this allocation of wealth points to ‘something more like demand sharing – a righteous claim for a due and proper share grounded in nothing more than membership (in a national collectivity) or even simply presence’. It is ‘this (emergent, only partially realized) politics’ that he locates as ‘the politics of the rightful share’ (ibid.: 184). A share in the sands While Accra’s mineral wealth is on an entirely different scale and degree, the city’s sand economy could point to a set of Africa 57 distributive claims: a rightful share to the unfolding wealth of a city from which many find themselves excluded. Beyond performing a service – such as preparing food or filling potholes – taking sand from the edges of the pit and simply demanding payments from passing trucks mark a different kind of claim on the values flowing from sand. Is this a rightful share, or is it something else? Does a rightful share map onto this space – onto a city in a country with a history that differs from those of the states of Southern Africa? Perhaps not. In this uncertain vein, far from providing a solution to historically produced condi- tions of poverty and inequality, I piece together a share in the sands as a tentative holding space for the many claims made on sand, arguing that they might be read as an emergent politics of claiming a share of the values flowing unevenly from the extraction of this grainy material. Tuning in to the sandy geographies of Accra has offered just one means of thinking through the way in which urban major- ities mark out a share in the uncertain ground of the city, yet it also hints at a broader point about the political potential embedded in urban materialities. I end with a call to stretch beyond sand, to consider the ways in which the materiality of the city more broadly emerges as a contingent platform upon which disparate claims to an income, a life and an urban future might be made. In this vein, a share in the sands becomes both a rallying call and a conceptual space for drawing together the always already existing political possibilities embedded in material city-making. Acknowledgements. I am incredibly grateful to those in Accra who generously shared their knowledge and time with me. In particular, my thanks go to Christabel Bannerman. Thank you to the reviewers, who pushed the article forward in important directions. 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https://openalex.org/W4362490998	https://figshare.com/articles/journal_contribution/Supplementary_Table_S5_from_Gene_Expression_Changes_in_an_Animal_Melanoma_Model_Correlate_with_Aggressiveness_of_Human_Melanoma_Metastases/22520739/1/files/39983379.pdf	English	null	Supplementary Table S2 from Gene Expression Changes in an Animal Melanoma Model Correlate with Aggressiveness of Human Melanoma Metastases	null	2,023	cc-by	2,635	s s 713 Yes 09 s related 606 Yes s ved s s 713 Yes 09 s related 606 Yes s ved 0066 Yes es es 713 Yes 009 es related 606 Yes es ived _TITLE RANK IN GENE LIST RANK METRIC SCORE RUNNING ES CORE ENRICHMENT b-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) 153 3.205331326 0.020510066 Y trophin, beta 2 (dystrophin-associated protein A1, 59kDa, basic component 2)" 263 Yes ocadherin 9 328 2.898431063 0.063289575 Yes th arrest-specific 7 510 2.676970482 0.07801228 Yes "ST3 beta-galactoside alpha-2,3-sialyltransferase 6" 560 2.627544403 0.09828084 Yes th arrest-specific 7 584 2.610097885 0.119575776 Yes glyceride lipase /// monoglyceride lipase 585 2.609040022 0.14190169 Yes eine and glycine-rich protein 1 657 2.534763098 0.1603815 Yes r-alpha (globulin) inhibitor H5 727 2.489469767 0.17856416 Yes th arrest-specific 7 897 2.374185801 0.19123852 Yes 9 "cadherin 19, type 2" 1038 2.290059805 0.20450433 Yes 1A "bone morphogenetic protein receptor, type IA" 1623 2.016437054 0.19535182 Yes C "brain and acute leukemia, cytoplasmic" 1681 1.992638707 0.20982566 Yes L2 "elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2" 833152 Yes AL6 "ST3 beta-galactoside alpha-2,3-sialyltransferase 6" 1864 1.927804232 0.23464713 Y apolipoprotein D 1908 1.912388921 0.2490673 Yes NULL 1918 1.909553885 0.26500067 Yes 83824 hypothetical protein LOC283824 1984 1.886804819 0.27820712 Yes B glycoprotein M6B 2001 1.879844189 0.2935697 Yes 7 G protein-coupled receptor 37 (endothelin receptor type B-like) 2007 1.877470493 "syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)" 2009 Yes 1 mitochondrial tumor suppressor 1 2119 1.843311548 0.33626252 Yes growth arrest-specific 7 /// growth arrest-specific 7 2236 1.804213524 0.3464561 Yes NF3 C1q and tumor necrosis factor related protein 3 /// C1q and tumor necrosis factor relat 0.35837108 Yes GRB2-associated binding protein 2 2331 1.775336146 0.3727037 Yes deleted in liver cancer 1 2518 1.71906662 0.3790034 Yes 2L1 tumor protein D52-like 1 2972 1.588901043 0.37211603 Yes 2 thrombospondin 2 3076 1.561524987 0.38082072 Yes 1 mitochondrial tumor suppressor 1 3225 1.526270151 0.38718894 Yes 1A ankyrin repeat and sterile alpha motif domain containing 1A 3231 1.524038911 T1 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 3395 1.490264416 0.40538606 Y 2L1 tumor protein D52-like 1 3422 1.48570013 0.41692373 Yes lipoprotein lipase 3437 1.481234312 0.4289658 Yes B glycoprotein M6B 3560 1.452906489 0.43588188 Yes 6 "ATP-binding cassette, sub-family A (ABC1), member 6" 3715 1.418496609 0.44105655 Yes "gelsolin (amyloidosis, Finnish type)" 3779 1.404004931 0.45022207 Yes B glycoprotein M6B 4092 1.339407325 0.44757548 Yes INF1 "serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived 1.301893115 0.45089322 Yes B glycoprotein M6B 4310 1.294229865 0.46002373 Yes lipoprotein lipase 4336 1.288612962 0.46992013 Yes frizzled homolog 7 (Drosophila) 4550 1.248874784 0.47097543 Yes "carboxypeptidase N, polypeptide 1, 50kD" 4589 1.242635131 0.47989053 Yes 4 2 ed, 042 tum 5 184 7 igm 798 2.62 90169 Yes Yes 2.01 82566 UR4/E 1864 20712 ciate Yes 1.80 nd t Yes Yes Yes 1A 3395 Yes 1.41 22207 , pi Yes 0.47 2566 R4/E 1864 0712 iate Yes 1.80 nd t Yes Yes Yes 1A 3395 Yes 1.41 2207 pi Yes 0.47 419 Y 6 Y 3 098 SUR 782 ke) soc 2 Y 6 an Y 3 Y 4 Y g 1 3 Y 5 502 in, 3 Y 31 23 2098 SUR 2782 ike) ssoc 52 Y 36 q an 7 Y s 03 Y 94 Y ng 1 73 Y 15 4502 min, 43 Y 131 17 es es 870 1/E se 481 pe fac 33 // 37 34 37 38 ont ase 41 es 493 nti es 47 24 1228 ase 7577 .60 6309 6976 3852 0433 e IA .99 ds ( nsf es .88 es ptor ell 1154 cifi tei 3614 .37 0104 es 7015 mai ans 0013 658 es mber .40 es ha- es 2013 7478 589 .99 s ( nsf es .88 es tor ll 154 ifi tei 614 .37 104 es 015 mai ans 013 58 es ber .40 es ha- es 013 478 589 2 347 94 912 045 typ 1 cid tra Y 1 Y ecep ce 33 spec pro 53 2 0 89 2 Y 62 do otr 57 89 8 Y mem 9 1 8 Y alp 3 Y 699 88 4 tran 85 0. s s 713 Yes 09 s related 606 Yes s ved e 58 2. 2. 01 0. 05 0. ptor, " 16 atty sialy 9067 19 3569 lin can 1. rest lated 1. 9066 1. 0820 1. moti O-sul 1. 12 0. 5881 BC1) 37 7575 de F 0023 62 0. 1. kD" ase 7 7 80 80 cep ic" fa 3-s 249 s 293 hel lyc 19 arr rel 31 719 72 380 25 ha 3-O 22 31 435 (AB )" 447 lad 460 96 50 50k 3-s 24 s 29 hel lyc 19 arr rel 31 71 72 38 25 ha 3-O 22 431 43 (AB )" 44 lad 46 296 50 50k 2,3 0.2 Ye 24 0.2 oth ogl 21 h a r r 233 1. 29 0.3 322 lph 3 342 34 0. A pe) 0. cl 0. s s 713 Yes 09 s related 606 Yes s ved 12 455 , 5 in 1 r H5 2 8 2 prot cy long e al 123 650 LOC 798 r 37 ate esso /// is f rote 2 ke 1 6152 esso teri cosa ke 1 7 1 529 b-fa inni 394 hibi 9422 6 1 phil pept 8 2 pro cy lon e a 123 650 LO 798 37 te sso // is ote 2 ke 615 sso ter cos ke 7 1 529 -fa nni 394 hib 942 6 1 hil ept -li 1.5 pre d s glu -li 343 1.4 sub Fi 1.3 in Yes 1.2 433 sop lyp 103 ic mia ry sid 1.9 0.2 ein 1.8 tor lfa pre 7 / ros pr 1 -li 1.5 pre d s glu -li 343 1.4 sub Fi 1.3 in Yes 1.2 433 sop lyp 08 885 pro 01 ece n s su fic ne din nce D5 76 su t a te D5 60 te, sis 92 das 22 10 (Dr , p act 08 88 pro 01 ece n s su fic ne din nce D5 76 su a e D5 60 te, sis 92 das 22 10 (Dr p D 1 955 cal B 2 ed par umo pec umo bi r c tein 2 3 umo epe ulf tein ase B 3 sse oid B 4 ept 089 B 4 ase g 7 se ype pho e l on -ga D 1 095 cal B 2 ed par umo pec umo bi r c tei 2 3 umo epe ulf tei ase B 3 sse oid B 4 ept 508 B 4 ase g 7 se rpho ute l ion a-ga n D 1 9095 ical M6B 2 pled hepar tumo -spec tumo s ed bi ver c otei n 2 3 tumo repe sulf otei ipase M6B 3 casse yloid M6B 4 pept 4508 M6B 4 ipase log 7 dase hep tu -sp tum s ed ver ot n 2 tu re su ot ipa M6B cas ylo M6B pe 450 M6B ipa log das rotei 18 1 pothe tein n-cou n 2 drial rrest q and 08 Y ociat in li mor p pondi drial kyrin paran mor p ein l tein ding n (am tein erpin 115 0 tein ein l homo pepti globu -spec n 19 one m nd a longa s T3 be rote 18 1 pothe tein n-co n 2 dria rres q and 08 Y ocia in l mor p pond dria kyrin paran mor p ein tein ding n (am tein erpin 115 0 tein ein hom pept TITLE RAN -b2 eryth rophin, b Yes cadherin h arrest- "ST3 bet h arrest- lyceride ine and g -alpha (g h arrest- "cadheri A "bo "brain a 2 "el 33152 Yes L6 "ST apolipop NULL 191 3824 hyp glycoprot G protei "syndeca Yes mitochon growth a F3 C1q 0.358371 GRB2-ass deleted L1 tum thrombosp mitochon A ank 1 hep L1 tum lipoprot glycoprot "ATP-bin "gelsoli glycoprot NF1 "se 1.301893 glycoprot lipoprot frizzled "carboxyp TITLE -b2 ery rophin, Yes cadheri h arres "ST3 b h arres lycerid ine and -alpha h arres "cadhe A "brain 2 33152 L6 apolip NULL 3824 glycop G prot "synde Yes mitoch growth F3 0.3583 GRB2-a delete L1 thromb mitoch A 1 L1 lipopr glycop "ATP-b "gelso glycop NF1 1.3018 glycop lipopr frizzl "carbo arr "ST3 arr ycer ne a alph arr "cadh "brai 3152 6 apoli NULL 824 glyco G pro "synd Yes mitoc growt 3 0.358 GRB2- delet 1 throm mitoc 1 lipop glyco "ATP- "gels glyco F1 1.301 glyco lipop frizz "carb ne a alph arr cadh brai 3152 6 poli ULL 824 lyco pro synd es itoc rowt 3 .358 RB2- elet 1 hrom itoc 1 ipop lyco ATP- gels lyco F1 .301 lyco ipop rizz carb endothelin receptor type B 4656 1.230809331 0.48743832 Yes 1 "protein tyrosine phosphatase, receptor-type, Z polypeptide 1" 4823 1.197096586 BH-protocadherin (brain-heart) 5308 1.108523726 0.47777602 Yes 6 zinc finger protein 106 homolog (mouse) 5410 1.08943212 0.4825314 Yes frizzled homolog 7 (Drosophila) 5648 1.051532865 0.4808128 Yes desmuslin 5650 1.051147938 0.4897624 Yes "S100 calcium binding protein, beta (neural)" 5687 1.043883204 0.49706718 Yes A "Fc fragment of IgG, low affinity IIa, receptor (CD32)" 5699 1.042750478 0.50549275 Yes 7 transmembrane protein 47 5713 1.040534019 0.5138089 Yes A2 "solute carrier family 12 (sodium/potassium/chloride transporters), member 2" 5860 Yes dishevelled associated activator of morphogenesis 2 5963 0.993700147 0.51977843 Yes A3 "aldehyde dehydrogenase 1 family, member A3" 6129 0.967625737 0.5205975 Yes "proteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated)" 65745 Yes mitochondrial tumor suppressor 1 6281 0.941609859 0.53010786 Yes "syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)" 6309 Yes "ATP-binding cassette, sub-family A (ABC1), member 8" 7071 0.813984275 0.5094684 No endothelin receptor type B 7139 0.804488301 0.51332283 No ral guanine nucleotide dissociation stimulator-like 1 7535 0.742437303 0.5018148 No Kruppel-like factor 9 7914 0.684244037 0.49057752 No 1 hyaluronan and proteoglycan link protein 1 8364 0.614485502 0.47553277 No 19 chromosome 4 open reading frame 19 8690 0.568432331 0.465701 No "endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2" 8828 No "gelsolin (amyloidosis, Finnish type)" 9088 0.511487246 0.45688778 No 2 metallophosphoesterase domain containing 2 9183 0.497603148 0.45689532 No 1 ly A) 9200 0.495482981 0.46041176 No 1 hyaluronan and proteoglycan link protein 1 9670 0.42460528 0.44283783 No 12 collectin sub-family member 12 /// collectin sub-family member 12 9789 0.406908274 F19 chromosome 20 open reading frame 19 11106 0.214803383 0.38331503 No B "ropporin, rhophilin associated protein 1B" 11157 0.207264721 0.38282773 No decorin 11236 0.197755247 0.38099292 No microphthalmia-associated transcription factor 11304 0.189152434 0.3795819 No alpha-2-macroglobulin 11382 0.17748335 0.37761885 No decorin 11461 0.166248903 0.37551445 No regulator of G-protein signalling 5 11563 0.152520835 0.37225255 No aryl-hydrocarbon receptor nuclear translocator 2 12078 0.075370975 0.34965536 No "retinoid X receptor, gamma" 12271 0.050202079 0.34140307 No regulator of G-protein signalling 5 12372 0.033695679 0.3371696 No decorin 12449 0.023446416 0.33393365 No glycoprotein (transmembrane) nmb 12597 6.65E-04 0.32729226 No 6 syntaxin binding protein 6 (amisyn) 12821 -0.030485388 0.31746948 No nucleoredoxin 12958 -0.05097048 0.31175596 No regulator of G-protein signalling 5 14346 -0.253598481 0.25120842 No FXYD domain containing ion transport regulator 3 15295 -0.407211602 0.21182616 No "secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 5 Ye 0 )" 9 8 74 on Yes 8 "ATP-binding cassette, sub-family A (ABC1), member 8" 7071 0.813984275 0.5094684 No B endothelin receptor type B 7139 0.804488301 0.51332283 No ral guanine nucleotide dissociation stimulator-like 1 7535 0.742437303 0.5018148 No Kruppel-like factor 9 7914 0.684244037 0.49057752 No N1 hyaluronan and proteoglycan link protein 1 8364 0.614485502 0.47553277 No F19 chromosome 4 open reading frame 19 8690 0.568432331 0.465701 No "endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2" 8828 No "gelsolin (amyloidosis, Finnish type)" 9088 0.511487246 0.45688778 No D2 metallophosphoesterase domain containing 2 9183 0.497603148 0.45689532 No S1 ly A) 9200 0.495482981 0.46041176 No N1 hyaluronan and proteoglycan link protein 1 9670 0.42460528 0.44283783 No C12 collectin sub-family member 12 /// collectin sub-family member 12 9789 0.406908274 RF19 chromosome 20 open reading frame 19 11106 0.214803383 0.38331503 No 1B "ropporin, rhophilin associated protein 1B" 11157 0.207264721 0.38282773 No decorin 11236 0.197755247 0.38099292 No microphthalmia-associated transcription factor 11304 0.189152434 0.3795819 No alpha-2-macroglobulin 11382 0.17748335 0.37761885 No decorin 11461 0.166248903 0.37551445 No regulator of G-protein signalling 5 11563 0.152520835 0.37225255 No 2 aryl-hydrocarbon receptor nuclear translocator 2 12078 0.075370975 0.34965536 No "retinoid X receptor, gamma" 12271 0.050202079 0.34140307 No regulator of G-protein signalling 5 12372 0.033695679 0.3371696 No decorin 12449 0.023446416 0.33393365 No B glycoprotein (transmembrane) nmb 12597 6.65E-04 0.32729226 No P6 syntaxin binding protein 6 (amisyn) 12821 -0.030485388 0.31746948 No nucleoredoxin 12958 -0.05097048 0.31175596 No regulator of G-protein signalling 5 14346 -0.253598481 0.25120842 No 3 FXYD domain containing ion transport regulator 3 15295 -0.407211602 0.21182616 No "secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 58 cate 63 No No 88 6908 No ivat 8 Ye 42750 mem 19778 20597 2, brog 75 0. s s 713 Yes 09 s related 606 Yes s ved 03 0. 75532 cept 56895 42837 97 3 No 82827 79581 49655 17469 2 No 0. mpho es .4825314 es .49706718 699 1.04 es porters), 0147 0.51 5737 0.52 raplegia es ated, fib .81398427 .74243730 5502 0.47 1 No upled rec 778 No 3148 0.45 528 0.44 mber 12 .38331503 4721 0.38 2434 0.37 255 No 0975 0.34 96 No o 0.31 .25120842 11602 rly T-lym ) 56 89 Ye transp .99370 .96762 tic pa 786 Ye associ 071 0 o 535 0 .61448 .46570 ein-cou .45688 .49760 .42460 ly mem 3383 0 .20726 .18915 .37225 .07537 o .33716 226 No 85388 0 0.4072 I, ea 432 812 883 32) 808 e t 0. 0. sti 107 -as 70 No 75 0. 0. tei 0. 0. 0. mil 803 0. 0. 0. 0. No 0. s s 713 Yes 09 s related 606 Yes s ved 292 048 1 -0 n 08 48 04 (C 51 ori 63 29 sp 53 fac 283 64 233 -pr 724 83 70 b-f 21 15 30 083 07 307 567 32 .0 984 29 ote lik 752 1 56 aci 51 2 1 tin 110 B" 885 15 2 34 03 4 282 o 0.2 3 ial lyc (AB 80 sti 403 nk e 1 osp )" con 176 nk // me d p 292 ion 335 445 ans 05 365 259 isy 31 re tin n s te, ty ide 9 d p ope ent sis oes 495 d p -fa op oph 197 cia in 166 ein cep r, ein 023 mem ing 958 ein ing ote 6 z frizzle desmusl "S100 c A " 7 t A2 " Yes disheve A3 " "proteo 65745 Y mitocho "syndec Yes "ATP-bi endothe ral gua Kruppel 1 h 19 c "endoth No "gelsol 2 m 1 l 1 h 12 c F19 c B " decorin microph alpha-2 decorin regulat aryl-hy "retino regulat decorin glycopr 6 s nucleor regulat FXYD do "secret A A2 Yes dishe A3 "prot 5745 mitoc "synd Yes "ATP- endot ral g Krupp 9 "endo No "gels 2 19 B decor micro alpha decor regul aryl- "reti regul decor glyco nucle regul FXYD "secr dishe 3 prot 5745 mitoc synd Yes ATP- endot ral g Krupp 9 endo No gels 2 19 decor micro alpha decor regul aryl- reti regul decor glyco nucle regul FXYD secr 0.20056434 No dic acid G-protein-coupled 071 0.19527239 No 5369464 No n) 18287 -0.977017939 2282883 No man) 19014 -1.170336962 0.10661836 No n) 19810 -1.416904926 in) 19810 -1.416904926 2 d 2 3369 upl o 369 -c 9 939 170 926 No tein 27239 70179 -1.1 No 69049 .19 o 0.9 o 901 836 1.4 3 No 19 06618 10 -1 883 .10 981 c aci 1 69464 1828 82883 n) 0.10 1981 .20 aci 9464 828 2883 .10 981 828 n) 0.1 198 n) 0. 19
https://openalex.org/W2999502435	https://journals.ispan.edu.pl/index.php/cs-ec/article/download/cs.2007/5462	Kirghiz, Kyrgyz	null	Hybridity and the linguistic landscape	Cognitive Studies	2,019	cc-by	6,095	Abstract This article argues that a linguistic landscape can be considered a hybrid when many languages and scripts simultaneously work within it. Being heterogeneous, urban signage (shop signs, busi- ness signs, outdoor advertising etc.) is open to hybridization, particularly in moments of historical and geopolitical transformation and at the intersections of diﬀerent cultures. Analyzing the lin- guistic landscape of Kiev’s Podil district, conscious, unconscious, explicit, and implicit hybridity are identiﬁed and examined. Linguistic hybridity, as an element of cultural hybridity, is closely related to everyday practices associated with work, food, clothes, hygiene, health, leisure, etc. Organic/unconscious and intentional/conscious forms of hybridization occur in linguistic creati- vity. The article shows that three languages (Ukrainian, Russian, and English), and two scripts (Latin and Cyrillic), participate in the hybridization process, and examples are cited. During the Soviet period, Russian was the dominant language in Ukraine and Kiev. The Soviet authorities reinforced Russian and weakened Ukrainian. The consequences of this colonial policy can be ob- served today, and one can see these results in the Ukrainian-Russian hybrid city-text. Since the restoration of Ukrainian independence in 1991, Ukraine has transformed from a post-colonial state to a European state, and has become part of a globalized world which uses English as a lingua franca. The eﬀects of this transformation are visible in the linguistic landscape in the form of Ukrainian-Russian-English, Ukrainian-English, and Russian-English hybrid signs. Keywords: linguistic landscape; hybridity; Kiev Podil; city-text; languages Keywords: linguistic landscape; hybridity; Kiev Podil; city-text; languages Landscape is . . . neither nature nor culture, neither mind nor matter. It is the world as known to those who have dwelt in that place, those who currently dwell there, those who will dwell there, and those whose practical activities take them through its many sites and journey along its multiple paths. (Urry, 2007, p. 32) Landscape is . . . neither nature nor culture, neither mind nor matter. It is the world as known to those who have dwelt in that place, those who currently dwell there, those who will dwell there, and those whose practical activities take them through its many sites and journey along its multiple paths. (Urry, 2007, p. 32) ORYSIA DEMSKA National University of Kyiv-Mohyla Academy, Kiev, Ukraine ordemska@gmail.com https://orcid.org/0000-0002-8915-0456 COGNITIVE STUDIES \| ÉTUDES COGNITIVES, 19 Warsaw 2019 Article No.: 2007 DOI: 10.11649/cs.2007 Citation: Demska, O. (2019). Hybridity and the lin- guistic landscape. Cognitive Studies \| Études cognitives, 2019(19). https://doi.org/10.11649/cs.2007 Citation: Demska, O. (2019). Hybridity and the lin- guistic landscape. Cognitive Studies \| Études cognitives, 2019(19). https://doi.org/10.11649/cs.2007 1More precise explanation of lingual hybridity is in Bakhtin’s Russian text: “Мы называем гибридной конструк- цией такое высказывание, которое по своим грамматическим (синтаксическим) и композиционным признакам принадлежит одному говорящему, но в котором в действительности смешаны два высказывания, две речевые манеры, два стиля, два «языка», два смысловых и ценностных кругозора. Между этими высказываниями, стилями, языками, кругозорами, повторяем, нет никакой формальной – композиционной и синтаксической – границы; раздел голосов и языков проходит в пределах простого предложения, часто даже одно и то же слово принадлежит одновременно двум языкам, двум кругозорам, скрещивающимся в гибридной конструкции, и, следовательно, имеет два разноречивых смысла, два акцента...” (Bakhtin, 1975, p. 118). 1 Introduction 1.1 A cityscape usually forms part of the past, present, and future. A city’s architecture and its language, or languages, are the best demonstrators of this mix of past-present-future. Humans do not only know and speak a language, but they also live in a verbalized space, especially in – 2/10 – Orysia Demska Hybridity and the linguistic landscape the contemporary city. Every time a person leaves their home they step into the linguistic world of public signs, advertisements, billboards etc. The linguistic picture painted by of all these signs depends on the time and place; on the history of the region, state and city; on cultural and language policies; on the type (oﬃcial, commercial, private); and on the values, education, native language and bi-/multilingualism (or lack of) of the author. These parameters have an inﬂuence on the content of signage and determine its heterogeneity. Being heterogeneous, urban signs are open to hybridization, particularly in moments of historical and geopolitical transformation, and at the intersections of diﬀerent cultures. The ﬁrst aim of this article is to show that the postcolonial and globalized linguistic landscape is a hybrid landscape. For this purpose, an analysis was conducted of the contemporary linguistic landscape of Kiev’s Podil district. The second aim of this article is to prove that the linguistic landscape hybridity of the Podil district is the consequence of mixing Ukraine’s colonial past, its present independence, and its ongoing Europeanization/westernization as part of the wider process of globalization. 1.2 In contemporary Ukrainian society, the words hybrid / hybridity / hybridization, popularized by the military conﬂict on the Ukrainian-Russian border, have a strong negative connotation. Meanwhile, the world on the border has always been, and still is, hybrid. Amar Acheraïou points out that ‘hybridity has been much discussed in postcolonial theory over the last three decades’ (Acheraïou, 2011, p. 5; see also Bhabha, 2004; Burke, 2009, 2012; Joseph, 1995, 1999; Kraidy, 2005; Spivak, 1999; Young, 1995). However, the idea “that cultures are not pure but mixed is not a new one. It was the Belgian classicist Franz Cumont who launched the idea of syncretism in his book Les religions orientales dans le paganisme romain” (Burke, 2012, p. 4) in 1906 (for more detail see Acheraïou, 2011; Burke, 2012). The contemporary notion of hybridity as a ‘historical fact and theoretical tool’ is rooted in 19th century colonial discourse. 1 Introduction At the end of the 20th century Homi Bhabha rethought and adapted this term to the ﬁeld of post-colonial studies: Bhabha rethought and adapted this term to the ﬁeld of post colonial studies: “Bhabha adopted the term ‘hybridity’ and divested it of its colonial connotations of on- tological and racial degeneration. With its adoption by Bhabha and, more generally, by postcolonial scholars, the concept of hybridity has seen its semantics rehabilitated and wi- dely inﬂected to stand for inclusiveness, dialogism, subversion, and contestation of grand narratives.” (Acheraïou, 2011, p. 5) “Bhabha adopted the term ‘hybridity’ and divested it of its colonial connotations of on- tological and racial degeneration. With its adoption by Bhabha and, more generally, by postcolonial scholars, the concept of hybridity has seen its semantics rehabilitated and wi- dely inﬂected to stand for inclusiveness, dialogism, subversion, and contestation of grand narratives.” (Acheraïou, 2011, p. 5) Language makes cultural hybridity visible while also being an element of it. This is why linguis- tic hybridity should be treated as part of cultural hybridity, rather than as a separate phenomenon. 1.3 The concept of Linguistic hybridity was introduced by Mikhail Bakhtin at the beginning of the 20th century. “What is hybridization? – Bakhtin asks and answers. – It is a mixture of two social langua- ges within the limits of a single utterance, an encounter, within the arena of an utterance, between two diﬀerent linguistic consciousnesses, separated from one another by an epoch, by social diﬀerentiation or by some other factor. (Bakhtin, 1981, p. 358)1 Bakhtin then explains the concept of a ‘linguistic hybrid’ as ‘it is obligatory for two linguistic consciousnesses to be present, [...], with each belonging to a diﬀerent system of language’ (Bakhtin, 1981, p. 359) and he distinguishes between unconscious and intentional/conscious hybrids. The former ‘is a mixture of two impersonal language consciousnesses’, and the latter is ‘a mixture – 3/10 – Orysia Demska Hybridity and the linguistic landscape of two individualized language consciousnesses as well as two individual language-intentions’ (see Bakhtin, 1975, 1981). of two individualized language consciousnesses as well as two individual language-intentions’ (see Bakhtin, 1975, 1981). 1.4 Unconscious and intentional/conscious linguistic hybridity can be implicit or explicit. Im- plicit linguistic hybridity is usually unconscious and exists in any natural human language. Such hybridity is not easy to identify or verify. It is mostly interpreted as a neutral phenomenon, as a fact, for instance. 2 Linguistic Landscape 2.1 The concept of linguistic landscape started its own multidisciplinary career at the moment when the smartphone, equipped with camera, became widely available to researchers. “Signs are part of the textual decor that surrounds us every day, as we walk, ride, or drive through urban environments.” (Gorter, 2013, p. 190). The language or languages of this ‘decor’ have become important not only for diﬀerent scholars (geographers, sociologists, linguists, historians etc.) but also for authorities, politicians, and businesses. Durk Gorter provides an excellent overview of the concept of ‘linguistic landscape’ which covers the scope, history, trends and researchers of modern linguistic landscape studies (Gorter, 2013; see also Garvin, 2010, pp. 252–253; Jaworski & Thurlow, 2010a, pp. 2–5; Pavlenko, 2009, pp. 248, 249), starting with Rodrigue Landry and Richard Bourhis’ well-known 1997 deﬁnition of this notion: Richard Bourhis well-known 1997 deﬁnition of this notion: “The language of public road signs, advertising billboards, street names, place names, commercial shop signs, and public signs on government buildings combines to form the linguistic landscape of a given territory, region, or urban agglomeration.” (Landry & Bour- his, 1997, p. 25) Such an understanding and deﬁnition have made the linguistic landscape a part of culture and a research tool. Such an understanding and deﬁnition have made the linguistic landscape a part of culture and a research tool. 2.2 According to Landry and Bourhis, the linguistic landscape ‘serves as a marker of the geo- graphical territory inhabited by a given language community’ and ‘delineate the territorial limits of the language group it harbors relative to other linguistic communities inhabiting adjoining ter- ritories’. As a part of culture, it has a symbolic function ‘where language has emerged as the most important dimension of ethnic identity’ (see Landry & Bourhis, 1997, pp. 25–27). Today, it is hard to ﬁnd ‘pure’ monocultural and monolingual geographical territories and communities. Territories have become more and more amorphous, borders more and more diaphanous, and communities more and more heterogeneous. ‘Pure’ linguistic landscapes are more and more diﬃcult to ﬁnd, es- pecially in regions where colonial past, independent present, and Western / European future meet. In this mixed space, the linguistic landscape uses many languages which identify many cultures. Several languages often coexist and work simultaneously in the same community. 1 Introduction The lexicon of any language is always hybrid, created by native and alien elements; furthermore, natural language is the product of this hybridization, the end point of this process. Explicit linguistic hybridity may be unconscious or conscious. Explicit linguistic hybri- dity is usually regarded as the starting point of the hybridization process. It aﬀects the recipient, provokes conﬂict and rejection, and creates a negative connotation of the sign. Visibility in the linguistic landscape is a key feature of such hybridity. 3 The Linguistic Landscape of Kiev’s Podil District 3.1 Aneta Pavlenko, in the article Language Conﬂict in Post-Soviet Linguistic Landscapes, twice mentions that ‘only a few studies have examined post-Soviet linguistic landscapes’ (Pavlenko, 2009, pp. 248, 254). This is true. There have only been a handful of works dealing with the Ukrainian linguistic landscape (see Bele˘ı, 2012; Bever, 2010; Matsiuk, 2017; Oli˘ınyk, 2013; and the most famous Pavlenko, 2009, 2010, 2012, 2017). Moreover, the notion of the linguistic landscape of the Podil district of Kiev as a hybrid space has never been taken into consideration. The linguistic landscape of Podil is worthy of investigation because it is one of the oldest parts of Kiev. The history of Podil dates back to the end of the 15th century (when Kiev was granted Magdeburg Rights), but some artefacts from this area date back to the 9th century or even older. Podil has been the city’s political, commercial, intellectual, and cultural centre for 400 years. During the Soviet period, the city centre shifted from Podil to Chreshchatyk and Sovietskaja / Kalinina / October Revolution square. Nowadays, the shift of the centre from Chreshchatyk and Maidan back once more to Podil and Soﬁjska square is underway. Eastern Europe’s oldest university, the Kyiv-Mohyla Academy, and other high schools, institutions, multinational corporation oﬃces, museums, global, regional and Ukrainian restaurant chains, hotels and hostels, boutiques etc. are now located in Podil. The district’s pedestrian streets and squares are the stage for both traditional and modern performances and events. Ukraine, Kiev, and Podil are fruitful objects for investigation ‘because, in the past two decades, post-Soviet symbolic landscapes have undergone drastic changes reﬂecting both nation-building eﬀorts and the transition to the new capitalist and global economies’ (Pavlenko, 2009, p. 253). 3.2 Podil’s linguistic landscape, as a ‘public use of written language’ (Pavlenko, 2010, p. 133), reveals its hybridity at both the micro and macro levels. The micro level encompasses single texts (the names of institutions, companies, stores, and restaurants, advertising and posters, etc.) which are interlinked with only one extra-lingual object. The macro level covers the whole of Podil’s tex- tual space. Microtexts usually mix two (Ukrainian and Russian/Ukrainian and English/Russian and English) or three (English/Ukrainian/Russian) languages, and two scripts (Latin and Cyril- lic) in one narrative. However, not all microtexts here are hybrid. Depending on the ‘information arrangement’ of the text, scholars identify “. . . 2“Similar observations are made by Backhaus (2007, p. 90) and Reh (2004, pp. 8–15) using diﬀerent terminology. Backhaus calls texts homophonic when they are complete translations of each other (i.e. the same message is conveyed in two or more diﬀerent codes); mixed, where there is a partial overlap of messages but the content conveyed is not identical in the diﬀerent codes, and polyphonic, where the messages are diﬀerent. Reh uses the terms duplicating (for complete translations), fragmentary (where translation is partial), overlapping and complementary” (Sebba, 2012, p. 36; see also Pavlenko, 2017). 2 Linguistic Landscape Under such ci- rcumstances one ‘impersonal language consciousness’ mixes with another ‘impersonal language consciousness’, or one ‘individualized language consciousness’ mixes with another ‘individualized language consciousness’, or these two impersonal and individualized consciousnesses mix mutu- ally. As a result, one can observe examples of unconscious and conscious linguistic hybridity in the city-text of the Podil district of Kiev, where more than two languages are present in one sign. Three languages are mainly used: the language of the former metropole (Russian), the oﬃcial language (Ukrainian), and the lingua franca (English). – 4/10 – Orysia Demska Hybridity and the linguistic landscape 3 The Linguistic Landscape of Kiev’s Podil District (i) duplicating, (ii) fragmentary, (iii) overlapping, and (iv) complementary, where diﬀerent types of information are provided in each language, trans- mitting somewhat diﬀerent messages to diﬀerent audiences” (Reh, 2004, pp. 8–15); or “Equivalent texts are those that have similar content in two or more languages [. . . ]. Disjoint texts have diﬀerent content [. . . ]. It is also possible to have overlapping language content, a mixed type in which some of the content is repeated in the other language” (Sebba, 2012, p. 36).2 Overlapping and complementary texts are deﬁnitely hybrid. As a general rule, such features are characteris- tic of commercial signs and some private signs in Podil. Oﬃcial signs, with the oﬃcial language (Ukrainian) duplicated into English, and private announcements on city notice boards, mainly in Russian, are usually non-hybrid and lie beyond the scope of this article. 3.3 As mentioned above, Ukrainian, Russian, and English, along with both the Latin and Cy- rillic scripts, are involved in the hybridization process of Podil’s commercial linguistic landscape. For instance, there are signs in which Ukrainian-Russian-English are blended: ЖЕЛТОК / Київ-дайнер Жовток / Київ 2012 / DINER CAFE; Cалон краси / Tamriko / Вiдчинено без вихiдних / L‘oreal Proﬀessionnel / Парикмахерские услуги. . . ; Premium coﬀee / Если кофе – 3.3 As mentioned above, Ukrainian, Russian, and English, along with both the Latin and Cy- rillic scripts, are involved in the hybridization process of Podil’s commercial linguistic landscape. For instance, there are signs in which Ukrainian-Russian-English are blended: ЖЕЛТОК / Київ-дайнер Жовток / Київ 2012 / DINER CAFE; Cалон краси / Tamriko / Вiдчинено без вихiдних / L‘oreal Proﬀessionnel / Парикмахерские услуги. . . ; Premium coﬀee / Если кофе – 3.3 As mentioned above, Ukrainian, Russian, and English, along with both the Latin and Cy- rillic scripts, are involved in the hybridization process of Podil’s commercial linguistic landscape. For instance, there are signs in which Ukrainian-Russian-English are blended: ЖЕЛТОК / Київ-дайнер Жовток / Київ 2012 / DINER CAFE; Cалон краси / Tamriko / Вiдчинено без вихiдних / L‘oreal Proﬀessionnel / Парикмахерские услуги. . . ; Premium coﬀee / Если кофе – 3.3 As mentioned above, Ukrainian, Russian, and English, along with both the Latin and Cy- rillic scripts, are involved in the hybridization process of Podil’s commercial linguistic landscape. 3 The Linguistic Landscape of Kiev’s Podil District For instance, there are signs in which Ukrainian-Russian-English are blended: ЖЕЛТОК / Київ-дайнер Жовток / Київ 2012 / DINER CAFE; Cалон краси / Tamriko / Вiдчинено без вихiдних / L‘oreal Proﬀessionnel / Парикмахерские услуги. . . ; Premium coﬀee / Если кофе – – 5/10 – Orysia Demska Hybridity and the linguistic landscape Orysia Demska y Hybridity and the linguistic landscape то только премиум / Їж пий насолоджуйся / Холодные коктейли / Термiнал тимчасово не працює / Чаевые на Одессу; Ноябрь / Ресторан подiльської кухнi / авторський проект Iллi Ноябрьова / Menu / Смачно та атмосферно... / Welcome to the Noyabr; Руккола / итальян- ское кафе для друзей / щасливi години Tasty! Examples of Ukrainian-Russian blending are: Мир Пола и Декора / ПАРКЕТ / ШПАЛЕРИ / ШТОРИ ; Горячая выпечка / Гаряча випiч- ка / Завiтайте до нас; ЗРУЧНО ТА ВИГIДНО / МАМА ДАРАГАЯ / ТАК! МИ ДОРОГО ОЦIНЮЄМО ЗОЛОТО I ТЕХНIКУ / НОВА ПРОГРАМА / Реальна цiна; Ремонт взут- тя, шкiргалантереї / Виготовлення ключiв, металокерамiки, автодзеркал / Пошив штор / Фотокерамика / Ремонт одежды любой сложности / Замена молний / Подгонка одежды по фигуре; Будинок №35 належить ЖЕК-802 / Товарищи жильцы! Надстройка балконов запрещается; The mixing of Ukrainian-English occurs: Клiринговий дiм / Цiнуємо бiльше / Privat Banking; LONDON / кавовий дiм; #BLINSTORY . . . твоя iсторiя смаку; STAR BURGER / БАР ОРИГIНАЛ БУРГЕР / я твiй бургер назважди; PROcosmetics / професiй- на косметика та аксесуари / Косметика; Golden Company / Innovation System / Професiйна косметика; Optica.ua: Eyewear and sunglasses / Контактна корекцiя / Комп’ютерна дiагно- стика / Виготовлення та ремонт окулярiв; and Russian-English hybrid microtexts can also be found: DECORATION CLUB / ОБОИ, ТКАНИ, МЕБЕЛЬ, КОВРЫ, СВЕТ; Beauty Ave- nue / шоу-рум / косметология / Мы открыты для Вас / Без выходных; Enjoy smoke / vape shop: Замените вредный дым вкусным паром; ЭйнШтейн coﬀee / All you need is love / a good cup of COFFEE; Coﬀee dream / Mocco / Frappe / Хороший день начните с чашечки хорошего кофе. Typical practices of hybridization include combining (i) the common name in Ukrainian and the proper name in English, such as PENCILVANIA Мережа канцелярських магазинiв; ROSHEN Фiрмовий магазин; The FLEXX Iталiйське взуття; (ii) the proper name in English or Russian and additional information in Ukrainian, e.g. Iмперрiя Хутра / Империя Меха / Пн-Вт-Ср- Чт-Пн-Сб-Нд (недiля instead of воскресенье); Николай / Пироговая / Бар-буфет / Очень вкусные пироги! 3 The Linguistic Landscape of Kiev’s Podil District / Щоденно 10-23. Ukrainian-English and, to a lesser degree, Russian-English hybrids belong to the commer- cial signage of mid-market and upmarket businesses, oriented towards the wealthy middle class and foreigners; Russian-Ukrainian hybrids are typical for the commercial signage of downmarket businesses, oriented to a poorer clientele. 3.4 Ukrainian language legislation still is in progress. the Constitution of Ukraine, the Declara- tion of the Nationality of Ukraine, the “Principles of the State Language Policy” Bill (which has now been cancelled, leading to the return of the Soviet-era “Languages in the Ukrainian SSR” law), the “Languages in Ukraine” Bill, the “Prohibition of Narrowing the Spheres of the Use of Regional Languages and the Languages of National Minorities of Ukraine” Bill, and the European Charter for Regional or Minority Languages (ECRML) are the legislative documents which regulate lan- guage usage in Ukraine. All these documents determine diﬀerent aspects of language usage in the oﬃcial and public spheres. According these documents, Ukrainian, as the oﬃcial state language has prerogative. Russian is classiﬁed as one among other minority languages. Consequently, the use of Ukrainian and the Cyrillic script in the commercial sector, being a public space, is obliga- tory. Nevertheless, the examples from the Podil district show the subversion of the oﬃcial norms of language choice. The ‘unique characteristic of today’s Kiev is [with] the discontinuity between the language of the cityscape (predominantly Ukrainian) and the language of everyday interaction (predominantly Russian)’ (Pavlenko, 2010, p. 133). Russian, as the main language of the former colony, still circulates in Ukraine, and for many citizens it is their native or ﬁrst language and is used in the public space. Due to the Russian-Ukrainian military conﬂict, the shift from Russian to Ukrainian, the upgrading of Ukrainian and the downgrading of Russian, the creation of a new Ukrainian identity, and the rethinking of history (especially that of the Soviet era), the Russian language is increasingly interpreted as a marker of the colonial past. In place of Russian, English has been aggressively conquering space in Ukrainian cities and villages, displacing not only Rus- – 6/10 – Orysia Demska Hybridity and the linguistic landscape sian, but also Ukrainian. Such language perturbations are a slow process, and the rival languages exist in the cityscape simultaneously, regardless of legislation. 3.5 Errors are a natural occurrence in any linguistic landscape, and Podil is no exception. There are many examples of microtexts containing errors, e.g. Wake Up Навчання iноземним мо- вам в Українi (correctly iноземних); VVS Fashion. Вiд українського виробника Зроблено з любовью (correctly з любов’ю); ONE MORE PIZZA ще одна пiцерiя another на ПОДОЛЄ (correctly ПОДОЛI ). The ﬁrst and second are examples of grammar mistakes which arise as a result of the inﬂuence of Russian grammar and orthography. The third is more a verbal game, or an example of conscious hybridization, widespread in Podil, and achieved through the combina- tion of not only the languages, but also alphabets, e.g. BestПАР (electronic cigarettes); ОХОТА NA OVETS МЯСО И АЗИЯ (Figure 1); НеВинное Leto; Цветочный FLOдом; РИБАLOVE; Cheбурек; ковZанка, ЛюбиStock. All these examples, with the exception of РИБАLOVE, are explicit hybrids. РИБАLOVE (Figure 2) is both an explicit and an implicit hybrid. Explicitly, 3 The Linguistic Landscape of Kiev’s Podil District y p y, g g Figure 1: Restaurant sign in Podil, Kiev 2018 (Reproduced with the permission of the author, Anna Lishchynska) Figure 2: Restaurant sign in Podil, Kiev 2018 (Reproduced with the permission of the author, Anna Lishchynska) Figure 1: Restaurant sign in Podil, Kiev 2018 (Reproduced with the permission of the author, Anna Lishchynska) Figure 1: Restaurant sign in Podil, Kiev 2018 (Reproduced with the permission of the author, Anna Lishchynska) Figure 2: Restaurant sign in Podil, Kiev 2018 (Reproduced with the permission of the author, Anna Lishchynska) igure 2: Restaurant sign in Podil, Kiev 2018 g g (Reproduced with the permission of the author, Anna Lishchynska) 3.5 Errors are a natural occurrence in any linguistic landscape, and Podil is no exception. There are many examples of microtexts containing errors, e.g. Wake Up Навчання iноземним мо- вам в Українi (correctly iноземних); VVS Fashion. Вiд українського виробника Зроблено з любовью (correctly з любов’ю); ONE MORE PIZZA ще одна пiцерiя another на ПОДОЛЄ (correctly ПОДОЛI ). The ﬁrst and second are examples of grammar mistakes which arise as a result of the inﬂuence of Russian grammar and orthography. The third is more a verbal game, or an example of conscious hybridization, widespread in Podil, and achieved through the combina- tion of not only the languages, but also alphabets, e.g. BestПАР (electronic cigarettes); ОХОТА NA OVETS МЯСО И АЗИЯ (Figure 1); НеВинное Leto; Цветочный FLOдом; РИБАLOVE; Cheбурек; ковZанка, ЛюбиStock. All these examples, with the exception of РИБАLOVE, are explicit hybrids. РИБАLOVE (Figure 2) is both an explicit and an implicit hybrid. Explicitly, – 7/10 – Orysia Demska Hybridity and the linguistic landscape the Ukrainian риба and English love are joined into one word, риболов [rybolov], which means ﬁsherman; additionally, the semantics ‘someone, who likes to eat ﬁsh’ is contained in the restau- rant’s name. Implicitly, the name of the restaurant is a trilingual hybrid. In Ukrainian, there is no such word as риболов, it is a Russian word. The Ukrainian equivalent is рибалка [rybalka]. Therefore, the casual observer can see the conscious and explicit Ukrainian-English hybrid, while a philologist or anyobody else who knows more about the words рибалка and риболов can discern the implicit Ukrainian-English-Russian hybrid. 3.6 A Hybrid macrotext is an integral continuum of microtexts, joined by a common space (city, district, street etc.), type (oﬃcial, commercial, private) and functional purpose (object name, an- nouncement, prohibition etc.). Only one diﬀerence exists between microtexts and macrotexts – the author of a microtext is personalized (an owner, seller, buyer, resident of Podil, etc.). A ma- crotext is written by a so-called collective author. The main feature of this collective author is polyphony, which mainly predetermines unconscious hybridity. Again, three languages, Ukrainian, Russian and English, are the actors at the macro level of Podil’s linguistic landscape, with pre- dominance belonging to English and the Latin script, for instance, Vagabond café, Living room, #SexEdMuseum / Art centre / Art-Café, Ranch / Burger state, Concept store and Hair design studio / Esthetic syndicate / In esthetics we trust, Podil East India Company, English school Speak up, Tequila House, Magic Snail, Irish Pub / O‘Connor‘s, Andrew‘s Irish pub, Star Burger, Tarantino / Wine Bar / Steak is here, Sl Talking / Fresh and healthy take away, FlyBAr / Eat. 3 The Linguistic Landscape of Kiev’s Podil District Constructions which mix English, Ukrainian or Russian words in the Latin script and/or English spelling or transliteration are also examples of conscious hybridization: Bochka Art Pub, Ars Kerylos; Bursa Gallery; Coﬀe in the MISTO; Uspikh / agricultural corporation; buterbrod V stakane / vegan café, Illinsky / bisness centre and conference hall. Figure 3: National Bank, former St. Katherine church and Greek Monastery, Podil, Kiev 2019 (Author’s photograph) Figure 3: National Bank, former St. Katherine church and Greek Monastery, Podil, Kiev 2019 (Author’s photograph) 3.6 A Hybrid macrotext is an integral continuum of microtexts, joined by a common space (city, district, street etc.), type (oﬃcial, commercial, private) and functional purpose (object name, an- nouncement, prohibition etc.). Only one diﬀerence exists between microtexts and macrotexts – the author of a microtext is personalized (an owner, seller, buyer, resident of Podil, etc.). A ma- crotext is written by a so-called collective author. The main feature of this collective author is polyphony, which mainly predetermines unconscious hybridity. Again, three languages, Ukrainian, Russian and English, are the actors at the macro level of Podil’s linguistic landscape, with pre- dominance belonging to English and the Latin script, for instance, Vagabond café, Living room, #SexEdMuseum / Art centre / Art-Café, Ranch / Burger state, Concept store and Hair design studio / Esthetic syndicate / In esthetics we trust, Podil East India Company, English school Speak up, Tequila House, Magic Snail, Irish Pub / O‘Connor‘s, Andrew‘s Irish pub, Star Burger, Tarantino / Wine Bar / Steak is here, Sl Talking / Fresh and healthy take away, FlyBAr / Eat. – 8/10 – Orysia Demska Hybridity and the linguistic landscape Hybridity and the linguistic landscape Figure 4: Shop sign in Podil, Kiev 2019 (Author’s photograph) Figure 4: Shop sign in Podil, Kiev 2019 (Author’s photograph) Drink. Fly, Laura Ashley, PR Bar. The most frequently used word in the Latin script is coﬀee, e.g. CoﬀeeDoor / brew bar & Coﬀee shop; Coﬀee club, Sex Ed Coﬀee; Coﬀee stop / best in city; Coﬀee Dream; Coﬀee to go; Maryland / coﬀee blend; Coﬀee Guru; Hot Dogs Coﬀee; CoﬀeeBox etc. This is a feature of globalization and a manifestation of the culture of public consumption of coﬀee, which is typical of the contemporary European city. 3.7 Several other languages reinforce English and the Latin script in Podil’s linguistic lands- cape. 3 The Linguistic Landscape of Kiev’s Podil District Italian: Dolce caﬀe; Silvio D’Italia; Gastro di Italia / Club; Roberto Boticelli; Cipollino; Pizza; Piatto / Pasta Bar; Spain: Festival de Cocina Espanola; Viva la revolution!; or French: CafeBoutique; Reprisa / Artisanale Boutique Patisserie. Additionally, Church Slavonic, Greek (Figure 3), Turkish, with an exotic Arabic script ® (halal / Turkish restaurant / халяль), and Japanese hieroglyphs (Figure 4) strengthen the displacement of not only Russian, but also of Ukrainian in the commercial segment of contemporary Podil’s linguistic landscape. 4 Conclusions 4.1 Contemporary hybridity/cultural hybridity has shifted from the periphery into the centre of human life and has become ‘one of the emblematic notions of our era’ (Kraidy, 2005, p. 1). This shift has been conditioned by a ‘moment of historical transformation’ (Bhabha, 2004, p. 1) in Eastern Europe and Ukraine (the end of colonial subordination, the beginning of independence and globalization). As an ‘emblematic notion of our era’, hybridity, or more precisely cultural hybridity, is deﬁnitely a neutral phenomenon, an ‘eﬀort to maintain a sense of balance among practices, values, and customs of two or more diﬀerent cultures’ (Albert & Páez, 2012) or ‘an association of ideas, concepts, and themes that at once reinforce and contradict each other’ (Kraidy, 2005, p. VII). 4.2 During the Soviet period, Russian was the dominant language in Ukraine and Kiev. The Soviet authorities reinforced Russian and weakened Ukrainian. Today, the consequences of this colonial policy can be observed and one can see them in the Ukrainian-Russian hybrid city-text. Since the restoration of independence in 1991, Ukraine has been undergoing a transformation from a post-colonial state to a European state, and has become a part of a globalized world with English as a lingua franca. This process is also visible in the linguistic landscape, through Ukrainian-Russian-English, Ukrainian-English, and Russian-English hybrid signage. 4.3 The commercial segment of Podil’s linguistic landscape is characterized by a downgrading of Russian and an upgrading of Ukrainian and, especially, English. Linguistic hybridity, as an – 9/10 – Orysia Demska Hybridity and the linguistic landscape element of cultural hybridity, is closely connected to everyday practices of work, food, clothes, hygiene, health, leisure, etc. Organic/unconscious and intentional/conscious hybridization occur in linguistic creativity. Some contradictions occur when spelling, stylistic or semantic mistakes emerge in microtexts, or when the macrotext is too variegated. element of cultural hybridity, is closely connected to everyday practices of work, food, clothes, hygiene, health, leisure, etc. Organic/unconscious and intentional/conscious hybridization occur in linguistic creativity. 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https://openalex.org/W2172011590	https://europepmc.org/articles/pmc3952606?pdf=render	English	null	Reply to Thomas et al	Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)	2,014	cc-by	1,119	Reply to Thomas et al TO THE EDITOR—We agree with Thomas et al that classifying patients with dengue can be difﬁcult [1]. However, dengue shock syndrome (DSS) is a unique clini- cal entity that follows a very typical course; after 4–5 days of general systemic symptoms, previously healthy children and young adults present around the time of defervescence with substantial plasma leakage and hypovolemic shock, sometimes accompanied by bleeding. Ur- gent intervention is crucial to prevent progression to profound or refractory shock, which is often complicated by se- vere hemorrhage, organ failure, and death [2]. We use the standard deﬁnition for DSS that is familiar to most physicians working in endemic areas—a history consistent with dengue, with hemody- namic compromise deﬁned as either narrowing of the pulse pressure or hypo- tension for age together with evidence of impaired perfusion—with the caveat that the deﬁnition refers to hypovolemia due to vascular leakage rather than due to blood loss [3]. Although gastrointestinal losses may result in some degree of dehy- dration during the initial febrile phase, it is highly unlikely that “straightforward dehydration” could explain these clinical signs. First, patients presenting with DSS usually look surprisingly well. They do not exhibit any of the conventional signs of dehydration, such as dry lips, sunken eyes, or reduced skin turgor, and yet in our series the median percentage hemoconcentration at presentation was 35% (interquartile range, 27%–45%). This level of dehydration should be im- mediately obvious to the treating physi- cian, and indeed it is partly because Thomas et al also suggest addition of complex criteria to the diagnosis of DSS, including a number of laboratory investigations, use of intensive care unit severity scoring systems, and presence of “indisputable diagnostic features of plas- ma leakage.” However, the vast majority of DSS cases are managed in healthcare facilities without immediate access to blood gas analysis, coagulation screening, or urgent radiological investigations, and none of the scoring systems mentioned are relevant to children (the primary DSS population) or have been evaluated in dengue-endemic areas to assess their utility. Finally, “indisputable evidence of leakage” is rarely apparent until after re- suscitation has commenced. We feel strongly that the diagnosis of DSS should remain practical and simple [2], designed to be immediately within the grasp of everyday clinicians working in environ- ments where they are faced with identify- ing and treating hundreds of such cases annually. most patients look tired but otherwise unremarkable that close attention to pulse pressure and general cardiovascular status are so strongly encouraged, to allow prompt identiﬁcation during the early phase of compensated shock. Second, al- though pleural and peritoneal effusions are rarely present at onset of DSS, within 12–24 hours of commencing ﬂuid resus- citation, approximately 30% of our sub- jects developed clinically detectable effusions [3], and >95% of those assessed radiologically had detectable effusions (unpublished data); this is not consistent with hypovolemia due to dehydration alone. most patients look tired but otherwise unremarkable that close attention to pulse pressure and general cardiovascular status are so strongly encouraged, to allow prompt identiﬁcation during the early phase of compensated shock. Second, al- though pleural and peritoneal effusions are rarely present at onset of DSS, within 12–24 hours of commencing ﬂuid resus- citation, approximately 30% of our sub- jects developed clinically detectable effusions [3], and >95% of those assessed radiologically had detectable effusions (unpublished data); this is not consistent with hypovolemia due to dehydration alone. Reply to Thomas et al In our case series of direct ad- missions, patient outcomes were excel- lent, largely due to prompt diagnosis and skilled management by experienced personnel. Undoubtedly, mortality is higher in referred cases or those diag- nosed late in the illness evolution [4–6]. Potential conﬂicts of interest. Both authors: No reported conﬂicts. Note Potential conﬂicts of interest. Both authors: No reported conﬂicts. CORRESPONDENCE • CID 2014:58 (1 April) • 1039 Both authors have submitted the ICMJE Form for Disclosure of Potential Conﬂicts of Interest. Conﬂicts that the editors consider relevant to the content of the manuscript have been disclosed. Phung Khanh Lam1 and Bridget Wills1,2 1Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam; and 2Centre for Tropical Medicine, Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, United Kingdom Tropical Diseases, Ho Chi Minh City, Vietnam; and 2Centre for Tropical Medicine, Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, United Kingdom 1040 • CID 2014:58 (1 April) • CORRESPONDENCE References 1. Thomas L, Cabié A, Teyssou R. Dengue shock syndrome or dehydration? The importance of considering clinical severity when classifying patients with dengue. Clin Infect Dis 2014; 58:1038–9. 2. World Health Organization. Dengue: guide- lines for diagnosis, treatment, prevention and control. Geneva, Switzerland: WHO, 2009. 2. World Health Organization. Dengue: guide- lines for diagnosis, treatment, prevention and control. Geneva, Switzerland: WHO, 2009. 3. Lam PK, Tam DT, Diet TV, et al. Clinical characteristics of dengue shock syndrome in Vietnamese children: a 10-year prospective study in a single hospital. Clin Infect Dis 2013; 57:1577–86. 3. Lam PK, Tam DT, Diet TV, et al. Clinical characteristics of dengue shock syndrome in Vietnamese children: a 10-year prospective study in a single hospital. Clin Infect Dis 2013; 57:1577–86. 4. Trung DT, Thao Le TT, Dung NM, et al. Clin- ical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than chil- dren. Plos Negl Trop Dis 2012; 6:e1679. 4. Trung DT, Thao Le TT, Dung NM, et al. Clin- ical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than chil- dren. Plos Negl Trop Dis 2012; 6:e1679. 5. Bunnag T, Kalayanarooj S. Dengue shock syn- drome at the emergency room of Queen Sirikit National Institute of Child Health, Bangkok, Thailand. J Med Assoc Thai 2011; 94(suppl 3): S57–63. 6. Anders KL, Nguyet NM, Chau NVV, et al. Ep- idemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Viet- nam. Am J Trop Med Hyg 2011; 84:127–34. Correspondence: Phung Khanh Lam, MD, Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam (lampk@oucru.org). Correspondence: Phung Khanh Lam, MD, Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam (lampk@oucru.org). Clinical Infectious Diseases 2014;58(7):1039–40 Clinical Infectious Diseases 2014;58(7):1039 40 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Crea- tiveCommonsAttributionLicense(http://creativecommons.org/ licenses/by/3.0/),whichpermitsunrestrictedreuse,distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciu017
https://openalex.org/W2017581610	http://juti.if.its.ac.id/index.php/juti/article/download/387/276	Indonesian	null	SEGMENTASI PENYAKIT PADA CITRA DAUN TEBU MENGGUNAKAN FUZZY C MEANS – SUPPORT VECTOR MACHINE DENGAN FITUR WARNA a*	Juti/JUTI (Jurnal Ilmiah Teknologi Informasi)	2,015	cc-by-sa	4,943	Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* ABSTRACT The disease in sugarcane agriculture must be addressed quickly to ensure a high productivity. Detection of the disease manually by an expert requires substantial time and cost. Therefore an automated system for detection the disease in sugarcane is required. This research aims to develop a system which able to automatically perform image segmentation disease in sugarcane leaves image with Fuzzy C Means (FCM)-Support Vector Machine (SVM) with color feature a. FCM-SVM com- bination can improve the accuracy of the segmentation process with characteristic leaf disease of sugarcane with not balance lighting due to the outdoor collection. Image segmentation in sugarcane leaf image consists of several phases, namely preprocessing, selection of region of interest (ROI), feature extraction, and the segmentation. Preprocessing stage capture the bone and removing the leaf veins, then the selection of ROI as the dominating presence of disease areas on the leaves using an overlapping window as wide as 100 x 100 pixels. The Combination of FCM and SVM used for diseased sugarcane leaf segmentation, where the FCM is used for the segmentation of sugarcane leaves on the training data. The segmentation results are used as the data labels in the second stage testing along with the data using SVM classification method. The proposed segmentation method shows a high rate accuracy of 76%. The system that has been built, further used in the process of disease detection as a reference to the precision agriculture system which requires early disease detection system. Keywords: disease segmentation, sugarcane leaf, FCM, SVM. ABSTRAK Penyakit pada pertanian tebu harus segera diatasi agar diperoleh peningkatan produktivitas. Deteksi penyakit yang secara manual dilakukan oleh ahli membutuhkan waktu dan biaya yang tinggi. Oleh karena itu, diperlukan otomatisasi sistem untuk mendeteksi penyakit pada tanaman tebu. Penelitian ini bertujuan untuk membangun sistem yang secara otomatis mampu melakukan segmentasi citra daun tebu berpenyakit menggunakan Fuzzy C Means (FCM)-Support Vector Machine (SVM) dengan fitur warna a. Kombinasi FCM-SVM dapat meningkatkan akurasi pada proses segmentasi dengan karakteristik penyakit daun tebu dengan pencahayaan yang tak seimbang akibat pengambilan secara outdoor. Segmentasi citra daun tebu berpenyakit memiliki beberapa tahapan yaitu praproses, pemilihan region of interest (ROI), ekstraksi fitur, dan segmentasi. Tahap praproses melakukan pengambilan bagian tulang daun serta penghapusan bagian tulang daun, kemudian pemilihan ROI menunjukan dominasi area penyakit pada daun menggunakan overlapping window seluas 100x100 pixel. Metode kombinasi FCM dan SVM digunakan untuk segmentasi daun tebu berpenyakit, dimana FCM digunakan untuk segmentasi daun tebu pada data training. Hasil segmentasi tersebut digunakan sebagai label data pada tahap kedua bersama dengan data testing menggunakan metode klasifikasi SVM. Metode segmentasi yang diusulkan mampu menunjukkan rata-rata akurasi yang tinggi pada 30 citra daun tebu berpenyakit, yaitu sebesar76%. Sistem yang dibangun selanjutnya digunakan pada deteksi penyakit sebagai referensi untuk ketepatan permasalahan pertanian yang membutuhkan sistem deteksi penyakit sejak dini. Kata Kunci: Segmentasi penyakit, daun tebu, FCM, SVM Kata Kunci: Segmentasi penyakit, daun tebu, FCM, SVM Mustika Mentari1), R.V. Hari Ginardi2), dan Chastine Fatichah3) 1, 2,3)Institut Teknologi Sepuluh Nopember Jurusan Teknik Informatika, Fakultas Teknologi Informasi, ITS Surabaya 60111 e-mail: must.mentari@gmail.com1), hari.ginardi@gmail.com2), chastine.fatichah@gmail.com3) Mustika Mentari1), R.V. Hari Ginardi2), dan Chastine Fatichah3) 1, 2,3)Institut Teknologi Sepuluh Nopember Jurusan Teknik Informatika, Fakultas Teknologi Informasi, ITS Surabaya 60111 e-mail: must.mentari@gmail.com1), hari.ginardi@gmail.com2), chastine.fatichah@gmail.com3) Jurusan Teknik Informatika, Fakultas Teknologi Informasi, ITS Surabaya 60111 e-mail: must.mentari@gmail.com1), hari.ginardi@gmail.com2), chastine.fatichah@gmail.com3) I. PENDAHULUAN enyakit yang menyerang tebu di Indonesia dapat menurunkan produktivitas. Penyebaran penyakit tebu me- nyebabkan dampak penurunan ekonomi yang kuat karena berdampak pada jumlah penurunan panen. Selain masalah umum tersebut penanganan penyakit tebu perlu segera dideteksi dan diatasi untuk mengurangi biaya penggunaan pestisida dan pencemaran lingkungan akibat obat penyakit tersebut [1]. Penyakit pada tanaman tebu dapat diketahui dari bercak yang terdapat pada bagian daun. Bercak tersebut perlu dideteksi dengan menggunakan metode segmentasi. P P Penelitian penyakit pada daun tebu dilakukan oleh Patil [1]. Penyakit ini menjelaskan pengaruh jamur pada tebu menjadi penyakit yang jika tidak diatasi tepat waktu dapat menyebabkan kerugian. Penggunaan pestisida terlalu banyak dapat meningkatkan biaya dan polusi lingkungan. Hal ini dapat diperoleh dengan mengetahui daerah pen- yakit, dengan kuantitas kesesuaian dan konsentrasi pestisida dengan mengestimasi penyakit menggunakan teknik image processing. Metode thresholding sederhana dan triangle thresholding digunakan untuk melakukan segmen- tasi daerah daun dan daerah berpenyakit secara berturut-turut. Penyakit dikategorikan dengan perhitungan hasil 45 JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 bagi daerah penyakit dan area keseluruhan daun. Hasil akurasi pada penelitian ini adalah 98.60%. Penelitian ini mengindikasikan metode usulan penyakit daun dilakukan secara kuat, cepat dan akurat. Metode ini mengeliminasi cara tradisional yang subjektif dan rawan akan kesalahan manusia. bagi daerah penyakit dan area keseluruhan daun. Hasil akurasi pada penelitian ini adalah 98.60%. Penelitian ini mengindikasikan metode usulan penyakit daun dilakukan secara kuat, cepat dan akurat. Metode ini mengeliminasi cara tradisional yang subjektif dan rawan akan kesalahan manusia. Penyakit yang dapat deteksi pada daun juga diteliti oleh Al-Trawneh dan Mokhled [2]. Penelitian ini melakukan investigasi analisis gambar dan teknik klasifikasi untuk mendeteksi tingkat keparahan daerah penyakit pada daun zaitun. Sample daun zaitun dikumpulkan dari lahan yang diambil secara outdoor di bawah pencahayaan yang tidak terkotrol. Resolusi gambar dirubah menjadi 256 x 256 piksel dan ditransformasikan dari Red Green Blue (RGB) menjadi Lab* colorspace (karena Lab* dianggap paling dekat dengan persepsi manusia). Citra hasil transfor- masi dipotong secara poligonal untuk kemudian disegmentasi pada bagian ROI (Region of Intereset) dan diklasifi- kasi menggunakan Fuzzy C Means (FCM) untuk penggunaan metode statistika dalam penentuan area terdeteksi penyakit pada daun. Perbaikan citra penelitian ini menggunakan median filter. Hasil yang didapatkan menunjukkan susunan yang baik antara FCM dan manual scoring dan menggunakan analisis gambar dengan rata rata akurasi 86% dibandingkan dengan K-mean Clustering (KCM) 66%. I. PENDAHULUAN Penelitian mengenai pengembangan metode segmentasi juga dilakukan oleh Wang, dkk [3]. Penelitian ini mem- presentasikan segmentasi warna menggunakan klasifikasi piksel secara otomatis menggunakan Support Vector Machine (SVM). Pertama, fitur warna pada piksel diekstraksi sesuai sensitivitas visualisasi manusia sesuai fitur warna dan fitur tekstur pada pixel menggunakan steerable filter. Kedua fitur tersebut digunakan sebagai input SVM, kemudian SVM melakukan proses training menggunakan FCM dengan mengekstrak fitur pada piksel. Ter- akhir dilakukan segmentasi citra testing menggunakan training SVM. Hasil dari penelitian ini menunjukkan metode usulan sangat efektif melakukan segmentasi serta memiliki perilaku komputasi yang baik pula. Waktu komputasi dan kualitas segmentasi menjadi lebih baik dari pada metode terdahulu (mean shift, graph and cuts, dan Edison). Metode usulan pada penelitian ini memliliki kelemahan tidak mempunyai ketahanan terhadap derau. Berdasarkan penelitian yang sudah dilakukan, maka dapat diketahui bahwa metode segmentasi memiliki akurasi lebih baik apabila dilakukan dengan klasifikasi karena adanya informasi model yang didapatkan dari data training. Akan tetapi pada segmentasi citra menggunakan metode klasifikasi memerlukan adanya pelabelan data training yang tidak bisa dilakukan secara manual atau inisialisasi random seperti penelitian [3]. Penelitian ini bertujuan untuk membangun sistem yang secara otomatis mampu melakukan segmentasi citra daun tebu berpenyakit dengan metode FCM-SVM. SVM mendapatkan inputan data training dari metode FCM, untuk kemudian dilajutkan dengan klasifikasi data testing. Pengujian kuantitatif metode segmentasi ini dilakukan dengan akurasi segmentasi. Dengan penggabungan metode FCM-SVM diharapkan daun tebu berpenyakit dapat disegmentasi dengan baik dan menunjukkan karakteristik penyakit dengan baik sehingga memudahkan proses lanjutan yaitu prediksi penyakit pada tanaman tebu. II. SEGMENTASI CITRA MENGGUNAKAN FCM-SVM Penelitian ini melakukan proses segmentasi dengan mengkombinasikan metode klastering dengan metode klas- ifikasi. Segmentasi citra daun tebu berpenyakit melalui beberapa proses mulai pengolahan data sampai dengan segmentasi. Tahap pertama yang dilakukan adalah pengolahan data, pengolahan ini akan memilih bagian citra hanya pada bagian daun saja tanpa daerah sekitar yang bukan bagian dari daun. Kemudian setelah hanya bagian daun saja, citra tersebut diproses menggunakan threshold untuk menghilangkan daerah tulang daun. Threshold dalam hal ini masih ditentukan secara manual dari channel red color space RGB berdasarkan gelap terang dari citra yang sudah berhasil dikumpulkan menggunakan beberapa jenis alat yang berbeda. Tahap kedua adalah pemilihan ROI menggunakan moving window berukuran 100x100 piksel, setelah itu dari beberapa window akan dipilih bebebrapa window yang mempunyai nilai standar deviasi maksimal. Hasil dari ROI ini yang akan masuk pada tahap ketiga ekstraksi fitur warna menggunakan channel a* dari color space Lab. Pemilihan jenis fitur ini dilakukan berdasarkan percobaan yang dilakukan sebelumnya. Tahap terakhir yaitu segementasi data label menggunakan metode FCM. Segmentasi data uji coba dilakukan menggunakan metode SVM berdasarkan data label tahap sebelumnya. Gambar 1 mendeksripsikan tahap-tahap segmentasi yang sudah dijelaskan sebelumnya. Kombinasi FCM-SVM menggunakan kelebihan FCM sebagai data label sehingga SVM mampu bekerja lebih maksimal dengan adanya informasi data training yang sudah dilakukan terlebih dahulu. A. Dataset Penelitian ini menggunakan empat jenis penyakit yang ada pada daun tebu, yaitu yellow spot, rust spot, ring spot, dan eye spot. Penyakit yellow spot pada citra daun tebu disebabkan oleh salah satu jenis jamur yang disebut dengan mycovellosiella koepki. Yellow spot berakibat pada penurunan hasil panen dan kadar sukrosa tanaman tebu. Penyakit ini akan semakin menyebar dan merugikan dengan kondisi kelembapan dan curah hujan yang tinggi. Sindrom penyakit ini pada awalnya mempunyai diameter sepanjang 1-2 mm. Warna penyakit ini mulai dari kuning sampai dengan merah, selain itu pada bagian bawah daun terdapat kabur keabu-abuan. Contoh citra daun tebu yang mempunyai penyakit yellow spot dijelaskan pada Gambar 2 (a)[4]. 46 Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* ROI Ekstraksi fitur warna Segmentasi data label dengan metode klastering (FCM) Segmentasi dengan metode klasifikasi SVM Pengambilan dan Pengolahan data Gambar 1 Alur Penelitian Segmentasi Citra Daun Tebu Berpenyakit Ekstraksi fitur warna Gambar 1 Alur Penelitian Segmentasi Citra Daun Tebu Berpenyakit (a) (b) (c) (d) Gambar 2 Penyakit Tebu (a) Yellow Spot, (b) Rust Spot, (c) Ring Spot, (d) Eye Spot (b) Gambar 2 Penyakit Tebu (a) Yellow Spot, (b) Rust Spot, (c) Ring Spot, (d) Eye Spot Penyakit karat (sugarcane rust diseases) kebanyakan mewabah pada bagian daun tanaman tebu. Gejala awal yang dapat dideteksi pada penyakit ini adalah tanaman kerdil dan terdapat bercak berwarna kuning. Bercak ini akan melebar dan menjadi coklat sampai orange-coklat atau merah-coklat. Rata-rata bercak ini berukuran antara 2 – 10 mm tapi dalam kasus tertentu dapat mencapai 30 mm. Penyakit karat pada tanaman tebu dijelaskan pada Gambar 2(b) [5]. Selain ciri-ciri yang sudah disebutkan di atas, penyakit tebu rust spot menyebabkan daerah sekitar penyakit pada daun juga ikut berubah warna kekuningan atau kecoklatan. Penyakit tebu ring spot (Gambar 2 (c) [6]) biasanya muncul pada daun tebu yang berumur dewasa atau sudah mendekati usia panen. Oleh karena itu, panyakit ini tidak terlalu berpengaruh pada penurunan hasil panen. Penyakit ini muncul karena jamur yang disebut dengan leptosphaeria sacchari. B. Ekstraksi Fitur Warna B. Ekstraksi Fitur Warna Fitur warna pada penilitian ini akan menggunakan warna dasar citra berdasarkan hasil pemotretan di lapangan yang dikonversi ke dalam channel Lab. Ruang warna tersebut adalah RGB. Selanjutnya color space tersebut akan dikonversi menjadi color space CIELAB (Lab). Channel L* mendefinisikan lightness, a* mendefinisikan nilai red/green, dan b* mendefinisikan nilai yellow/blue [9]. Sumbu a* digambarkan dari arah kiri ke kanan. Pergeseran ke arah positif menggambarkan pergeseran ke arah warna merah. Sedangkan sumbu b* yang digambarkan dari bawah ke atas. Pergeseran ke arah positif merupakan pergeseran ke warna kuning. Pusat diagram tersebut merupakan L* yang menunjukkan L=0 untuk daerah total penyerapan (bagian bawah), dan di daerah sekitar tengah menunjukkan warna netral atau abu-abu. Perumusan untuk mendapatkan color space Lab* didapatkan pada persamaan (1). Dari nilai RGB terlebih dahulu dikonversi pada XYZ. Dimana X, Y dan Z nilai referensi warna putih untuk pencahayaan yang digunakan sesuai keputusan pengamat. ܮ∗= 116 (ܻ/ܻ௡)ଵ/ଷ−16, ܽ∗= 500[ቀ௑ ௑೙ቁ భ య−( ௒ ௒೙)ଵ/ଷ], ܾ∗= 200[ቀ௒ ௒೙ቁ భ య−( ௓ ௓೙)ଵ/ଷ] (1) ܮ∗= 116 (ܻ/ܻ௡)ଵ/ଷ−16, ܽ∗= 500[ቀ௑ ௑೙ቁ భ య−( ௒ ௒೙)ଵ/ଷ], ܾ∗= 200[ቀ௒ ௒೙ቁ భ య−( ௓ ௓೙)ଵ/ଷ] (1) ܮ∗= 116 (ܻ/ܻ௡)ଵ/ଷ−16, ܽ∗= 500[ቀ௑ ௑೙ቁ భ య−( ௒ ௒೙)ଵ/ଷ], ܾ∗= 200[ቀ௒ ௒೙ቁ భ య−( ௓ ௓೙)ଵ/ଷ] (1) A. Dataset Jamur tersebut membutuhkan kondisi hangat dan lembab yang biasanya terjadi pada saat musim panas untuk berkembang biak. Penyebaran jamur tersebut menggunakan media hujan atau angin. Pada usia muda terjangkit, maka penyakit ini akan berwujud bintik-bintik berwarna bronze brown. Kemudian semakin parah tingkat keparahannya, maka penyakit ring spot akan memanjang dengan tepi berwarna kuning. Spot tersebut berbentuk oval dengan ukuran mulai dari 1-5 mm sampai dengan 4 – 18 mm ([6]). Penyakit tebu eye spot (Gambar 2 (d) [7]) adalah salah satu penyakit tebu yang penting untuk dipelajari dan juga disebabkan oleh jamur. Jamur pada penyakit ini dinamakan dengan bipolaris sacchari. Tingkat keparahan panyakit ini tergantung dari kondisi lingkungan lahan pertanian. Kecepatan penyebaran akan semakin didukung dengan kelembapan tinggi serta adanya embun yang jernih dan deras. Tingkat keparahan penyakit ini ditandai dengan adanya spot berwarna kemerahan. Spot tersebut mempunyai lebar 0,5-0,4 mm, 0,5-2 mm berwarna kekuningan dengan daerah tepi berwana coklat. Pada saat daun masih baru saja terjangkit penyakit ini, maka akan muncul spot bintik-bintik yang kemudian berbentuk memanjang menyerupai bentuk mata manusia. Penyebaran penyakit eye spot adalah menggunakan media angin [8]. Data citra uji coba secara keseluruhan terdapat pada Tabel 1. 47 47 JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 TABEL I DAFTAR JENIS PENYAKIT DATA UJI COBA Citra Jenis Penyakit Citra Jenis Penyakit 1 Yellow Spot 16 Yellow spot 2 Rust Spot 17 Yellow spot 1 Yellow Spot 18 Yellow spot 4 Yellow Spot 19 Yellow Spot 5 Rust Spot 20 Rust spot 6 Yellow Spot 21 Yellow spot 7 Ring spot 22 Ring spot 8 Yellow Spot 23 Yellow spot 9 Yellow Spot 24 Ring spot 10 Eye spot 25 Rust Spot 11 Yellow Spot 26 Yellow Spot 12 Yellow Spot 27 Rust Spot 13 Eye Spot 28 Yellow Spot 14 Ring spot 29 Ring spot 15 Yellow Spot 30 Ring Spot D. Support Vector Machine SVM merupakan sebuah teknik klasifikasi yang muncul pada tahun 1995. Banyak bidang yang telah mengimplementasikan metode ini seperti bidang ekonomi, cuaca, kedokteran, dll. SVM menemukan solusi secara global optimal dan solusi training yang sama jika dibandingkan dengan metode klasifikasi lain. Untuk melakukan proses klasifikasi menggunakan SVM, dibutuhkan suatu pemisah yang dapat dibagi menjadi dua macam. Pemisah tersebut dapat berupa fungsi linear ataupun non linear. Fungsi linear didefinisikan pada persamaan (6). Pada fungsi linier ditentukan pemisah (hyperplane) yang memisahkan dua macam objek saja. Hyperplane yang memisahkan dua objek tersebut mengalami solusi yang terbaik di saat posisi berada di tengah kedua objek [10]. ݃(ݔ) ≔ݏ݃݊൫݂(ݔ)൯ , ݂(ݔ) = ݓ்ݔ+ ܾ, (6) Beberapa fungsi kernel yang dapat digunakan adalah Linear, Polynomial, Radial basis function (RBF), Tangent hyperbolic (sigmoid). Pilihan kernel ini dapat menangani pemisahan kasus yang tidak dapat diselesaikan dengan metode linear biasa. Ketepatan pemisahan bergantung pada jenis data yang digunakan dalam penelitian. Beberapa fungsi kernel yang dapat digunakan adalah Linear, Polynomial, Radial basis function (RBF), Tangent hyperbolic (sigmoid). Pilihan kernel ini dapat menangani pemisahan kasus yang tidak dapat diselesaikan dengan metode linear biasa. Ketepatan pemisahan bergantung pada jenis data yang digunakan dalam penelitian. III. HASIL AND DISKUSI C. Fuzzy C-Means ߤ௜௞ = ଵ ∑ ቆ ฮೣೖష ೐೔ฮ ቛೣೖష ೐ೕቛቇ మ ೘షభ ೎ ೕసభ (4) ݁௜= ∑ (ఓ೔ೖ)೘௫ೖ ೙ ೖసభ ∑ (ఓ೔ೖ)೘ ೙ ೖసభ , 1 ≤݅≤ܿ (5) ߤ௜௞ = ଵ ∑ ቆ ฮೣೖష ೐೔ฮ ቛೣೖష ೐ೕቛቇ మ ೘షభ ೎ ೕసభ ݁௜= ∑ (ఓ೔ೖ)೘௫ೖ ೙ ೖసభ ∑ (ఓ೔ೖ)೘ ೙ ೖసభ , 1 ≤݅≤ܿ (4) (5) C. Fuzzy C-Means FCM adalah salah satu jenis metode yang digunakan untuk membagi-bagi kelas suatu data secara tidak terawasi (unsupervised). Metode ini memperhitungkan tiap data pada klaster untuk berada sesuai derajat keanggotaannya. Metode ini diawali dengan penentuan pusat klaster, kemudian pusat klaster dan derajat keanggotaan tersebut akan diperbaharui setiap iterasi secara berulang. Proses perulangan ditentukan dengan meminimalkan fungsi objektif yang merupakan penggambaran jarak dari posisi data yang diuji dengan pusat klaster yang sudah terboboti oleh derajat keanggotaan data uji. Fungsi objektif FCM (2) menjelaskan bahwa variabel m adalah suatu konstanta yang bernilai lebih dari satu. Jumlah klaster diwakili oleh variabel ݁௜ yang berisi minimal lebih dari satu [2]. Derajat keanggotaan diantara sample k dan cluster i dituliskan sebagai ߤ௜௞ yang dijabarkan pada persamaan (3). ܬ௠(ܷ, ܧ) = ∑ ∑ (ߤ௜௞)௠‖ݔ௞− ݁௜‖ଶ ௖ ௜ୀଵ ௡ ௞ୀଵ (2) ߤ௜௞∈{0,1}, ∀݅, ݇; ∑ ߤ௜௞= 1, ∀݇ ௖ ௜ୀଵ (3) ܬ௠(ܷ, ܧ) = ∑ ∑ (ߤ௜௞)௠‖ݔ௞− ݁௜‖ଶ ௖ ௜ୀଵ ௡ ௞ୀଵ ߤ௜௞∈{0,1}, ∀݅, ݇; ∑ ߤ௜௞= 1, ∀݇ ௖ ௜ୀଵ (2) (3) Update nilai membership function pada FCM dilakukan dengan persamaan (4), sedangkan perhitungan pusat klaster ditentukan dengan persamaan (5). Secara keseluruhan mulai dari awal proses FCM dimulai dengan menen- tukan jumlah klaster c, variabel m, inisialisasi matriks untuk derajat keanggotaan, dan penentuan jumlah maksimal iterasi. Tahap kedua adalah melakukan perhitungan pusat klaster (derajat keanggotaan matrix). Pada tahap tiga dilakukan perhitungan matriks keanggotaan berdasarkan beberapa pusat klaster. Tahap terakhir pada proses FCM adalah pengulangan tahap dua dan tiga sampai jumlah maksimum iterasi atau kondisi yang sudah konvergen, yaitu 48 Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* kondisi pusat klaster yang tidak berubah dan nilai error yang kurang dari threshold. Setelah iterasi berhenti, klaster untuk setiap data ditentukan dengan derajat keanggotaan terbesar dari suatu data. kondisi pusat klaster yang tidak berubah dan nilai error yang kurang dari threshold. Setelah iterasi berhenti, klaster untuk setiap data ditentukan dengan derajat keanggotaan terbesar dari suatu data. JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 TABEL 2 HASIL UJI COBA SEGMENTASI DENGAN METODE FCM Citra Akurasi Model (Data Training) (%) Rata- rata (%) 1 2 3 4 5 6 7 8 9 10 1 81 46 46 87 74 92 94 80 94 77 2 90 39 39 92 89 98 99 89 96 81 3 86 38 38 92 73 88 90 84 99 76 4 88 43 43 97 68 80 82 99 91 77 5 88 42 42 94 75 83 84 99 90 77 6 94 29 29 99 79 88 89 96 96 77 7 81 67 56 72 96 87 86 66 79 77 8 63 66 84 66 60 61 61 67 64 66 9 98 95 33 96 98 99 99 95 97 90 10 94 76 19 87 98 96 96 73 93 81 11 92 95 59 94 84 89 90 95 92 88 12 94 88 19 91 98 97 96 88 93 85 13 95 86 20 20 99 96 96 84 94 76 14 95 91 22 22 99 97 96 91 94 79 15 94 97 54 41 86 91 92 97 94 83 16 99 98 22 22 98 100 100 98 99 82 17 84 63 23 23 74 93 90 61 82 66 18 75 53 31 31 61 85 82 52 71 60 19 96 83 34 34 87 84 99 82 93 77 20 69 36 28 28 43 93 83 35 61 53 21 91 83 36 36 86 95 95 83 89 77 22 95 88 30 30 91 98 98 88 94 79 23 97 89 36 36 92 90 98 89 95 80 24 92 88 27 27 89 97 94 87 91 77 25 77 85 37 37 82 67 73 74 79 68 26 93 97 30 30 100 78 87 88 95 78 27 85 97 41 41 97 63 73 76 89 74 28 96 96 54 54 98 63 83 85 95 80 29 91 87 26 26 89 97 94 93 86 77 30 89 64 58 58 72 99 93 93 63 77 Rata- rata (%) 89 82 35 37 86 85 89 89 82 89 76 A. Hasil Percobaan Segmentasi citra dengan metode klastering dan klasifikasi dilakukan sesuai dengan penelitian [11] yang menyatakan bahwa penggabungan metode unsupervised FCM dan supervised SVM akan menghasilkan solusi yang cukup baik dalam pemisahan data dalam kasus ini yaitu segmentasi yang memisahkan derah penyakit dan daerah daun sehat. Pelabelan data pada uji coba ini dilakukan menggunakan metode klastering FCM, yang kemudian dikombinasikan dengan metode SVM untuk melakukan segmentasi data testing. Metode FCM-SVM menggunakan parameter c=2, m=2, rata-rata iterasi sebanyak 20 kali, error rate sebesar 0,001, dan kernel polynomial. Pengujian segmentasi dengan metode FCM-SVM dilakukan pada 30 data berupa citra daun tebu berpenyakit yang mempunyai 10 model. Seluruh model yang digunakan ini didasarkan pada pembagian data training yang digunakan dari ke-10 data. Masing- masing model memiliki 3 citra yang digunakan sebagai pembelajaran pada metode klasifikasi. Mas- ing-masing model berisi citra yang berbeda-beda antara satu dengan lainnya. Hasil uji coba pada skenario ini dapat dilihat pada Tabel 2. Setiap baris pada tabel tersebut yang tidak mempunyai nilai dan mempunyai sel yang berwarna sama dengan judul model nya diartikan bahwa sel tersebut tidak dilatih dengan data training pada model tersebut. Hal tersebut karena pengenalan suatu data terhadap dirinya sendiri tidak ikut diperhitungkan dalam analisis validitas metode klasifikasi pada penelitian ini. Rata-rata akurasi untuk seluruh data pada kesepuluh model yang sudah diuji pada skenario ini adalah 76%. 49 49 B. Diskusi Penelitian ini melakukan perhitungan rata-rata uji coba seluruh data pada seluruh model dan juga uji coba seluruh model pada setiap data dengan rata-rata akurasi total kurang dari 80%. Hal ini disebabkan nilai akurasi terendah percobaan tiap data pada seluruh model yang ditentukan mempunyai rata-rata akurasi terendah sejumlah 35% yaitu pada model 3 diikuti rata-rata akurasi terendah sejumlah 37% pada model ke-4. Hal ini dikarenakan model 3 mempunyai kombinasi 3 jenis penyakit yang mempunyai ciri minoritas di dalam data. Penyakit tersebut adalah Ring Spot, Rust Spot, dan juga Yellow Spot. Dalam keseluruhan 30 data yang digunakan, hanya 1 citra yang menunjukkan ring spot, sedikit citra yang mempunyai ciri rust spot dan yellow spot seperti gambar tersebut. Hal inilah yang menyebabkan akurasi rata-rata dari keseluruhan data yang diuji coba sangat rendah. Ciri warna yang dimiliki oleh model 3 yang terdiri dari tiga citra ini terlampau berbeda dengan yang lain. Citra yang mempunyai ciri penyakit ring spot dengan daerah tengah yang berwarna coklat muda-keabuan dan daerah tidak terlalu tebal berwarna coklat tua seperti citra ke-7 hanya dimiliki oleh satu citra ini. Data training kedua pada model ini adalah jenis penyakit rust spot dengan ciri berwana kuning muda tanpa bagian pusat spot ini berbeda dengan jenis penyakit yang sama yang ada pada data uji coba. Demikian pula dengan citra ke-9 yaitu yellow spot yang mempunyai proyeksi berwarna putih di sekitar pusat spot ini berbeda dengan jenis penyakit yang sama pada data uji coba penelitian ini. Hal inilah yang menyebabkan model 3 memiliki akurasi terendah daripada model lain. 50 Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Mentari, Ginardi, dan Fatichah — Segmentasi Penyakit pada Citra Daun Tebu Menggunakan Fuzzy C Means-Support Vec- tor Machine dengan Fitur Warna a* Gambar 3 Perbandingan akurasi segmentasi FCM dengan FCM-SVM Perbandingan akurasi metode segmentasi dimana kedua metode tersebut diterapkan pada data uji coba yang sama yang digunakan dalam penelitian ini. Khusus untuk metode FCM-SVM akurasi diambil dari akurasi tertinggi dari keseluruhan model yang ada. Jika akurasi tertinggi ini dibandingkan dengan metode segmentasi FCM, maka rata- rata akurasi keseluruhannya adalah 89,37% untuk metode FCM, dan 96,33% untuk metode FCM-SVM. Dalam Gambar 3 dapat dilihat bagaimana perbadingan akurasi kedua metode tersebut. Hampir dari keseluruhan data me- raih akurasi tertinggi dengan metode segmentasi kombinasi FCM-SVM. JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 JUTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 UTI: Jurnal Ilmiah Teknologi Informasi - Volume 13, Nomer 1, Januari 2015: 45 – 52 IV. KESIMPULAN Penggunaan fitur warna channel a dari colorspace Lab* membantu metode yang ada pada skenario penelitian ini melakukan segmentasi dengan baik dibandingkan dengan channel RGB, B, atau I. Penggunaan dua metode dalam segmentasi, yaitu metode klastering FCM dan metode klasifikasi SVM mampu melakukan segmentasi pada citra daun tebu berpenyakit dengan akurasi rata-rata 76%. Metode segmentasi FCM-SVM mampu mensegmentasi citra daun tebu berpenyakit dengan akurasi tertinggi pada sebagian besar data uji coba (15 citra pada total 30 citra). Segmentasi penyakit pada citra daun tebu menggunakan metode FCM-SVM mampu mengatasi terutama untuk jenis penyakit yang mempunyai proyeksi warna kekuningan atau kecoklatan di sekitar daerah penyakit. DAFTAR PUSTAKA 1] Patil, Sanjay B., and Bodhe, Shrikant K. (Oktober - November 2011). Leaf Disease Severity Measurement Using Image Proc [1] Patil, Sanjay B., and Bodhe, Shrikant K. (Oktober - November 2011). Leaf Disease Severity Measurement Using Image Processing. International l f i i d h l O li 3 h l 29 301 di h // j l /ij /d / 11 03 05 01 df [1] Patil, Sanjay B., and Bodhe, Shrikant K. (Oktober - November 2011). Leaf Disease Severity Measurement Using Image Processing. International Journal of Engineering and technology. [Online]. 3 (5), hal. 297-301. Tersedia : http://www.enggjournals.com/ijet/docs/IJET11-03-05-017.pdf. g g gy ( ) p ggj j p [2] Al-Trawneh, Mokhled. (2013). An empirical Ivestigation of Olive Leave Spot Disease Using Auto-Cropping Segemention Fuzzy C-Means Classfica- tion. World Applied Science Journal. [Online]. 23 (9), hal. 1207-1211. Tersedia : http://www.idosi.org/wasj/wasj23(9)13/12.pdf. [2] Al-Trawneh, Mokhled. (2013). An empirical Ivestigation of Olive Leave Spot Disease Using Auto-Cropping Segemention Fuzzy C-Means Classfica- tion. World Applied Science Journal. [Online]. 23 (9), hal. 1207-1211. Tersedia : http://www.idosi.org/wasj/wasj23(9)13/12.pdf. [3] Wang, Xiang-Yang., Wang, Qin-Yang., Yang, Hong-Ying., and Bu, Juan. (November 2011). Color image segmentation using automatic pixel classi- fication with support vector machine. Neurocomputing. [Online]. 74 (18), hal. 3989-3911. Tersedia : http://www.sciencedirect.com/science/article/pii/S0925231211004474. [3] Wang, Xiang-Yang., Wang, Qin-Yang., Yang, Hong-Ying., and Bu, Juan fication with support vector machine. Neurocomputing. [Online]. 74 (18 http://www.sciencedirect.com/science/article/pii/S0925231211004474. [4] Ranghoo-Sanmukhiya, V.M., Dookhun-Saumtally, A. (February 2007) . Molecular characterisation of Mycovellosiella koepki, the causal agent of yellow spot disease of sugarcane. African Journal of Biotechnology. [Online]. 6 (3), hal. 198-203. Tersedia : https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CBoQFjAA&url=http%3A%2F%2Fwww.ajol.in fo%2Findex.php%2Fajb%2Farticle%2Fdownload%2F56137%2F44584&ei=1mv0U8TeI8Pi8AXu0YGgBQ&usg=AFQjCNEmn98Pq5UOivqrCrS6p SfSOIU-4A&sig2=U10H_eqdsMKSIWqiLqLUGA&bvm=bv.73231344,d.dGc. [4] Ranghoo-Sanmukhiya, V.M., Dookhun-Saumtally, A. (February 2007) . Molecular characterisation of Mycovellosiella koepki, the causal agent of yellow spot disease of sugarcane. African Journal of Biotechnology. [Online]. 6 (3), hal. 198-203. Tersedia : https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CBoQFjAA&url=http%3A%2F%2Fwww.ajol.in fo%2Findex.php%2Fajb%2Farticle%2Fdownload%2F56137%2F44584&ei=1mv0U8TeI8Pi8AXu0YGgBQ&usg=AFQjCNEmn98Pq5UOivqrCrS6p SfSOIU-4A&sig2=U10H_eqdsMKSIWqiLqLUGA&bvm=bv.73231344,d.dGc. y p g gy [ ] ( ), https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CBoQFjAA&url=http%3A%2F%2Fwww.ajol.in fo%2Findex.php%2Fajb%2Farticle%2Fdownload%2F56137%2F44584&ei=1mv0U8TeI8Pi8AXu0YGgBQ&usg=AFQjCNEmn98Pq5UOivqrCrS6p SfSOIU-4A&sig2=U10H_eqdsMKSIWqiLqLUGA&bvm=bv.73231344,d.dGc. https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CBoQFjAA&url=http%3A%2F%2Fwww.ajol.in fo%2Findex.php%2Fajb%2Farticle%2Fdownload%2F56137%2F44584&ei=1mv0U8TeI8Pi8AXu0YGgBQ&usg=AFQjCNEmn98Pq5UOivqrCrS6p SfSOIU-4A&sig2=U10H_eqdsMKSIWqiLqLUGA&bvm=bv.73231344,d.dGc. g q q q [5] Raid, RN., Comstock, JC. (2007) . Sugarcane Rust Disease 1. [Online]. University of Florida IFAS Extension. Tersedia : https://edis.ifas.ufl.edu/pdffiles/SC/SC00700.pdf. [5] Raid, RN., Comstock, JC. (2007) . Sugarcane Rust Disease 1. [Online]. University of Florida IFAS Extension. Tersedia : https://edis.ifas.ufl.edu/pdffiles/SC/SC00700.pdf. p p p [6] Ring Spot. Sugar Research Australia. [Online]. Tersedia [6] Ring Spot. Sugar Research Australia. [Online]. Tersedia : http://www.sugarresearch.com.au/icms_docs/164894_Ring_spot_IS13117.pdf [7] Eye spot Disease. [Online]. Tersedia : http://www.vsisugar.com/india/agriculture_divisions/plantpathology/eyespotdisease-sugarcane.htm [8] Plant Pathology Section (Agriculture Division). Tersedia : [7] Eye spot Disease. [Online]. B. Diskusi Sejumlah lima data uji coba memiliki akurasi tertinggi dalam segmentasi menggunakan metode FCM. Sisa-nya 25 data memiliki akurasi tertinggi dalam segmentasi citra menggunakan metode kombinasi FCM dan SVM. Akurasi tertinggi yang diraih oleh metode FCM disebabkan karena kombinasi data citra yang digunakan dalam tiap model. Akan tetapi kelima data ini tidak mempunyai jarak signifikan yang terlalu jauh, sehingga tetap saja metode FCM-SVM mendominasi akurasi tertinggi. Dari ke-25 akurasi tertinggi yang diraih pada segmentasi menggunakan FCM-SVM memiliki per- bandingan yang cukup jauh dengan metode FCM. Misalkan saja pada citra no. 19. Metode SVM mencapai akurasi 71,25%, sedangkan metode FCM-SVM mampu mencapai akurasi 92,96%. g p p Visualisasi perbandingan metode FCM dan FCM-SVM dapat dilihat pada Tabel 3. Groundtruth penyakit pada citra daun tebu dibuat menggunkan Adobe Photoshop dengan brush tool dan paint bucket tool. Dalam tabel tersebut terdapat dua citra yaitu citra no.26 yang mempunyai jenis penyakit yellow spot dan citra no. 27 yang mempunyai jenis penyakit rust spot. Jenis penyakit yellow spot lebih baik tersegmentasi menggunakan metode FCM-SVM. Penyakit ini memiliki ciri-ciri penyakitnya menyebar dan tidak terlalu berkumpul secara jelas. Se- dangkan citra yang kedua merupakan jenis penyakit rust spot dimana mempunyai karakteristik menyerupai garis tipis atau tebal, serta memiliki daerah proyeksi berwarna kekuningan di daerah sekitar penyakit. TABEL 3 HASIL SEGMENTASI No Citra GroundTruth FCM-SVM FCM 26 27 51 51 DAFTAR PUSTAKA Tersedia : http://www.vsisugar.com/india/agriculture_divisions/plantpa [7] Eye spot Disease. [Online]. Tersedia : http://www.vsisugar.com/india/ [8] Plant Pathology Section (Agriculture Division). Tersedia : de to Understanding Color Communication. [Online]. Tersedia : http://www.xrite.com/documents/literature/en/L10-001_Under- 9] X-rite. (2007). A Guide to Understanding Color Communication. [Online]. Tersedia : http://www.xrite.com/documents/literatu stand_Color_en.pdf [9] X-rite. (2007). A Guide to Understanding Color Communication. [Online]. Tersedia : http://www.xrite.com/d stand_Color_en.pdf [10] Santosa, Budi. “Tutorial Support Vector Machine”. Teknik Idustri, ITS. [Online]. 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Engineering Application of Artificial Intelligence . [Online]. 25 (8), hal. 1656-1669. Tersedia : http://www.sciencedirect.com/science/article/pii/S0952197612002412 p q y g [11] Yang, Hong-Ying., Wang, Xiang-Yang., Zhang, Xian-Yin., and Bu, Juan. (Ddecember 2012). Color texture segmentation based on image pixel classification. Engineering Application of Artificial Intelligence . [Online]. 25 (8), hal. 1656-1669. Tersedia : http://www.sciencedirect.com/science/article/pii/S0952197612002412 ., Zhang, Xian-Yin., and Bu, Juan. (Ddecember 2012). Color textu 52 52
https://openalex.org/W4318777944	https://riviste.fupress.net/index.php/rifp/article/download/2020/1332	English	null	Politics in the Anthropocene: Non-human Citizenship and the Grand Domestication	Rivista italiana di filosofia politica	2,023	cc-by	13,746	Copyright: © 2022 Gianfranco Pellegrino. This is an open access, peer-reviewed article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY-4.0). Il tema: Antropocene e post-antropocene Il tema: Antropocene e post-antropocene Firenze University Press www.fupress.com/rifp Rivista Italiana di Filosofia Politica SOCIETÀ ITALIANA FILOSOFIA POLITICA Firenze University Press www.fupress.com/rifp Rivista Italiana di Filosofia Politica SOCIETÀ ITALIANA FILOSOFIA POLITICA Rivista Italiana di Filosofia Politica Rivista Italiana di Filosofia Politica Firenze University Press www.fupress.com/rifp Firenze University Press www.fupress.com/rifp 1 Davies, The Birth of the Anthropocene; Ellis, Anthropocene; Lewis and Maslin, The Human Planet. 2 Zalasiewicz et al., The Anthropocene as a Geological Time Unit. 3 Hamilton, Defiant Earth, chap. 1; Horn and Bergthaller, The Anthropocene; Merchant, The Anthropocene and the Humanities. 4 Rockström et al., “Planetary Boundaries.” 5 I’ll come back to this topic, i.e., a socio-political definition of the Anthropocene, in §2 below. See also the references in that section. I thank an anonymous referee of this journal for having La politica nell’Antropocene. Cittadinanza non umana e la grande domesticazione Gianfranco Pellegrino Luiss University gpellegrino@luiss.it ORCID: 0000-0002-8029-3936 Gianfranco Pellegrino Luiss University gpellegrino@luiss.it ORCID: 0000-0002-8029-3936 Luiss University gpellegrino@luiss.it ORCID: 0000-0002-8029-3936 Abstract. The article has two aims. First, it provides a view of why the standard liberal-democratic political theory is unfit for the Anthropocene. Then, it defends two claims: that the fittest politics for the Anthropocene is to be fully non-anthro- pocentric and that the best model of a non-anthropocentric political theory is to be grounded in the notion of ‘ecological citizenship’, which can be easily extended to non-human living beings and even to non-living objects, such as ecosystems. The latter claim is defended by endorsing and enlarging Sue Donaldson and Will Kymlicka’s views of animal citizenship rights and by putting forward a view of the Anthropocene as an age of massive domestication of non-human nature. Keywords: anthropocene, politics, anthropocentrism, citizenship, domestication, Sue Donaldson, Will Kymlicka. Riassunto. L’articolo ha due scopi. In primo luogo, spiegare perché la politica libe- ral-democratica tradizionale è inadatta alle condizioni dell’Antropocene. Quindi, difendere due tesi: la teoria politica più adatta all’Antropocene dev’essere totalmente non antropocentrica e il miglior modello di teoria politica non antropocentrica si fonda sulla nozione di ‘cittadinanza ecologica’, che si può facilmente estendere agli esseri viventi non umani e persino a oggetti non viventi, come ad esempio gli eco- sistemi. Quest’ultima tesi viene difesa riprendendo ed estendendo la teoria di Sue Donaldson e Will Kymlicka sui diritti di cittadinanza degli animali e descrivendo l’Antropocene come età della domesticazione massiccia della natura non umana. ISSN 2785-3330 (print) \| DOI: 10.36253/rifp-2020 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Copyright: © 2022 Gianfranco Pellegrino. This is an open access, peer-reviewed article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY-4.0). 132 Gianfranco Pellegrino Parole chiave: Antropocene, politica, antropocentrismo, cittadinanza, domestica- zione, Sue Donaldson, Will Kymlicka. Parole chiave: Antropocene, politica, antropocentrismo, cittadinanza, domestica- zione, Sue Donaldson, Will Kymlicka. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 1 Davies, The Birth of the Anthropocene; Ellis, Anthropocene; Lewis and Maslin, The Human Planet. 2 l l Th h l l 1. Introduction. The Anthropocene as a Political Issue According to many, the Anthropocene is a state shift of the Earth system, which may amount to a novel geological age and a rupture in the functioning of the Earth system.1 From the geological point of view, the Anthropocene is supposed to be a novel geological age, i.e., a time in which relevant changes occur at the stratigraphic level. However, geolo- gists are still discussing (at least) two issues: first, whether at the strati- graphic level relevant changes are really occurring, and whether they are substantial enough to label a new geological age; second, when and where these changes happened.2 However, a non-geological definition of the Anthropocene can be framed – and it has been framed.3 According to this definition, the Anthro- pocene is a new epoch in which the size of human impacts on non-human nature is unprecedented, and the state-shift of our world mentioned in many discourses about the Anthropocene amounts to this novel and increased impact of humans on non-human nature.4 So understood, the Anthropocene is the epoch in which the usual equilibrium between nature and society, or nature and culture, changes, in at least two ways. First, it is no longer the case that the working of human society can happen on the background of an unchanging, inert, and inexhaustible nature. Second, it is no longer the case that the standard conceptual distinctions between non-human nature and society or culture hold. Indeed, most of the natural resources on which human societies have been based in the last two centuries are doomed to deplete, the environmental impacts of the fossil fuels-based economy are increasingly dangerous for human beings, and the pervasive impacts of humans on non-human nature made the latter more a human creation than an independent force. This new cultural and social condition is unprecedent- ed, and this very fact licenses calling it a new social age.5 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 133 Of course, there is no logical connection between the social defini- tion of the Anthropocene and the geological understanding of it. Or, at least, there is no biconditional implication. While, if changes at the strati- graphic level are so great to authorize labelling a new geological age, then social changes could be relevant as well; the contrary does not hold. pushed me to clarify at this stage what a socio-political definition of the Anthropocene would amount to. 6 I thank an anonymous referee of this journal for having attracted my attention to the lack of logical connection between the geological and the sociological definitions of the Anthropo- cene. 7 Braje and Erlandson, “Looking Forward, Looking Back.” 8 Chakrabarty, “The Human Condition in the Anthropocene”; Chakrabarty, The Climate of History, chap. 1. 9 Ellis, Anthropocene, 130. On the discussion concerning the Anthropocene as a geological period, Hamilton, Defiant Earth, 6–7; Zalasiewicz et al., The Anthropocene as a Geological Time Unit. On the debates concerning the nature of the Anthropocene and the very notion of ‘Anthropocene’, Altvater et al., Anthropocene or Capitalocene?; Bonneuil and Fressoz, The Shock of the Anthropocene; Malm and Homborg, “The Geology of Mankind?” On the distinction between a geological and a social Anthropocene, Haff, “The Technosphere,” 138. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 6 I thank an anonymous referee of this journal for having attracted my attention to the lack of logical connection between the geological and the sociological definitions of the Anthropo- cene. 10 I focus mainly on political theory. On environmental politics, Dobson, Environmental Poli- tics. 11 Thanks to an anonymous referee for this journal for having pushed me to clarify my notion of ‘Holocene politics’. 12 On anthropocentrism and non-anthropocentrism in environmental ethics, Attfield, “Beyond Anthropocentrism”; Thompson, “Anthropocentrism. Humanity as Peril and Promise.” 13 Donaldson and Kymlicka, Zoopolis. 14 On the unavoidability of anthropocentrism in the Anthropocene, Hamilton, Defiant Earth, chap. 2. On Anthropocene and chauvinism, Haff, “Purpose in the Anthropocen,” 55; Haff, “The Technosphere.” 1. Introduction. The Anthropocene as a Political Issue It is possible that the increased human impacts on non-human nature, despite being relevant, are not enough to set a new geological age.6 However, for our present purposes, I assume that a social definition of the Anthropo- cene is plausible, whatever the decision will be about the Anthropocene as a geological time unit. According to many, the change deriving from the increasing human impacts on Earth is also a matter of politics, for at least two reasons. First, the Anthropocene and its consequences are anthropogenic, i.e., they have been caused by human (collective) action.7 Indeed, in the Anthropocene, humans act as a planetary geological force (much like the biosphere in the Earth system dynamics), thereby going beyond their biological standard behaviour in pre-Anthropocene conditions. In the Anthropocene, humans become a geological power, i.e., a collective agent able to willfully affect the conditions of life on Earth. Politics is the realm where human collec- tive and individual action and power lie, and political theory is the field in which it is to be assessed. Hence, the Anthropocene is a subject of politics and of political theory.t Second, the consequences of the Anthropocene will deeply, and often negatively, impact our associated life and its settled forms. Indeed, the Anthropocene is an unprecedented and deep change in human history and condition.8 Thus, the Anthropocene is a political problem and a polit- ical theory issue. The Anthropocene is not only a new geological age (if it is, as this is still a contested claim, as said above). It is also a new social and historical epoch, a real ‘epochalipse’.9 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 134 Gianfranco Pellegrino However, the Anthropocene is neither a traditional political sub- ject nor a standard problem of politics. Indeed, Anthropocene politics is a radical departure from the political traditions settled in the Holocene. Of course, the Holocene hosted a panoply of different political forms, and the State-based, post-Westphalian politics standardly considered in politi- cal theory belongs only to a small fraction of the Holocene. In what fol- lows, I argue that the political forms, and above all the political theories that have been standard during the last two centuries are unfitting to the new conditions we face in the Anthropocene.10 (Of course, this is a fur- ther ungrounded generalization. In the last two centuries, different styles of political theories flourished. 1. Introduction. The Anthropocene as a Political Issue For present purposes, I am assuming that these differences can be neglected.) I call the standard form of politics and political theory which we need to discard the ‘Holocene politics’, to emphasize that the new politics, the Anthropocene politics, is motivated by the epoch change in front of us. (I say more on the Holocene politics in the next sections.)11 In this article, I defend two claims. First, a political theory for the Anthropocene should be fully non-anthropocentric, i.e., it should give full moral standing to non-human beings.12 Second, a non-anthropocentric pol- itics for the Anthropocene should focus on the relations between humans and non-humans and this could be done by applying the notion of ‘citizen- ship’ to non-human creatures and ecosystems and by extending Sue Don- aldson and Will Kymlicka’s view of animal rights to other living beings and ecosystems, thereby framing a theory of ‘ecological citizenship’.13 The politics of the Anthropocene should be a politics for non-human citizens. Contrary to what many claim, the Anthropocene should mark the end of anthropocentrism. The Anthropocene has been the age of Man (chauvinism intended!), but it should become the age of the recognition of non-humans, the age of the demotion of human beings from centre stage.14 I defend these claims by giving a view of the Anthropocene as the age when the relations between humans and non-human nature took the shape of a Grand Domes- tication. The latter has obvious ethical and political consequences, that a politics for the Anthropocene should duly consider. 10 I focus mainly on political theory. On environmental politics, Dobson, Environmental Poli- tics. 11 Th k f f h l f h h d l f Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 135 The paper develops as follows. In §2, I show why the traditional lib- eral-democratic politics that has been standard in the last two centuries (from now on, ‘Holocene politics’) is unfit for the Anthropocene and why a politics for the Anthropocene should be fully non-anthropocentric. This argument for a fully non-anthropocentric Anthropocene politics rests on Dipesh Chakrabarty’s well-known four theses on history after anthropo- genic climate change.15 Drawing on Chakrabarty’s theses, I put forward four theses about how politics should look in the Anthropocene. 2. Politics in the Anthropocene In this section, I defend two claims. First, Holocene politics is unfit for the Anthropocene. Second, the Anthropocene demands a fully non- anthropocentric politics. In the next section, I will claim that the best non-anthropocentric politics for the Anthropocene should be based on the notion of citizenship – a non-human, or ecological, citizenship. 2.1. Holocene politics is unfit for the Anthropocene 1. Introduction. The Anthropocene as a Political Issue In §3, I defend the view that a non-anthropocentric politics fitting the Anthropo- cene condition should be based on the idea of non-human, or ecological, citizenship. This section articulates the idea of the Anthropocene as the age of the Grand Domestication of non-human nature and of the ethical and political consequences of it. §4 concludes. 15 Chakrabarty, The Climate of History, chap. 1. 16 On this assumption, its credentials and its functions, Latour, We Have Never Been Modern; Latour, Politics of Nature. yh f y p 16 On this assumption, its credentials and its functions, Latour, We Have Never Been Modern; Latour, Politics of Nature. 2.1. Holocene politics is unfit for the Anthropocene Holocene politics rested on the following assumptions: Holocene politics rested on the following assumptions: 1. Nature is outside politics: The state of the Earth system is a mere background condition of politics, whose stability and endurance are not at risk and are not impacted by human action. Natural laws are not political laws, nor can they be changed by political acts of will. Human deeds and human societies occupy centre stage, and natural environments, the background. Hence, non-human nature lies outside of political theory, nor is it a political problem16 2. Agency: Political communities are constituted by political agents, i.e., by human beings who initiate and conduct political action and whose inter- ests are what matters politically. These agents are mutually accountable Rivista Italiana di Filosofia Politica 3 (2022): 131-160 136 Gianfranco Pellegrino 3. Responsibility: Individuals can be regarded as uniquely (causally and morally) responsible for certain outcomes and accountable for themi 3. Responsibility: Individuals can be regarded as uniquely (causally and morally) responsible for certain outcomes and accountable for themi 3. Responsibility: Individuals can be regarded as uniquely (causally and morally) responsible for certain outcomes and accountable for themi y 4. Politics is artificial or cultural: Because of 1.-3., politics lies in the realm of culture, human artifice, and social construction, and it is insulated from the mechanics of nature, and political theory is a social or human science, to be distinguished from the natural sciences that provide the best descriptions available of non-human and (a part of) human nature. 4. Politics is artificial or cultural: Because of 1.-3., politics lies in the realm of culture, human artifice, and social construction, and it is insulated from the mechanics of nature, and political theory is a social or human science, to be distinguished from the natural sciences that provide the best descriptions available of non-human and (a part of) human nature. The Anthropocene challenges the above assumptions. In the Anthro- pocene, the natural conditions of politics are unsettled, and the equi- librium that led to the current stability of world politics is doomed. The Anthropocene had an impact on current liberal-democratic politics and on some of its deficits. In addition, liberal-democratic politics itself can be a cause of the rupture of the natural equilibrium of the Holocene. 17 Chakrabarty, The Climate of History, 32. On the responsibility of standard politics and polit- ical theory and culture for the ecological crisis, Plumwood, Environmental Culture; White Jr, “The Historical Roots of Our Ecologic Crisis.” On the history of fossil fuel-based economy in the Anthropocene, and its impact on democratic politics and its deficits, Malm, Fossil Capital; Sachs, Common Wealth; Mitchell, Carbon Democracy. 18 Ellis, Anthropocene, chap. 1; Hamilton, Defiant Earth, 62; Lynas, The God Species. 19 Hamilton, Earthmasters, 2; Hamilton, Defiant Earth, 85; Latour, Facing Gaia. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 2.1. Holocene politics is unfit for the Anthropocene As Dipesh Chakrabarty famously intimated, “the mansion of modern free- doms stands on an ever-expanding foundation of fossil-fuel use.”17 In the Anthropocene, nature is no longer outside politics. In the Anthropocene, agency chains become dispersed, indirect, and globally diffused: the unintended consequences of collective human actions on the natural environment and their feedback make the conditions of politics and associated life more troubled and unstable than ever. In addi- tion, the impact of political action on nature is unprecedented, and human political agents and natural non-agents are increasingly entangled. We are no longer inhabitants of the planet but Earth-changers, or world-makers.18 In the Anthropocene, “the fate of nature has come to depend on the ‘good- will’ of humans”, and “the Earth system itself has acquired a moral force”: the dormant forces of the Earth system have been activated by an unprec- edented human power.19 If humans, their behaviour, and human societies are the main subject matter of politics, now politics becomes the history and a study of how humans changed their world. In the Anthropocene, politics is no longer a purely human or social science. It is also natural history, as it were, or a view of the place of human beings in the natural world. Moreover, in the Anthropocene, new forms of agency emerge. Collec- tive agents – human collectives, such as multinational corporations, global political and economic institutions, and non-human entities, such as eco- Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 137 systems, the biosphere, and non-human species – are now centre stage. Indeed, what earlier appeared as mere behaviour (for instance, the behav- iour of non-human animals, plants, artificial intelligences, or even of the ‘technosphere’) now increasingly qualifies as full-fledged acting.20 Finally, merely possible agents – for instance, future people – acquire relevance since our actions can have dangerous and pervasive impacts on them.21 In the Anthropocene, the Earth system at large becomes a political actor, nature itself acts as a political agent.22 In the Anthropocene, standard collective action problems, such as the tragedy of the commons and other issues concerning public goods or vot- ing, acquire a planetary and an intergenerational extension. 20 On animal and plant agency and their political impact, see Donaldson and Kymlicka, Zoo- polis; Hall, Plants as Persons; Marder, Plant-Thinking. On collective and corporate agency, List and Pettit, Group Agency. On the agency of the technosphere and non-human agency in the Anthropocene, Haff, “Purpose in the Anthropocene;” Haff, “The Technosphere.” 21 De-Shalit, Why Posterity Matters; González-Ricoy and Gosseries, Institutions For Future Gen- erations; Mulgan, Future People; Page, Climate Change, Justice And Future Generations; Schef- fler, Why Worry About Future Generations? 22 Chakrabarty, The Climate of History, 3; Hamilton, Defiant Earth. 23 Chakrabarty, The Climate of History, 37; Gardiner, A Perfect Moral Storm; Hamilton, Defiant Earth, 129; Jamieson, Reason in a Dark Time; Malm, Fossil Capital. 24 Pellegrino, “Robust Individual Responsibility”; Jamieson, “Climate Change, Responsibili- ty, and Justice”; Malm, The Progress of This Storm; Pattberg and Zelli, Environmental Politics Rivista Italiana di Filosofia Politica 3 (2022): 131-160 2.1. Holocene politics is unfit for the Anthropocene On the one hand, no one can fix the global environmental problems of the Anthro- pocene by acting alone, nor can they do it by activist influence on others; on the other hand, it is the cumulative impact of billions of single actions (such as consuming, driving, and so on) that creates the most dangerous environmental harms. This holds for the individual actions of people but also for the individual actions of companies, groups, states, and genera- tions: most of the individuals cannot prevent the most dangerous environ- mental effects of the cumulated pattern of conduct of which some of their actions themselves are a constituent part.23 Moreover, while anthropogenic processes cause environmental damag- es, no one is individually responsible for them. The causal chains are mani- fold, multiscale, indirect, and loose. They are often a matter of chances, tip- ping points, and background conditions. The Anthropocene is a matter of species’ action, not of individual or limited group action. Some of the most dangerous events in our future are the unintended consequences of various and seemingly innocuous events in the distant past. Individual action and responsibility happen, and are lost, within a sea of agency, where non-indi- vidual, non-human, and human entities join a complex network of causes and cumulative, non-linear effects. This makes the standard conception of political responsibility no longer viable in the Anthropocene.24 138 Gianfranco Pellegrino Given the above, in the Anthropocene the traditional dichotomies between political theory (and social and human sciences) and natural sci- ences blur. In the Anthropocene, human beings become planetary geologi- cal agents, while keeping their political agency – indeed, political agency becomes planetary agency. In the Anthropocene, an increasingly large part of nature is human-made, as politics has been used to be (regarded) in modernity. Moreover, human history and politics impacted and will be impacted by planetary processes.25 Hence, in the Anthropocene the long geological history mixes up with the short human political history, the human timescales will interact with non-human timescales in the same historical flux, and different, or even divergent, points of view are neces- sary to deal with planetary events.26 In the Anthropocene, we are part of the background of our history, and the background is no longer inert or unchangeable. Our action goes along with the impersonal operation of natural forces, and the effects of both are mixed. and Governance; Powers, “Individual Moral Responsibility”; Sinnott-Armstrong, “It’s Not My Fault.” 25 Chakrabarty, The Climate of History, 27. 26 Chakrabarty, The Climate of History, 6–7, 10–11, 15, 26; Kelly, Politics and the Anthropocene, 5; Malm, Fossil Capital, 17; McNeill and Engelke, The Great Acceleration; Steffen et al., “The Trajectory of the Anthropocene.” 27 Chakrabarty, The Climate of History, 29. On tipping points, Barnosky et al., “Approaching a State Shift”; Gladwell, The Tipping Point; Lenton et al., “Tipping Elements”; Lenton and Wil- liams, “On the Origin of Planetary-Scale Tipping Points”; Rockström et al., “Planetary Bound- aries.” and Governance; Powers, “Individual Moral Responsibility”; Sinnott-Armstrong, “It’s Not My Fault.” 25 h k b Th l f Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 2.1. Holocene politics is unfit for the Anthropocene Moreover, the speed of nature’s changes is increased, and when certain tipping points are over- come, irreversible changes follow.27 Therefore, politics and political the- ory need to deal with the political consequences and relevance of issues (such as climate change, the impacts of human technology on non-human nature, and so on) traditionally considered the exclusive field of the nat- ural sciences. In the Anthropocene, world politics becomes the politics of the world, understood as the politics of the planet we live in. In the Anthropocene, politics needs to access the level of Earth System thinking. In the Anthropocene, global and domestic liberal democratic institu- tions are very often gridlocked. Due to the global nature of current envi- ronmental problems, cooperation among states and new global institu- tions is absolutely necessary. But the working of the existing global insti- tutions, as well as the creation of new ones, is increasingly difficult as short-term interests diverge, and long-term interests are unable to affect daily politics. Domestically, many relevant nation states are gridlocked by veto players determined by constitutional arrangements and political practices that originated in the last two centuries, in the post-Westphali- an part of the Holocene, and are now unfit for the levels of flexibility and Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 139 international cooperation required in the Anthropocene. There is a strik- ing contradiction between a global economy and divided political socie- ties across the world. The Anthropocene makes contradictory requests to liberal democracies. On the one hand, it needs more cooperation and flexibility in governance, which may be obtained with less democratic and more centralized political procedures. On the other hand, the Anthropo- cene makes new political subjectivities (even non-human ones) emerge, thereby requiring new and larger procedures of democratic representation and interests’ composition. 28 In virtue of the above, politics and political theory can no long- er be the same in the Anthropocene. The Anthropocene requires us at least to update, but possibly to change radically, our views of the nature of politics, as well as of the kind of values and norms that politics deals with, and of the demos and the forms of government. 28 In the previous paragraphs, I borrowed from Dryzek, The Politics of the Anthropocene; Hale, Held, and Young, Gridlock; Jamieson and Di Paola, “Political Theory for the Anthropocene”; Sachs, Common Wealth; Wainwright and Mann, Climate Leviathan. 29 Hamilton, Defiant Earth, 15. 30 Gupta, The History of Global Climate Governance; Jamieson, Reason in a Dark Time. 2.1. Holocene politics is unfit for the Anthropocene As Clive Hamil- ton remarks, “the Anthropocene rupture will require original political thinking.”29 Standard forms of politics and political theory, as they have been developed and established in the last two centuries of human histo- ry, are unfit for the Anthropocene. Holocene politics is short-termist, too focused on nation-States and on human societies, and blind to the inter- action of human collective behaviour and non-human nature’s working. Moreover, it totally neglects the value and the demands of non-human subjects. This made it unfit for the new age. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 2.2. Four Theses About Anthropocene Politics Some have proposed substantial changes in Holocene politics to deal with the new features of the Anthropocene. For instance, according to some authors, like global climate change, the Anthropocene is a con- sequence of globalization, and it needs new global political institutions and new global policies, the only things able to ensure coordinated global action. Then, a politics for the Anthropocene should rely on the lessons learned in dealing politically with climate change, lessons concerning the intra- and intergenerational tragedy of the commons related to miti- gation, and adaptation and the need for long-sighted global institutions able to promote international agreements and their enforcement.30 The model for Anthropocene politics should be the Montreal Protocol and the Rivista Italiana di Filosofia Politica 3 (2022): 131-160 140 Gianfranco Pellegrino Paris Agreement: international governance mechanisms and institutions should address human alterations of the Earth system. The solution to the Anthropocene problems lies in global government, global institutions, and global regulation. 31 h This general view has been spelled out in many (sometimes contrast- ing) ways. Some authors claim that a politics for the Anthropocene should provide political and market incentives for better distribution of environ- mental goods and sustainable development. The primary means for this are global political cooperation, more flexible and long-sighted political institutions, and/or the development of mass movements promoting envi- ronmental protection goals and the global redistribution of environmental goods. Some claim that putting environmental rights into national consti- tutions or a global constitutional arrangement is the best way to imple- ment a politics for the Anthropocene. Protection goals are necessary to preserve ‘ecosystem services’ for humans.32 Others saw the Anthropocene (labelled as the Capitalocene) as the most recent and the worst outcome of commercial capitalism, colonialism, and racism, emerging in the commercial societies of the seventeenth cen- tury with the European colonization of the Americas. The exploitation of nature and the spoiling or waste of the Earth, which are the main features of modernity, are consequences of the exploitation of workers and the deep inequalities of modern capitalism. The rapacious attitude to nature is a counterpart and a result of patriarchal chauvinism. Responsible for our current ecological crisis is not the species, but male, Western, modern capitalists. 32 Dryzek, The Politics of the Anthropocene. On sustainable development and global political cooperation as a model of Anthropocene politics, Sachs, Common Wealth. On the role of lim- its, Hamilton, Defiant Earth, 132. On environmental justice and environmental citizenship, Di Chiro, “Environmental Justice and the Anthropocene Meme”; Dobson, Citizenship and the Environment; Holifield, Chakraborty, and Walker, The Routledge Handbook of Environmental Justice; Hayward, “Ecological Citizenship”; Sandler and Pezzullo, Environmental Justice and Environmentalism; Schlosberg, Defining Environmental Justice; Walker, Environmental Justice, Amirante and Bagni, Environmental Constitutionalism in the Anthropocene; Barry, “Towards a Green Republicanism”; Collins, The Ecological Constitution; Kotz, Global Environmental Con- stitutionalism; May, Global Environmental Constitutionalism. 31 A taxonomy of pre-Anthropocene environmental political theories is in Dryzek, Politics of the Earth.h Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 2.2. Four Theses About Anthropocene Politics Nature has been considered separate from Society, inert and subaltern, as a mere resource without limits and available at no price (this is the idea of ‘cheap Nature’), and through the dominion of Nature (and the underlying Cartesian ontological dualism), the dominion of subaltern classes and women, with its accompanying inequalities and injustices, has been established. Capitalism originated in the web of life: nature affected capitalism, and capitalism affected nature. Humans are inextricably entan- 31 A taxonomy of pre-Anthropocene environmental political theories is in Dryzek, Politics of the Earth.h Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 141 gled with nature, and nature is necessarily blended with humans. A poli- tics of the Anthropocene should be emancipatory politics, where social justice and freedom will be achieved both for humans and non-human nature, with awareness of their necessary blended metabolism. Environ- mental justice goes hand in hand with social justice and the discarding of the current capitalistic production modes.33h The views above rest on the assumption that the Anthropocene poli- tics should be a kind of green or emancipatory democracy. Some doubted that democratic regimes and procedures can be fit to the Anthropocene. According to some authors, the Anthropocene needs coordinated action, far-reaching and long-sighted planning, some sacrifices of the wealthy and present people to the benefit of poor or disadvantaged present and future people, efficient coordination to deal with the tragedy of the commons at various levels, and across generations, dealing with vast bodies of com- plex information. This goes against many features of democratic politics as we know – its short-termism, its dependence on voluble and ignorant constituencies, its liability to irrational biases, and its difficulty in man- aging complex information. Hence, we can successfully cope with the Anthropocene only by establishing an authoritarian regime of experts and enlightened élites, able to push people to coordinated action towards cer- tain specific goals. In the Anthropocene, any government, even a harsh- ly authoritarian one, is better than anarchy – where the Anthropocene’s anarchy is not merely civil war, but civil war derived from the shortage of natural resources and worsening of the world’s natural conditions.34h The views above share anthropocentric grounds. Even when they advocate respect and protection for non-humans, this is mostly ground- ed in the instrumental value of the latter for humans. 33 Benton, The Greening of Marxism; Foster, Marx’s Ecology; Foster, The Ecological Rift; Klein, This Changes Everything; Malm and Homborg, “The Geology of Mankind?” Malm, The Prog- ress of This Storm; Moore, Capitalism in the Web of Life. 34 Flannery, The Weather Makers; Lovelock, A Rough Ride to the Future, 119–20; Plumwood, Environmental Culture, 62–65; Radcliffe and Campling, Green Politics; Shearman and Smith, The Climate Change Challenge; Wainwright and Mann, Climate Leviathan. 35 Chakrabarty, The Climate of History, 24. Here, I do not embark on a detailed assessment or interpretation of Chakrabrarty’s theses, which have been extensively discussed since their orig- Rivista Italiana di Filosofia Politica 3 (2022): 131-160 inal appearance; Dube, Seth, and Skaria, Dipesh Chakrabarty and the Global South; Emmett and Lekan, “Whose Anthropocene?” 36 Chakrabarty, The Climate of History, 26, 31, 35, 43. 37 Hamilton, Defiant Earth, 51–52. 2.2. Four Theses About Anthropocene Politics It is my conten- tion here that these common anthropocentric presuppositions make these views unfit for the Anthropocene. A politics for the Anthropocene needs to be fully non-anthropocentric. This conclusion straightforwardly derives from a correct view of what the Anthropocene is and of its implications. The best view of the Anthropocene as a social phenomenon, and of its impacts on human culture, can be found in Dipesh Chakrabarty’s four theses on the impact of the Anthropocene on history as a discipline.35 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Gianfranco Pellegrino 142 Chakrabarty’s four theses are the following ones: Chakrabarty’s four theses are the following ones: 2. the idea of the Anthropocene severely qualifies humanist (i.e., whig- gish) histories of modernity/globalization 3. the age of the Anthropocene requires us to connect the global history of capitalism with the history of humans as a species 4. the above theses show that the current understanding of history (i.e., the Diltheyan model of historical writing) has limits and should be discarded.36 From the claims above the following theses can be derived: From the claims above the following theses can be derived: 1. in the Anthropocene, the standard distinction between human poli- tics (constituted of supposedly intentional human conduct in the realm of human freedom) and non-human behaviour, or events in the realm of natural necessity, collapsed37 2. the idea of the Anthropocene sets limits to the standard humanist and progressive view of politics. The non-human enters politics, and its flourishing may often contrast (and it often contrasted) with human flourishing l 3. the Anthropocene requires us to connect more closely human politi- cal communities and politics – whose main characters are capitalism and States – with non-human collectives and politics – whose main characters are evolution, co-evolutionary paths, niches, and the pros- pect of species extinction. The Anthropocene needs a non-humanistic and not exclusively human politics. It makes necessary interspecies politics, despite that in many cases, human interests contrast with non-human ones 4. the above theses show that the current understanding of politics (i.e., the model of Holocene politics) has limits and should be discarded. 4. the above theses show that the current understanding of politics (i.e., the model of Holocene politics) has limits and should be discarded. The theses above are implicated (even if not logically entailed) by Chakrabarty’s original theses. If natural history, i.e., the history of Earth and the forces acting on it, is no longer distinct from human history, as humans are now a geological force, then human politics (i.e., the inten- Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 143 tional behaviour of humans in governed societies) is no longer distinct from human action as a species. In the Anthropocene, the unintended consequences of human politics (for instance, its consequences on the environment and future generations) are to be considered a proper politi- cal topic, as well as the subject matter of a science of the by-product of human activities.38 Since human intentionality and awareness are no long- er necessary features of political action, then events and pieces of non- human conduct can also be considered genuine political actions.h The supposed progress of political freedom caused the current eco- logical crisis, being based on intensive fossil-fuel consumption: this is an instance of the general claim conveyed by theses 2 and 3 above. 38 Chakrabarty, The Climate of History, 34; Malm, Fossil Capital, 11. 39 Sachs, Common Wealth, chap. 6. 40 Baxter, A Theory of Ecological Justice, chap. 6, provides answers to these objections. 41 Ibid., chap. 5; Cochrane, Sentientist Politics; De Grazia, Taking Animals Seriously; Donald- son and Kymlicka, Zoopolis; Garner, A Theory of Justice for Animals; Zuolo, Animals, Political Liberalism and Public Reason. On policies, see art. 13 of the Treaty on the Functioning of the European Union (TFEU). 42 Baxter, A Theory of Ecological Justice, chaps. 5-6; Calvo, Planta Sapiens; Hall, Plants as Per- sons; Marder, Plant-Thinking; Rolston III, Environmental Ethics, chap. 5. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 42 Baxter, A Theory of Ecological Justice, chaps. 5-6; Calvo, Planta Sapiens; Hall, Plants as Per- sons; Marder, Plant-Thinking; Rolston III, Environmental Ethics, chap. 5. From the claims above the following theses can be derived: Applied to politics, this view amounts to emphasizing that Western liberties and their triumph had detrimental impacts not only on non-Western people or subaltern agents but also (and greatly) on non-humans and ecosystems.39 Human politics harms non-humans, and human progress often amounts to regress for non-humans. And this is thesis 2 above. Of course, thesis 2* is not a straightforward consequence of thesis 1, nor is it a direct coun- terpart of Chakrabarty’s thesis 2. It might be argued that, even though the human species is now one of the forces of nature, due to its unprec- edented impacts on Earth, this does not amount to harming, or better to wronging, anyone. For nature is inert, it is a mere thing, and non-human animals or other sentient beings have no moral standing. Thus, harm to them is not a wrong. Only human agents, or moral agents understood as reasonable persons capable of being autonomous sources of ends, can be entitled to justice. So, the very concept of a non-anthropocentric political theory and politics is a non-starter, and thesis 2 above is false.h The supposed progress of political freedom caused the current eco- logical crisis, being based on intensive fossil-fuel consumption: this is an instance of the general claim conveyed by theses 2 and 3 above. Applied to politics, this view amounts to emphasizing that Western liberties and their triumph had detrimental impacts not only on non-Western people or subaltern agents but also (and greatly) on non-humans and ecosystems.39 H l h h d h ft There is no space here to rebut these objections.40 For now, it is suf- ficient to say that the view that animals have moral standing is increas- ingly endorsed by theorists, and also by common sense morality. Indeed, this view is reflected in current policies.41 Moreover, there is an emerging trend to consider plants and ecosystems as sources of interests, or as hav- ing a good of their own.42 Thesis 2* rests on these views, which I do not defend here, simply taking their initial plausibility for granted. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 144 Gianfranco Pellegrino A rupture with the standard humanist history of politics is also impli- cated by the above, in three senses. First, in the Anthropocene, politics is no longer a humanistic discipline or a social science, but it is more and more related with the natural sciences. 43 Chakrabarty, The Climate of History, 36; Hamilton, Defiant Earth, 4, 61–62, 118, 119; Smail, On Deep History and the Brain; Zalasiewicz et al., “The New World of the Anthropocene.” 44 Baxter, A Theory of Ecological Justice; Gleeson and Low, Justice, Society and Nature; Wien- hues, Ecological Justice and the Extinction Crisis. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 From the claims above the following theses can be derived: (This is another corollary of the- sis 3 above.) Second, a politics focusing on the progressive advancement of humanistic values, i.e., of the good for human beings, is no longer enough in the Anthropocene because in it, politics should necessarily consider the good of non-human living and non-living beings, even allowing that some- times the latter can prevail over human good. Third, in the Anthropocene, politics is no longer an exclusively human story. There is a political history of the planet. Anthropocene politics is Gaian or planetary politics.h Thesis 3 above is the view that the consequences of the Anthropocene can be understood only by looking to “humans as a form of life”, or to “life in the human form,” and to “human history as part of the history of life on this planet,” or better by connecting human social life, and its capi- talist and globalization structure, with the history of humans as a species, as a universal Anthropos – the so-called ‘deep history’ of humankind, or better, a planetary history.43 Thesis 3* above extends this view to politics (and draws the consequences of thesis 2) by claiming that dealing with the consequences of the Anthropocene requires us to see non-humans as political partners and collectives, and their behaviour and life as a form of political life. Theses 2 and 3* vindicate the idea that a politics for the Anthropo- cene cannot but be a fully non-anthropocentric one, by giving non-human beings full political status and entitlements. If these theses are true, then a politics for the Anthropocene cannot be anthropocentric. Some non-anthropocentric models of Anthropocene politics have been recently put forward. For present purposes, we can distinguish two broad approaches. According to justice biocentrism, the community of justice should be extended, as all living beings can be holders of justice entitlements, and pollution, exploitation, degradation, and climate change are instances of misdistribution. Ecological justice is interspecies justice, i.e., the justice of the relations between humans and the rest of the world in sharing Earth’s material space. 45 Bennett, Vibrant Matter; Coccia, Metamorphoses; Descola, Par-delà nature et culture; Har- away, The Companion Species Manifesto; Haraway, Staying with the Trouble; Latour, Reassem- bling the Social; Morton, Hyperobjects; Morton, Dark Ecology; Morton, Humankind; Morton and Boyer, hyposubjects; Tsing, The Mushroom at the End of the World; Youatt, Interspecies Politics. From the claims above the following theses can be derived: Different kinds of relations between dif- ferent types of human and non-human beings give rise to different spheres of justice, and different kinds of entities have different kinds of entitle- ments to environmental resources.44 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 145 According to neo-materialism, the Anthropocene urges us to rediscov- er the common vitality, or the agency, running across the different entities on the planet. There are no different species, there is no human uniqueness, but rather a common life shared by each living being. This life is a continu- ous phenomenon, supervening upon both supposedly living and non-living beings. This is the real truth of the Darwinian evolutionary theory. This unity of life across different forms entails a democratic approach. No rank- ings and value differences can and should be established. Agency is not an exclusively human feature: agency can be distributed across nature – at least if seen as a matter of actants’ role and network of actors. There is no human nature, but rather human nature is the outcome of interspecies mutual rela- tions, where humans define and are defined, domesticate and are domesti- cated by non-humans. The Anthropocene is the age of hybrids or symbionts, of ‘weird’ and ‘uncanny’ mixtures of humans and non-humans, of new kinds of objects and subjects – hyper-objects and hypo-subjects. The Anthropocene is the realm of multiple ontologies, where the boundaries between nature and culture, and the extent of personhood, can be traced in various ways.45h The views listed above are fully non-anthropocentric, as they enlarge the sphere of politics and political subjects to non-human living beings and even to non-living beings such as ecosystems and the entire planet. As such, these views fit the Anthropocene condition. In particular, they conform with theses 2* and 3* above, and this makes them specifically suited to the Anthropocene. h i The argument I am giving here in favour of a non-anthropocentric politics of the Anthropocene can be spelled out as follows: i. The Anthropocene is a specific condition, where the traditional assumptions of Holocene politics no longer hold; then, i. The Anthropocene is a specific condition, where the traditional assumptions of Holocene politics no longer hold; then, ii. a new form of politics and political theory is needed, based on new assumptions; iii. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 From the claims above the following theses can be derived: one of these assumptions is that non-humans (be they non-human animals, or other sentient or even non-sentient beings, such as eco- systems) are to be considered when taking political decisions. To put it otherwise, these beings or collectives have moral standing, and this give them the status of objects of political concern; as a consequence, iv. a politics for the Anthropocene should give acknowledgment to non- human entities; a politics for the Anthropocene should be fully non- anthropocentric. iv. a politics for the Anthropocene should give acknowledgment to non- human entities; a politics for the Anthropocene should be fully non- anthropocentric. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 146 Gianfranco Pellegrino The two approaches listed above meet the requirement set by the anti- anthropocentrism argument. However, in the next section I argue that a view based on the notion of ‘ecological citizenship’ is the most plausible in the Anthropocene. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 3. Ecological Citizenship for the Anthropocene Justice biocentrism and neo-materialism rest on a common assump- tion, i.e., the idea that non-human sentient and non-sentient entities have moral status, and that they should be objects of political concern in virtue of their moral standing. This assumption may be problematic under many respects, as already said in § 2.2. However, here I take this assumption for granted. Nevertheless, these views are not fully fitting the Anthropocene condition. Justice biocentrism focuses on the distribution of environmental resources to human and non-human beings, while most of neo-materi- alists focus on the inclusion of non-human beings in the democratic dis- course and processes of representation in human politics. Despite these views being fully non-anthropocentric, they still miss capturing a specific feature of the Anthropocene, and this makes them not fully fitting for the Anthropocene. An unprecedented human impact on non-human nature is the main distinguishing feature of the Anthropocene. Of course, environ- mental degradation is (and has been for many years now) the main upshot of human impact. Then, it might be argued that the Anthropocene is not a real novelty: it is only the exacerbation of the ecological crisis that we are facing since a century, at least. But it is my contention that the Anthropo- cene, whatever its real starting point is, brings about a novelty which is ethically and politically relevant. First, in the Anthropocene the impact of our collective actions has an unprecedented size. Second, this size makes the difference, because humanity is no longer an agent acting on the back- ground of natural forces and events, but it acts as a new natural force, as a novel cause of unprecedented changes in non-human nature. Climate change is a case in point, here. Climate changes have always happened on Earth. Since their appearances, hominins and later humans have co- operated with the forces of nature in favouring or slowing down these changes. It might be argued that agriculture and deforestation contribut- ed to certain climatic changes. However, it was not the case that humans were a unique cause of these changes. What marks the current changes, instead, is their fully anthropogenic origin, i.e., the fact that they have been brought about by collective human action, the latter being a unique cause of them. 46 An anonymous referee for this journal took my view (in a previous draft) as a theory con- cerning the political representation of nature. Indeed, it is my contention that a politics for the Anthropocene is not simply a view of the representation of nature. Evidently, the first version of this article was unclear on this. 3. Ecological Citizenship for the Anthropocene Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 147 If the view above is correct, the impact of humans on non-human nature in the Anthropocene can be compared to human domestication of animals and plants. (I will come back to this issue in § 3.1. below.) It is a relation where human agency impacted non-humans, and sometimes this impact was harmful. This makes the relation between humans and non-humans in the Anthropocene ethically and politically relevant in a different way, as compared to the Holocene condition. Starting from this premise (in the Anthropocene, humans harmfully impacted non-humans in an unprecedented way), I defend two claims. First, a politics for the Anthropocene cannot be limited to issues concerning the just distribution of environmental resources and the political representation of nature;46 rather, it should take into account the ethically and politically relevant relations between humans and non-humans. Second, the best way to give a political view of the relations between humans and non-humans in the Anthropocene is by developing a view of non-human citizenship. Some clarifications are needed, though, on the premise for these views, i.e., the claim that the relations between humans and non-humans in the Anthro- pocene can be compared to human domestication of non-human nature. To this I turn in the next sub-section. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 3.1. The Anthropocene Grand Domestication As said, the Anthropocene is the age when the human impact on non-human nature reached an unprecedented size. This impact had sev- eral forms. It was a matter of environmental degradation of ecosystems, as well as of harming non-human animals in various ways related to envi- ronmental degradation, for instance with the destruction or deterioration of certain ecological niches, that caused losses and damages to flora and fauna settled in these niches. In the Anthropocene, then, humans caused non-humans to lose their habitats, to suffer various injuries, sometimes even to extinguish them. However, what is most typical of the Anthropo- cene is that the human impact very often determined irreversible chang- es in ecosystems and in non-human animals and plants living in them. The human impact on the Anthropocene can be compared to a gigan- tic domestication of the Earth. This is not only a metaphor. It might be argued that in the Anthropocene humans reproduced on a large scale the domestication processes they performed as a species since their very Rivista Italiana di Filosofia Politica 3 (2022): 131-160 148 Gianfranco Pellegrino appearance on Earth. Of course, this claim depends on the meaning of ‘domestication’ we assume. In recent discussions, ‘domestication’ took different meanings, and the very term had many different overtones in various disciplines.47 To present purposes, we can single three meanings out.48 Domestication can be consti- tutive, when it determines the breeding and other in-born characteristics of some animal and vegetal species. It can be locational, when it is a matter of animals and plants living (and having their habitat) in ecosystems greatly or fully affected by human activities. It can be dispositional or behavioural when it is a matter of certain conducts of animals or plants, namely, their being aggressive or fearful towards human beings, and so on. (Notice that the latter kind of domestication usually goes together with the first. Con- stitutively domesticated animals display certain patterns of behaviour, even though this is not necessarily the case. Sometimes, humans can create sup- posedly wild characteristics by purpose – for instance, aggressive dogs. Likewise, supposedly wild plants, for instance weeds, can be created, or artificially spread, for human purposes. This meaning of ‘domestication’ is not involved in my discussion below, though.) y g The Anthropocene greatly expanded locational domestication. 47 Cassidy, “Introduction: Domestication Reconsidered.” 48 Palmer, “Animal Ethics in Context,” 63-7. 49 McKibben, “The End of Nature.” Rivista Italiana di Filosofia Politica 3 (2022): 131-160 3.1. The Anthropocene Grand Domestication As Bill McKibben famously claimed, the Anthropocene is the age of the end of nature, i.e., the age where wild spaces (understood as pristine nature, or completely untouched ecosystems) disappeared from Earth.49 If so, some animals and plants, as already said, literally lost their ecological niche. This may yield three possible outcomes: i. adaptation, ii. maladaptation (i.e., imperfect adaptation, or adaptation at a suboptimal level of well-being, as compared to previous adaptation), iii. no adaptation, i.e., extinction. It might be argued that, assuming that animals and plants have a good of their own (and this is the assumption about the moral status of non-human beings I have often mentioned in this article), ii. is a wrong to them. It is not clear that iii. is also wrong to individuals or to groups, as it might be argued that complete extinction does not harm the members of the extinct species. However, the extinction of species can reduce biodiversity, and this in its turn may have several effects on remaining species. As a consequence, when locational domestication leads to extinction, then it may be regarded as harmful. Then, in expanding locational domestication, the Anthropocene is an instance of harmful relationship between humans and non-humans.h The Anthropocene increases constitutive domestication, as well. Or at least, some of the ways in which humans impacted non-human nature Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 149 in the Anthropocene involves constitutive domestication. For instance, mass domestication of animals for the sake of massive meat production is mainly based on constitutive domestication of animals. Intensive agri- culture, also, may be seen as a kind of massive constitutive domestication of plants, as it is based on the selection of certain varieties within species. However, it is far from clear that constitutive domestication is a harm to the domesticated species (even though it may be harmful to wild species, that can be crowded out by new domesticated ones, whose occupation of places and appropriation of food is warranted by human help, as it were). Indeed, domesticated animals can gain from domestication, in terms of chances to live, and of better conditions of living. It might be argued that, had it not been for certain human purposes (i.e., food production or other human-related usages), certain non-human individuals would have not lived. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 3.1. The Anthropocene Grand Domestication Assuming that living is better than not living, domestication can be a positive advantage for domesticated animals. Of course, this holds when the living conditions of animals and plants are good enough. Mass meat production is often led by keeping animals in very bad conditions. Inten- sive agriculture and monoculture may be detrimental to biodiversity, and to certain species, whose survival can be threatened by the privileging of the unique species that are farmed by humans. However, even assuming that domesticated animals and plants have good lives, constitutive domes- tication may create dependency and vulnerability. Their good lives heav- ily depend on human action. They can be unable to survive outside of a relation with humans (they lack self-sufficiency), and their very existence depended on human projects. Their dependency is permanent, enduring, and lifelong.h Three patterns of domestication, or co-existence, have been distin- guished in recent debates: mutualism (both parties benefit from the rela- tions), commensalism (animals or plants benefit, humans do not lose), contramensalism (animals or plants benefit at the expenses of humans). Animals’ and plants’ well-being depend on humans in each of these pat- terns. Even in contramensalism, if humans take measures to stop the situ- ation where animals or plants can parasitize them, very often the latter will be seriously damaged, for lack of viable alternatives – think of scaven- gers led to starving due to human migration and abandonment of towns. As a consequence of the above, the Anthropocene can be understood as a ground for increased constitutive and locational domestication of ani- mals and plants. However, there is another sense in which Anthropocene is a factor of domestication. The above patterns of domestication do not exhaust the modes in which human action can have an impact on non- human nature. Human activities can change the situation even when domestication as described above does not occur. Human-driven modifi- Gianfranco Pellegrino 150 cations of the habitat of wild animals does not make them domesticated, but still changes their condition, for better or worse. As Claire Palmer clearly intimated, there is a ‘contact zone’ between animals and humans that is wider than the place in which domestication happens.50 These kinds of impacts of humans on non-human animals are increasingly fre- quent in the Anthropocene. 50 Palmer, “Animal Ethics in Context,” 66-8. 51 There is a discussion about whether failed assistance amounts to harm. I do not consider the details of this discussion here. 51 There is a discussion about whether failed assistance amounts to harm. I do not consider the details of this discussion here. 3.1. The Anthropocene Grand Domestication Indeed, the Anthropocene can be seen as the age where this pattern – human action modifying ecosystems, and this modification having an impact on animals and plants within the modi- fied ecosystems – became ubiquitous. Assuming that this mode of human impact is a kind of domestication, the Anthropocene can be seen as a sort of global, or grand, domestication of non-human nature. In the Anthropocene, then, the traditional patterns of human domes- tication of animals and plants increased their extent and frequency. More- over, in the Anthropocene the human modifying impact on ecosystems increased its extent and frequency, with enhanced effects on flora and fau- na. As I will claim below, the relations between humans and non-humans embedded in the Anthropocene Grand Domestication are ethically and politically relevant in obvious ways. 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship domesticated animals live a life closely connected with humans; their well-being has been negatively affected by, and now depends on, human behaviour and social cooperation with humans; they have capacities to have and express a subjective good, to participate and to cooperate. As a consequence, they should have full membership rights, i.e., rights of res- idency, the right to have their interests counted when determining the collective or public good of the community, and the right to shape evolv- ing rules of interaction. ii. Liminal animals, i.e., the non-domesticated animals who live among us, should be granted denizenship, i.e., rights of residency short of full citizenship. iii. Wild animals, whose existence does not depend on, but can be adversely affected by, humans, should have sovereignty rights, i.e., rights to their own territory and autonomy on that territory; rules of international justice should regulate the inter- action between human communities and wild animals’ communities.54 B. Relational duties: even if animals have a unique moral standing, their rights yield different duties for humans, depending on the different relations humans have with different kinds of animals. In particular, i. domesticated animals live a life closely connected with humans; their well-being has been negatively affected by, and now depends on, human behaviour and social cooperation with humans; they have capacities to have and express a subjective good, to participate and to cooperate. As a consequence, they should have full membership rights, i.e., rights of res- idency, the right to have their interests counted when determining the collective or public good of the community, and the right to shape evolv- ing rules of interaction. ii. Liminal animals, i.e., the non-domesticated animals who live among us, should be granted denizenship, i.e., rights of residency short of full citizenship. iii. Wild animals, whose existence does not depend on, but can be adversely affected by, humans, should have sovereignty rights, i.e., rights to their own territory and autonomy on that territory; rules of international justice should regulate the inter- action between human communities and wild animals’ communities.54 Donaldson and Kimlicka’s view rests on the idea that certain rela- tions and interactions, some of them symbiotic, between humans and ani- mals have moral relevance, as they negatively affect animals’ well-being, and they can be rectified by making the terms of these relations just, fair, and mutually beneficial for both humans and animals. 52 Palmer, “Animal Ethics in Context,” chaps. 5-6. Here, I do not linger over the details of Palmer’s view. 53 Donaldson and Kymlicka, Zoopolis. Here, I do not linger over the details of Donaldson and Kymlicka’s view, especially their controversial conception of citizenship. 54 Ibid., 90, 169, 214. 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship As said in the section above, domestication can make domesticated animals or plants worse off or vulnerable to human actions. This fact has obvious ethical and political consequences. When an agent has a relation to a patient, whose outcome is that the latter is harmed or made vulner- able, the agent has specific duties. The agent has corrective justice, or repa- ration duties, when the relation is harmful. She has assistance duties when the relation makes the patient vulnerable or dependent.51h This thought has been used in animal ethics literature to mount a case for the claim that humans have differential obligations towards non- human animals, depending on their relations with them. Humans have stronger obligations to fully domesticated animals and weaker, or even no obligations at all to wild animals. For instance, Claire Palmer defend- ed the so-called laissez faire view, i.e., the claim that we have no duties towards wild animals, while we have duties towards semi- or fully domes- ticated animals, and those duties are directly proportional to the degree of our contact with them. We can also have duties of reparation towards Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 151 wild animals, Palmer claims, when our past behaviour damaged them in significant ways and our intervention is not counterproductive.52 i Sue Donaldson and Will Kymlicka took Palmer’s relation view as their starting point to extend to animals the notion of ‘citizenship’.53 Their main idea is that the standard animal rights theory can be better supported by seeing animal rights as citizenship rights, of various strength and scope, correlative to different relational duties towards different kinds of animals. Donaldson and Kymlicka’s reasoning is as follows: Donaldson and Kymlicka’s reasoning is as follows: A. Animal rights: since they have sentience and consciousness, animals have inviolable rights not to be used as mere means for human pur- poses and goods; p g B. Relational duties: even if animals have a unique moral standing, their rights yield different duties for humans, depending on the different relations humans have with different kinds of animals. In particular, i. 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship This view overlaps with Palmer’s view of the human relations with non-human, even though Palmer makes no use of the notion of ‘citizenship’. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 152 Gianfranco Pellegrino Donaldson and Kymlicka develop a notion of ‘ecological citizenship’, where not only humans can have rights of citizenship and the relative duties, but also non-human animals can be citizenship-right holders. This goes beyond the traditional notion of ‘environmental citizenship’, where the provision and distribution of environmental goods were connected to traditional citizenship rights and duties.55 However, Donaldson and Kylmlicka passingly claim that their view cannot be extended to living beings different from animals or even to eco- systems. Here’s a relevant passage: There are many good reasons to respect and protect nature, including instru- mental as well as non-instrumental ones. But it is wrong to characterize these reasons as protecting the interests of orchids or other non-sentient entities. Only a being with a subjective experience can have interests, or be owed the direct duties of justice that protect those interests. A rock is not a person. Neither is an eco-system, an orchid, or a strain of bacteria. They are things. They can be damaged, but not subject to injustice. Justice is owed to subjects who experience the world, not to things. Non-sentient things can rightfully be the objects of respect, awe, love, and care. But, lacking subjectivity, they are not rightfully the objects of fairness, nor are they agents of inter-subjec- tivity, the motivating spirit of justice.56 Here, Donaldson and Kymlicka echo the view according to which only sentient beings have moral status. As I said many times above, this view is now increasingly challenged. According to many recent views, plants have sentience and consciousness, and some plants can even inter- act and constitute communities.57 According to some authors, ecosystems also have moral standing, as they have a good of their own. 58 An argument can be mounted in favour of a wider notion of ‘Anthro- pocene ecological citizenship’, where citizenship rights are held by and extend to non-human, non-sentient individual beings and collectives. The argument can go as follows. i. the Anthropocene is the age of the Grand Domestication, in which humans impacted animals, plants, and ecosystems by changing their original condition and nature;t i. 55 Dobson, “Citizenship and the Environment.” 56 Donaldson and Kymlicka, Zoopolis, 36. 57 Calvo, Planta Sapiens; Chamovitz, What a Plant Knows; Maher, Plant Minds; Trewavas, Plant Behaviour and Intelligence. 58 Rolston III, Environmental Ethics, chap. 5. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 58 Rolston III, Environmental Ethics, chap. 5. 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship the Anthropocene is the age of the Grand Domestication, in which humans impacted animals, plants, and ecosystems by changing their original condition and nature;t ii. the Anthropocene’s domestication is often harmful and vulnerability- originating for the domesticated animals, plants, and ecosystems; ii. the Anthropocene’s domestication is often harmful and vulnerability- originating for the domesticated animals, plants, and ecosystems; iii. harmful and vulnerability-originating relations establish duties of rep- Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Politics in the Anthropocene 153 aration and assistance upon the agents whose action causes these rela- tions; aration and assistance upon the agents whose action causes these rela- tions; i i t f i iii aration and assistance upon the agents whose action causes these rela- tions; in virtue of i.-iii., iv. in the Anthropocene, humans have duties of reparation and assistance towards animals, plants, and ecosystems which have been harmfully impacted by their action, or whose vulnerability depends on human causes.hf v. These duties have differential scope, though. When human action had lesser, or no, impacts, reparation or assistance is not owed. In these cases, animals, plants, or ecosystems are entitled to respect, or to sov- ereignty, i.e., to no-harm or no-intrusion conduct. ‘Anthropocene ecological citizenship’ is the umbrella notion that cov- ers the various layers of duties and rights connecting humans, non-human animals and plants, and ecosystems in the Anthropocene. Notice that Anthropocene ecological citizenship is not simply an extension of animal citizenship. The latter can be extended to plants, but it is not so clear that ecosystems can also be directly included. Plants can have the three levels of rights that animals have, according to Donaldson and Kymlicka. There are domesticated plants, and we can consider them members of our com- munities. This amounts to saying that they are entitled to full assistance, i.e., to full protection and care, and their well-being counts as animal and human well-being, at least ceteris paribus. There are wild plants – for instance, trees in forests – and we can consider them sovereign in their communities: we owe them non-interference, not active protection and care. There are liminal plants, i.e., non-domesticated plants living with us (weeds, for instance), and we can grant them denizenship rights, i.e., some less demanding form of protection. Ecosystems are sites and conditions of morally relevant relations between humans, animals, and plants. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 3.2. The best political answer to the Anthropocene: Non-Human, or Ecologi- cal, Citizenship This may be the ground to protect them in virtue of their instrumental or conditional value. This is of course far from constituting citizenship, denizenship, or sovereignty. Ecosys- tems can be granted a sort of sovereignty, if they are seen as intrinsically valuable in virtue of their organismic life and capacity to have a subjec- tive good. This may require us to assign them some rights – for instance, rights to integrity and autonomy in the territories they ‘occupy’.h This view has it that the entire Earth system is a net of ethically and politically relevant relations. This relational citizenship rights-based view is the fittest model of politics for the Anthropocene, because it takes seri- ously the fact that the Anthropocene is the age of unprecedented human impact on Earth, an impact that changed and affected, often for the worse, the condition of non-human nature. Views exclusively focused on ecologi- 154 Gianfranco Pellegrino cal justice or non-human democracy are unable to account for the unprec- edented quality of the relations between humans and non-human nature in the Anthropocene. An ecological citizenship political theory, then, is the best politics for the Anthropocene. 4. Conclusions In this paper I claimed that the best politics for the Anthropocene should take into account the unprecedented human impact on non- human nature and its bad consequences for non-humans – an impact that can be regarded as a massive domestication project. As a consequence, a politics for the Anthropocene should acknowledge duties of reparation and assistance towards animals, plants, and ecosystems. I also claimed that a notion of ‘ecological citizenship’ is the best conceptual tool to acknowledge and ground these duties. Of course, many details of this view need to be settled. Let me men- tion two of them, as a way of conclusion. First of all, I mentioned many times a necessary assumption of a fully non-anthropocentric politics, i.e., the view that non-human and non-sentient beings have moral status. This view still needs full defense, even though current scholarship contains many good arguments in favour of it. The growing scholarship on plant intelligence and on the Earth system sciences can be used to this purpose. Second, the notion of ‘ecological citizenship’ requires broader boundaries of citizenship, and a relaxing of some traditional requirements of citizen- ship status – such as full rationality, full representation, and so on. This reframing of the concept of citizenship (partially attempted by Donaldson and Kymlicka) should still be fully achieved and defended. However, it is my contention that the project of a political theory of ecological citizen- ship for the Anthropocene is a promising one. Rivista Italiana di Filosofia Politica 3 (2022): 131-160 Rivista Italiana di Filosofia Politica 3 (2022): 131-160 References Amirante, Domenico, and Silvia Bagni. Environmental Constitutionalism in the Anthropocene: Values, Principles and Actions. 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https://openalex.org/W3198977402	https://europepmc.org/articles/pmc8465171?pdf=render	English	null	Pressurized Liquid Extraction Combined with Enzymatic-Assisted Extraction to Obtain Bioactive Non-Extractable Polyphenols from Sweet Cherry (Prunus avium L.) Pomace	Nutrients	2,021	cc-by	9,638	Citation: Domínguez-Rodríguez, G.; García, M.C.; Marina, M.L.; Plaza, M. Pressurized Liquid Extraction Combined with Enzymatic-Assisted Extraction to Obtain Bioactive Non-Extractable Polyphenols from Sweet Cherry (Prunus avium L.) Pomace. Nutrients 2021, 13, 3242. https://doi.org/10.3390/nu13093242 Academic Editors: Alejandro Cifuentes and Alberto Valdés Received: 27 July 2021 Accepted: 14 September 2021 Published: 17 September 2021 Academic Editors: Alejandro Cifuentes and Alberto Valdés Keywords: enzyme-assisted extraction; non-extractable polyphenols; pressurized liquid extraction; proanthocyanidins; sweet cherry pomace nutrients nutrients nutrients nutrients nutrients Pressurized Liquid Extraction Combined with Enzymatic-Assisted Extraction to Obtain Bioactive Non-Extractable Polyphenols from Sweet Cherry (Prunus avium L.) Pomace guez 1 , María Concepción García 1,2, María Luisa Marina 1,2 and Merichel Plaza 1,2,* Gloria Domínguez-Rodríguez 1 , María Concepción García 1,2, María Luisa Marina 1,2 a Gloria Domínguez-Rodríguez 1 , María Concepción García 1,2, María Luisa Marina 1,2 and Merichel Plaza 1,2,* 1 Universidad de Alcalá, Departamento de Química Analítica, Química Física e Ingeniería Química, Facultad de Ciencias, Ctra, Madrid-Barcelona Km 33.600, 28871 Alcalá de Henares, Madrid, Spain; gloria.dominguezr@uah.es (G.D.-R.); concepcion.garcia@uah.es (M.C.G.); mluisa.marina@uah.es (M.L.M.) 2 Universidad de Alcalá, Instituto de Investigación Química Andrés M. del Río (IQAR), Ctra, Madrid-Barcelona Km 33.600, 28871 Alcalá de Henares, Madrid, Spain * Correspondence: merichel.plaza@uah.es; Tel.: +34-91-885-6392 p * Correspondence: merichel.plaza@uah.es; Tel.: +34-91-885-6392 Abstract: Sweet cherry generates large amounts of by-products within which pomace can be a source of bioactive phenolic compounds. Commonly, phenolic compounds have been obtained by conventional extraction methodologies. However, a signiﬁcant fraction, called non-extractable polyphenols (NEPs), stays held in the conventional extraction residues. Therefore, in the present work, the release of NEPs from cherry pomace using pressurized liquid extraction (PLE) combined with enzyme-assisted extraction (EAE) using PromodTM enzyme is investigated for the ﬁrst time. In order to study the inﬂuence of temperature, time, and pH on the NEPs extraction, a response surface methodology was carried out. PLE-EAE extracts displayed higher TPC (75 ± 8 mg GAE/100 g sample) as well as, PA content, and antioxidant capacity than the extracts obtained by PLE (with a TPC value of 14 ± 1 mg GAE/100 g sample) under the same extraction conditions, and those obtained by conventional methods (TPC of 8.30 ± 0.05 mg GAE/100 g sample). Thus, PLE-EAE treatment was more selective and sustainable to release NEPs from sweet cherry pomace compared with PLE without EAE treatment. Besides, size-exclusion chromatography proﬁles showed that PLE-EAE allowed obtaining NEPs with higher molecular weight (>8000 Da) than PLE alone. Citation: Domínguez-Rodríguez, G.; García, M.C.; Marina, M.L.; Plaza, M. Pressurized Liquid Extraction Combined with Enzymatic-Assisted Extraction to Obtain Bioactive Non-Extractable Polyphenols from Sweet Cherry (Prunus avium L.) Pomace. Nutrients 2021, 13, 3242. https://doi.org/10.3390/nu13093242 Academic Editors: Alejandro Cifuentes and Alberto Valdés Received: 27 July 2021 Accepted: 14 September 2021 Published: 17 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations 1. Introduction Phenolic compounds have been extensively investigated because their potential activ- ity as antioxidants. Vegetables, fruits, and grains are the main sources of these compounds. The processing of these products originates a signiﬁcant amount of by-products containing high quantities of phenolic compounds [1]. In particular, sweet cherries are processed into different products, such as marmalades or juices, generating important amounts of by-products among which the pomace may be highlighted as the main residue [2]. Sweet cherry pomace represents an undervalued source of bioactive phenolic compounds like ﬂavonols, ﬂavan-3-ols, anthocyanins, or hydroxycinnamic acids that present antioxidant, anticarcinogenic, and antihypertensive capacities, among others [3]. These types of phe- nolic compounds have been recovered by different extraction methodologies from food by-products to be used in the pharmaceutical, cosmetic, or food industries. Conventional extraction is the most used technique to obtain phenolic compounds, but requires a high volume of solvents and long extraction times and provides low reproducibility and se- lectivity [4]. In order to mitigate these problems, advanced extraction techniques such as pressurized liquid extraction (PLE) have been used to increase the efﬁciency and speed to 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/). https://www.mdpi.com/journal/nutrients Nutrients 2021, 13, 3242. https://doi.org/10.3390/nu13093242 Nutrients 2021, 13, 3242 2 of 13 extract phenolic compounds using a lower volume of solvents resulting in a more sustain- able process [5]. PLE is based on the employ of solvents at high pressure and temperature maintaining the solvent in the liquid state and increasing its penetration into the food matrix to extract bioactive compounds [6]. However, conventional and advanced extraction techniques do not allow to extract an important fraction of bioactive phenolic compounds called non-extractable polyphenols (NEPs). NEPs have been shown to possess high antiox- idant, antihypertensive, and antidiabetic properties [7–9]. NEPs comprise different classes of phenolic compounds such as phenolics with high molecular weight or simple phenols associated with macromolecules, like proteins, in the cell wall [10]. These compounds remain in the residue obtained by conventional extraction due to their strong interactions with the cell wall. Recovery of bioactive NEPs requires an additional treatment to break these interac- tions. 1. Introduction In this sense, acid and alkaline hydrolysis are the most used treatments to release NEPs from the residue of conventional extractions [9]. Nevertheless, these hydrolysis treat- ments are non-speciﬁc and use aggressive pH values that might change the conformation of NEPs [11]. Thus, enzymatic-assisted extraction has emerged in the last years as one of the most selective and environmentally sustainable techniques to extract NEPs. Different enzymes have been employed to release NEPs from food matrices such as pectinases, cellulases, tannases, and glucuronidases, among others [12,13]. In the case of NEPs from sweet cherry pomace, EAE has proven to extract bioactive NEPs with higher molecular weight than acid hydrolysis. In fact, extracts obtained by EAE from sweet cherry pomace presented higher proanthocyanidin content and antioxidant capacity than the extracts ob- tained by acid and alkaline hydrolysis [9]. However, as far as we know, there are no studies based on the combination of PLE with EAE to release NEPs from the matrix. For instance, EAE and PLE were compared when used separately to extract bioactive compounds from lemon balm observing that extracts obtained by PLE presented higher bioactive phenolic compounds than EAE [14]. Besides, PLE has been used after EAE with the extraction residue of EAE to obtain phenolic compounds from Sargassum muticum seaweed, but the studies about the combination of both extraction techniques are very limited [15]. For this reason, this work aimed to increase the efﬁciency in the extraction of antioxi- dant NEPs from the residue of conventional extraction of sweet cherry pomace (Prunus avium L.) developing an optimum extraction method based on the combination of PLE with EAE. PromodTM enzyme with protease activity was selected because it was the most appropriate enzyme, based on previous studies, to extract NEPs from sweet cherry po- mace [9]. A Box-Behnken experimental design was used to determine the inﬂuence of time, temperature, and solvent pH on the extraction conditions to obtain high phenolic and proanthocyanidin contents with high antioxidant capacity. Additionally, extracts per- formed under the optimal extraction conditions to obtain antioxidant NEPs by PLE-EAE were compared with extracts obtained using the same conditions with PLE without EAE and with conventional extraction. Furthermore, a determination of the distribution of the molecular weight of NEPs extracted by PLE-EAE and PLE was carried out by HPLC-SEC. 2.1. Chemicals and Reagents Ethanol, hydrochloric acid (37%), and acetone of HPLC grade were purchased from Scharlab Chemie (Barcelona, Spain). Gallic acid, Folin-Ciocalteu reagent, epicatechin, vanillin, polyethylene glycol (8000 Da), polyethylene glycol (4000 Da), twin20 (1228 Da), ethylene glycol (62 Da), dextran (50,000 Da), iron(III) chloride, sodium carbonate, hydro- gen peroxide, 4-dimethylaminocinnamaldehyde (DMAC), 6-hydroxy-2,5,7,8-tetramethyl- chromane-2-carboxylic acid (Trolox), potassium persulfate, 2,2’-azinobis(3-ethylbenzothia zoline-6-sulphonic acid) and diammonium salt (ABTS)were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Sodium dihydrogen phosphate dihydrate and dipotassium hydro- gen phosphate were obtained from Merck (Hesse, Darmstadt, Germany). Nutrients 2021, 13, 3242 3 of 13 Methanol (99.99%), formic acid, acetonitrile, and butanol of HPLC grade were supplied by Fisher Scientiﬁc (Midlands, Leicestershire, UK). Ultrapure water (18.2 MΩ/cm) was obtained from a Millipore system (Millipore, Billerica, MA, USA). Promod 439 LTM enzyme was a kind donation of Biocatalysts Ltd. (Wales, Cardiff, UK). 2.3. Conventional Extraction of Extractable Polyphenols (EPPs) Extractable polyphenols were obtained from the cherry pomace through the method employed by Condezo-Hoyos et al. [16] with some modiﬁcations [9]. Twenty mL of methanol/water (50:50, v/v) acidiﬁed to pH 2 with 2 N of HCl were added to 15 g of cherry pomace and the mixture was shaken for 1 h at room temperature. To obtain the supernatant, the extract was centrifuged at 2100× g for 10 min. Then, extraction residue was re-extracted employing 20 mL of acetone/water (70:30, v/v) for 1 h at room temperature with shaking, followed by centrifugation at 2100× g for 10 min. Subsequently, methanol and acetone supernatants were mixed. Extract and extraction residue were stored at −20 ◦C until the analysis of extractable polyphenols and the NEPs extraction, respectively. The extraction was carried out in triplicate. 2.4. Release of Non-Extractable Polyphenols (NEPs) An experimental design was carried out using PLE combined with EAE employing Promod 439 LTM enzyme to optimize the NEPs extraction from the residue of conventional extraction of sweet cherry pomace. The Box-Behnken design was used as a second-order design with three levels and ﬁve central points. To investigate the effect of time (5–40 min), temperature (60–80 ◦C), and pH (6–10) on the NEPs extraction, MODDE 10.1 software (Sartorius Stedim Biotech, Malmö, Sweden) was used. A Dionex ASE 150 instrument (Thermo Fisher; Baviera, Germering, Germany) was used for the extractions. Extractions were achieved in 10 mL extraction cells, which were ﬁlled with 5.5 g of the dried residue of the conventional extraction of sweet cherry pomace on were added 140 µL of PromodTM enzyme/g of sample. Enzyme concentration as well as buffer phosphate (100 mM) as extraction solvent were selected for releasing NEPs from cherry pomace based on the results displayed in a previous study carried out by our research team [9]. HCl or NaOH were employed to adjust the pH of the buffer. This extraction solvent was sonicated for 30 min to remove dissolved oxygen. The cell was heated up for 6 min before each analysis. In total, 17 experiments were carried out in a random run order (Table S1). Total phe- nolic content (Folin-Ciocalteu assay), total proanthocyanidin content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity (Trolox Equivalent Antioxidant Capacity or TEAC) and inhibition of hydroxyl radical assays) were selected as response variables. The adequacy of ﬁtted models between time, temperature, and pH and response variables was evaluated by analysis of variance (ANOVA). Graphical and numerical analy- ses based on the response surface plots and the criteria of the desirability function were employed to calculate the theoretical optimal processing conditions. Finally, experimental extractions were carried out using the theoretical optimal extraction conditions to verify the study. Additionally, PLE without enzyme was also performed under the optimal extraction conditions achieved by the Box-Behnken experimental design. 2.2. Samples Sweet cherries from the Prunus avium L. genus, Early Lory variety, and Rosaceae family were selected from La Almunia de Doña Godina (Zaragoza, Spain) for this study. Fruits were washed, de-stemmed, de-stoned, and pressed manually to attain the pomace. Finally, the pomace was stored at −20 ◦C until its analysis. 2.6.2. Vanillin Assay Vanillin assay was employed according to Gu et al. [19] to measure the total PA content. Brieﬂy, 1.7 mL of a solution of 0.5% vanillin and 4% concentrated HCl in methanol was mixed with 100 µL of extract. After 20 min at room temperature, the absorbance was read at 500 nm. The amount of PAs was expressed as mg epicatechin/100 g sample. 2.6.1. DMAC Assay Total proanthocyanidin content (PA) was measured through DMAC method used by Montero et al. [18]. In order to prepare the DMAC solution, 0.1% DMAC reagent (w/v) was used on ethanol/water/HCl (75:12.5:12.5, v/v/v). The extract (140 µL) was mixed with 420 µL of DMAC solution and after 15 min at room temperature, the absorbance was read at 640 nm. Blanks with 140 µL of methanol instead of sample and control samples without DMAC solution were included. Results were expressed as mg of epicatechin/100 g sample. 2.5. Total Phenolic Content (TPC) A Folin-Ciocalteu (FC) assay was employed to determine TPC following the method described by Kosar et al. [17] with some modiﬁcations. Brieﬂy, 600 µL of water, 50 µL of undiluted FC reagent, and 10 µL of sample were mixed under shaken. After 1 min, 150 µL Nutrients 2021, 13, 3242 4 of 13 of 2% (w/v) Na2CO3 and 190 µL of water were added to the mixture and were shaking. After completing the reaction at 20 ◦C for 2 h, the absorbance was measured at 760 nm in a Cary 8454 UV-Vis spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). Finally, results were expressed as mg of gallic acid equivalents (GAE)/100 g sample. 2.6.3. HCl/Butanol Assay HCl/butanol assay described by Pérez-Jiménez et al. [20] with some modiﬁcations was applied. Brieﬂy, 200 µL of extract were added to 800 µL of HCl/butanol (5:95, v/v). They were let to react for 1 h at 100 ◦C. Then, tubes were centrifuged at 2500× g for 10 min and the supernatants were collected. Subsequently, the absorbance was measured at 555 nm and the results were expressed as mg epicatechin/100 g sample. 2.7. Antioxidant Capacity Determination 2.7. Antioxidant Capacity Determination 2.7.1. Trolox Equivalent Antioxidant Capacity (TEAC) Assay 2.7.1. Trolox Equivalent Antioxidant Capacity (TEAC) Assay 2.7.1. Trolox Equivalent Antioxidant Capacity (TEAC) Assay TEAC assay was utilized following the method described by Re et al. [21]. To form the ABTS radical cation (ABTS·+), a stock solution of 7 mM ABTS was made to react with 2.45 mM potassium persulfate during 12–16 h at room temperature and under darkness. The stock solution was diluted with 5 mM phosphate buffer (pH 7.4) to form the work solution until absorbance reached values of 0.70 (±0.02) AU at 734 nm. The reaction was started by adding 10 µL of different sample concentrations to 990 µL of work solution. The bleaching of ABTS was followed at 734 nm at room temperature until completely reacted (45 min). Trolox was used as a reference standard to express the results as TEAC values (µmol Trolox/g sample) employing four different concentrations of each extract giving a linear response between 20 and 80% comparing with the initial absorbance. 2.7.2. Capacity to Inhibit the Formation of Hydroxyl Radical Assay 2.7.2. Capacity to Inhibit the Formation of Hydroxyl Radical Assay The capacity to inhibit the formation of hydroxyl radicals was measured using the Ajibola et al. [22] method. Fifty µL of a 3 mM solution of 1,10 phenanthroline in 0.1 M of phosphate buffer (pH 7.4) was added to 50 µL of 3 mM FeSO4 in water, 50 µL of sample, and 50 µL of 0.01% H2O2. Then, the mixture was incubated for 1 h at 37 ◦C and 700 rpm in a Thermomixer Compact (Eppendorf AG, Hamburg, Germany). The capacity to inhibit the formation of hydroxyl radicals was obtained by measuring the absorbance at 536 nm. The % of hydroxyl radical formation inhibition was expressed through the following equation: % = Abs sample −Abs blank Abs control −Abs blank × 100 where Abs sample is the absorbance of the sample, Abs blank is the absorbance of the buffer and Abs control is the absorbance of the solution prepared with water instead of H2O2. Nutrients 2021, 13, 3242 5 of 13 2.8. High-Performance Liquid Size-Exclusion Chromatography (HPLC-SEC) Determination of Molecular Weight of NEPs from Sweet Cherry Pomace Extracts 2.8. High-Performance Liquid Size-Exclusion Chromatography (HPLC-SEC) Determination of Molecular Weight of NEPs from Sweet Cherry Pomace Extracts To determine the phenolic proﬁle of NEPs obtained by PLE with PromodTM en- zyme and PLE without enzyme under the optimal extraction conditions, size-exclusion chromatography (SEC) was employed an 1100 HPLC-DAD system from Agilent (Agi- lent Technologies, Santa Clara, CA, USA). Separation was carried out on a SEC column (PolySep-GFC-P2000, 300 × 7.8 mm, Phenomenex, Torrance, CA, USA) with a fractionation range of 100 Da-10 KDa. Separation conditions were applied according to Domínguez- Rodríguez et al. [9], where 100% water was used in isocratic mode at 0.3 mL/min as mobile phase for 60 min with a column temperature of 25 ◦C and an injection volume of 20 µL. Twenty microliters of extract were injected. The detection wavelength employed was 280 nm. In order to calibrate the molecular weight of the SEC column, polyethylene glycol (8000 Da), polyethylene glycol (4000 Da), twin20 (1228 Da), and ethylene glycol (62 Da) were employed as standards. The calibration curve obtained plotting molecular weight (MW) as a function of retention time (min) was employed to determine the MW of the extracted NEPs. 2.9. Statistical Analysis Statistical software Statgraphics Centurion version XVII (Statistical Graphics Corp, The Plains, VA, USA) was used to observe differences in TPC, PA contents, and antioxidant capacity between PLE with PromodTM enzyme extracts, PLE without enzyme extracts, and conventional extraction extracts. ANOVA by Fisher’s exact test allowed to determine statistically signiﬁcant differences (p ≤0.05) between mean values for different extracts at 95% conﬁdence level. All the analyses were carried out in triplicate for each extract. All the analyses were carried out in triplicate for each extraction. 2.7.2. Capacity to Inhibit the Formation of Hydroxyl Radical Assay Linear equation (y = −0.0028x + 37.043) with an R2 determination coefﬁcient value of 0.9122 was used to express the responses obtained. The void volume was determined with dextran (50,000 Da). 3. Results and Discussion This work describes for the ﬁrst time the use and optimization of the combination of two eco-friendly extraction methodologies based on PLE with EAE to release NEPs from the residue of conventional extraction of sweet cherry pomace. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE The extraction of NEPs was carried out from the residue obtained by the conventional extraction of polyphenols from sweet cherry pomace. PromodTM enzyme was selected because it was the most efﬁcient in the extraction of bioactive NEPs from cherry pomace compared with PectinaseTM and DepolTM enzymes in a previous work performed by our re- search group [9]. Other conditions consisted of the use of a phosphate buffer (100 mM) and an enzyme concentration of 140 µL of PromodTM enzyme/g of sample. This enzyme was added to the residue of conventional extraction before starting the PLE process. PromodTM enzyme presents protease and polygalacturonase activities and allows to modify the func- tionality of the proteins as well as to solubilize proteins and their aggregates improving the release of NEPs from the cell wall of the matrix and aggregates. The protease activity enables to break the proteic tonoplast surrounding cellular vacuoles contributing to the release of phenolic compounds contained in them. On the other hand, the polygalactur- onase activity enables to disrupt α-1,4-glycosidic bonds on polygalacturonic acid of pectins degrading the pectic chain and releasing phenolic compounds that interact with carboxyl and hydroxyl groups of pectin [23,24]. Once the enzyme was added, extraction processes were carried out according to Box Behnken experimental design to optimize the inﬂuence of time (5–40 min), temperature (60–80 ◦C), and solvent pH (6–10) on six response variables (FC, DMAC, vanillin, bu- tanol/HCl, TEAC, and hydroxyl radical assays). The time, temperature, and pH ranges Nutrients 2021, 13, 3242 6 of 13 to carry out the experimental design were established taken into account the results ob- tained by Domínguez-Rodríguez et al. [9] and according to the enzyme speciﬁcations. The 17 experiments established by Box Behnken design with their respective TPC, total PA content, and antioxidant capacity values are summarized in Table S1. Table 1 shows the analysis of variance, goodness of ﬁt, and the adequacy of the model. For instance, it can be observed that the regression of the model of PromodTM enzyme could explain a satisfactory developed model with a range from 81.6 to 97.1% of the results obtained by FC, DMAC, vanillin, butanol/HCl, TEAC, and hydroxyl radical assays. In addition, the standard error of the regression model expressed as relative standard deviation (RSD) was below 6.01% in all assays. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Besides, ANOVA analysis showed an adequate regression model for the responses of vanillin, butanol/HCl, TEAC, and inhibition of hydroxyl radical assays since it presented a p-value for the regression lower than 0.05. However, the regression model was not adequate for FC and DMAC assays since they showed p-values of 0.073 and 0.167, respectively. Additionally, all responses displayed an adequate p-value for the lack-of-ﬁt test, presenting values higher than 0.05 (Table 1). Furthermore, the ANOVA test was used to determine the main variables (time, temperature, and/or pH) that can affect the response factors with a signiﬁcant effect (p-value < 0.05) (see Table 1). The time followed by the temperature were the variables that presented less signiﬁcant effects on the different responses. That is why, as can be seen in Figure 1, to observe the effects of variables (time and pH) on the ﬁve responses, the counterplots were ﬁxed at the optimum extraction temperature for obtaining the highest response values (60 ◦C). The TPC values increased at high pH (p-value < 0.05) while the temperature and time did not have a signiﬁcant effect (p-value > 0.05) (Figure 1A and Table 1). The extraction time, temperature, and pH did not present a signiﬁcant effect on the extraction of PAs when these compounds were measured by DMAC and vanillin assays (p-value > 0.05) (Figure 1B,C and Table 1). However, when the PA content was determined by butanol/HCl assay, the pH presented a negative effect on their extraction (p-value < 0.05). Therefore, when a high pH was employed in the extraction of PAs, the PA content decreased (Figure 1D). Concerning antioxidant capacity, the time presented a negative effect while temperature and pH showed a positive impact on the TEAC value (see Table 1). In this sense, Figure 1E shows that the antioxidant capacity measured by TEAC assay was higher at lower extraction times but at higher temperatures and pH values (p-value < 0.05). As can be seen in Table 1, the positive and negative effects of temperature and pH, respectively, were observed on antioxidant capacity measured by the inhibition of hydroxyl radical assay. In fact, Figure 1F shows that high temperatures and low pH allowed to obtain extracts with a high capacity to inhibit the formation of hydroxyl radicals. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE g p y y y Therefore, 60 ◦C for 31 min at pH 10 were the optimal extraction conditions to obtain the highest TPC, PA content, and antioxidant capacity from cherry pomace extraction residue. These optimal extraction conditions were employed to release antioxidant NEPs from cherry pomace extraction residue. Table 2 shows the predicted theoretical values obtained under the optimal extraction conditions of TPC, total PA content (DMAC, vanillin, and butanol/HCl assays), and total antioxidant capacity (TEAC and percentage of in- hibition of hydroxyl radical formation assays). As can be seen in Table 2, experimental values of TPC, PA content, and antioxidant capacity were within the range of theoretical values obtained by Box-Behnken design and closer to optimum value excepting the total PA content values obtained by vanillin and butanol/HCl assays which were closer to the upper theoretical value. This means that the predictive model from experimental design allowed to attain a good prediction to release antioxidant NEPs from cherry pomace extraction residue by PLE in combination with EAE with PromodTM enzyme. 7 of 13 Nutrients 2021, 13, 3242 Table 1. Coefﬁcients of the multiple linear regression model from the experimental design for PLE combined with EAE with Promod enzyme that the best ﬁtted responses (Folin-Ciocalteu, DMAC, vanillin, butanol/HCl, TEAC, and inhibition of hydroxyl radical assays) with the extraction parameters (t: time, T: temperature and p: pH) and the analysis of variance (ANOVA). ar regression model from the experimental design for PLE combined with EAE with Promod enzyme that the best ﬁtted responses (Folin-Ciocalteu, nd inhibition of hydroxyl radical assays) with the extraction parameters (t: time, T: temperature and p: pH) and the analysis of variance (ANOVA). Table 1. Coefﬁcients of the multiple linear regression model from the experimental design for PLE combined with EAE with Promod enzyme that the best ﬁtted responses (Folin-Ciocalteu, DMAC, vanillin, butanol/HCl, TEAC, and inhibition of hydroxyl radical assays) with the extraction parameters (t: time, T: temperature and p: pH) and the analysis of variance (ANOVA). 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE a,b Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). obtained under the optimal extraction conditions of TPC, total PA content (DMAC, van- illin, and butanol/HCl assays), and total antioxidant capacity (TEAC and percentage of inhibition of hydroxyl radical formation assays). As can be seen in Table 2, experimental values of TPC, PA content, and antioxidant capacity were within the range of theoretical values obtained by Box-Behnken design and closer to optimum value excepting the total PA content values obtained by vanillin and butanol/HCl assays which were closer to the upper theoretical value. This means that the predictive model from experimental design allowed to attain a good prediction to release antioxidant NEPs from cherry pomace ex- traction residue by PLE in combination with EAE with PromodTM enzyme. Table 2. Theoretical and experimental values of TPC (Folin-Ciocalteu method), total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity (DPPH and TEAC methods) obtained under the optimal PLE-EAE condi- tions as well as experimental values obtained by PLE under the optimal PLE-EAE conditions but without enzyme. a,b Letters show the significant differences among extraction methods of NEPs (p ≤ 0.05). Optimal EAE Conditions Theoretical Values Experimental Values O ti u PLE ith P o odTM Optimal EAE Conditions Theoretical Values Experimental Values Optimum Value Lower Upper PLE with PromodTM Enzyme PLE without Enzyme TPC (mg GAE/100 g sample) 72.1 57.7 86.5 75 ± 8 a 14 ± 1 b DMAC (mg epicatechin/100 g sample) 1.04 0.65 1.44 0.97 ± 0.07 a 0.24 ± 0.03 b Vanillin (mg epicatechin/100 g sample) 66.2 53.8 78.6 76 ± 8 a 30 ± 8 b Butanol/HCl (mg epicatechin/100 g sample) 13.2 2.4 24.2 20 ± 2 a 11.6 ± 0.9 b TEAC (µmol Trolox/g sample) 0.0056 0.0039 0.0074 0.0051 ± 0.0006 a 0.0027 ± 0.0008 b Hydroxyl radical assay (% of hydroxyl radical inhibition) 22.8 10.8 34.9 20 ± 4 a 10 ± 2 b 3.2. Comparison of PLE Combined with EAE and PLE Alone to Release NEPs from Cherry Pomace Extraction Residue g p Theoretical Values Experimental Values O ti PLE ith P dTM 3.2. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Parameters Folin- Ciocalteu p-Value DMAC p-Value Vanillin p-Value Butanol/HCl p-Value TEAC p-Value Hydroxyl Radical p-Value Constant 76.5687 0.714975 50.4326 2.31323 0.00631004 12.5415 t 3.74087 0.0690312 0.0988549 0.0757302 1.48121 0.274243 −1.55757 0.259711 −0.00057974 0.023165 −0.226677 0.874268 T −0.642173 0.708234 −0.0983441 0.0768368 −2.6666 0.0781056 −1.7044 0.244879 0.0008498 0.0122791 3.93067 0.0248276 p 4.92316 0.0288109 0.0657091 0.197594 −0.972796 0.432818 −5.1847 0.0143429 0.00068073 0.0136543 −7.98952 0.00067382 t2 −5.30567 0.0321596 −0.12659 0.0499341 −1.84528 0.176053 1.47379 0.383005 0.00066431 0.0270325 −2.92402 0.0664017 T2 −4.74775 0.0463558 −0.020252 0.697806 4.16827 0.0162499 −0.925015 0.517185 0.00037737 0.0519364 4.04103 0.019886 p2 −3.79391 0.0892811 0.0230183 0.659731 7.39256 0.00278374 5.73637 0.0116925 −0.00072299 0.0206362 9.85783 0.00015956 tT −0.902904 0.624473 −0.0188704 0.70636 0.305061 0.84502 0.874684 0.510832 9.47 × 10−5 0.653919 −0.755402 0.601397 tp 2.94867 0.149462 −0.0406045 0.429809 −0.903755 0.458469 3.93605 0.0315481 0.00025249 0.140774 −2.08563 0.174755 T*p 0.182355 0.920256 −0.0668348 0.216721 4.09835 0.0148862 −1.64187 0.361947 −8.93 × 10−5 0.672088 −1.09081 0.455551 R2 0.876 0.816 0.943 0.931 0.971 0.941 RSD 6.063 0.1655 4.232 4.237 0.000517 5.524 p-value (test of regression) 0.073 0.167 0.012 0.05 0.009 0.002 p-value (lack of ﬁt) 0.65 0.346 0.946 0.109 0.213 0.492 8 of 13 8 of 14 Nutrients 2021, 13, 3242 Nutrients 2021, 13, x FO Figure 1. Contour plots presenting the effect of time (min) and pH at the optimum extraction temperature (60 °C) on the TPC (Folin-Ciocalteu method, mg GAE/g sample (A)), total PA content (DMAC (B), vanillin (C), and butanol/HCl (D) assays, mg epicatechin/100 g sample), and total antioxidant capacity (TEAC (µmol Trolox/g sample) (E) and capacity to inhibit the formation of hydroxyl radical (% inhibition) (F) methods) from PLE-EAE extracts. Figure 1. Contour plots presenting the effect of time (min) and pH at the optimum extraction temperature (60 ◦C) on the TPC (Folin-Ciocalteu method, mg GAE/g sample (A)), total PA content (DMAC (B), vanillin (C), and butanol/HCl (D) assays, mg epicatechin/100 g sample), and total antioxidant capacity (TEAC (µmol Trolox/g sample) (E) and capacity to inhibit the formation of hydroxyl radical (% inhibition) (F) methods) from PLE-EAE extracts. Figure 1. Contour plots presenting the effect of time (min) and pH at the optimum extraction temperature (60 °C) on the TPC (Folin-Ciocalteu method, mg GAE/g sample (A)), total PA content (DMAC (B), vanillin (C), and butanol/HCl (D) assays, mg epicatechin/100 g sample), and total antioxidant capacity (TEAC (µmol Trolox/g sample) (E) and capacity to inhibit the formation of hydroxyl radical (% inhibition) (F) methods) from PLE-EAE extracts. Figure 1. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Contour plots presenting the effect of time (min) and pH at the optimum extraction temperature (60 ◦C) on the TPC (Folin-Ciocalteu method, mg GAE/g sample (A)), total PA content (DMAC (B), vanillin (C), and butanol/HCl (D) assays, mg epicatechin/100 g sample), and total antioxidant capacity (TEAC (µmol Trolox/g sample) (E) and capacity to inhibit the formation of hydroxyl radical (% inhibition) (F) methods) from PLE-EAE extracts. Therefore, 60 °C for 31 min at pH 10 were the optimal extraction conditions to obta the highest TPC, PA content, and antioxidant capacity from cherry pomace extraction re idue. These optimal extraction conditions were employed to release antioxidant NEP from cherry pomace extraction residue. Table 2 shows the predicted theoretical valu Table 2. Theoretical and experimental values of TPC (Folin-Ciocalteu method), total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity (DPPH and TEAC methods) obtained under the optimal PLE-EAE conditions as well as experimental values obtained by PLE under the optimal PLE-EAE conditions but without enzyme. a,b Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). Therefore, 60 °C for 31 min at pH 10 were the optimal extraction conditions to obta the highest TPC, PA content, and antioxidant capacity from cherry pomace extraction re idue. These optimal extraction conditions were employed to release antioxidant NEP from cherry pomace extraction residue. Table 2 shows the predicted theoretical valu Table 2. Theoretical and experimental values of TPC (Folin-Ciocalteu method), total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity (DPPH and TEAC methods) obtained under the optimal PLE-EAE conditions as well as experimental values obtained by PLE under the optimal PLE-EAE conditions but without enzyme. a,b Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). Therefore, 60 °C for 31 min at pH 10 were the optimal extraction conditions to obta the highest TPC, PA content, and antioxidant capacity from cherry pomace extraction re idue. These optimal extraction conditions were employed to release antioxidant NE from cherry pomace extraction residue. Table 2 shows the predicted theoretical valu Table 2. Theoretical and experimental values of TPC (Folin-Ciocalteu method), total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity (DPPH and TEAC methods) obtained under the optimal PLE-EAE conditions as well as experimental values obtained by PLE under the optimal PLE-EAE conditions but without enzyme. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Comparison of PLE Combined with EAE and PLE Alone to Release NEPs from Cherry Pomace Extraction Residue tions Optimum Value Lower Upper PLE with PromodTM Enzyme PLE without Enzyme ample) 72.1 57.7 86.5 75 ± 8 a 14 ± 1 b n/100 g 1.04 0.65 1.44 0.97 ± 0.07 a 0.24 ± 0.03 b in/100 g 66 2 53 8 78 6 76 8 30 8 b The efﬁciency of the combination of both advanced extraction techniques, PLE and EAE with PromodTM enzyme, for the recovery of NEPs from the extraction residue of sweet cherry pomace (obtained after the recovery of EPPs) was next evaluated. For that purpose, PLE without EAE was carried out under the optimal extraction conditions achieved by Box-Behnken experimental design (60 ◦C for 31 min at pH 10) and results were compared to the obtained when using simultaneously PLE and EAE. / g 66.2 53.8 78.6 76 ± 8 a 30 ± 8 b As can be seen in Figure 2, results obtained for the EPPs extract, and NEPs extract by PLE with and without enzyme showed statistically signiﬁcant differences. As can be observed, EPPs extract presented the lowest TPC and PA content values obtained by 9 of 13 spe- mon- Nutrients 2021, 13, 3242 be because cific to dete Folin-Ciocalteu, DMAC, and vanillin assays. However, PA content using butanol/HCl assay was higher in EPPs extract than in PLE-EAE and PLE extracts (Figure 2B). This fact may be because each assay has a different reaction mechanism since butanol/HCl is more speciﬁc to determine polymeric polyphenols while DMAC and vanillin assay react with monomeric polyphenols [9]. hand, TPC, PA content obtained by vanillin, DMAC, and butanol/HCl idant capacity measured by TEAC and percentage of inhibition of hy- ays showed higher values in PLE combined with EAE with PromodTM an PLE without EAE extracts. Folin-Ciocalteu, DMAC, and vanillin assays. However, PA content using butanol/HCl assay was higher in EPPs extract than in PLE-EAE and PLE extracts (Figure 2B). This fact may be because each assay has a different reaction mechanism since butanol/HCl is more speciﬁc to determine polymeric polyphenols while DMAC and vanillin assay react with monomeric polyphenols [9]. r hand, TPC, PA content obtained by vanillin, DMAC, and butanol/HCl xidant capacity measured by TEAC and percentage of inhibition of hy- says showed higher values in PLE combined with EAE with PromodTM han PLE without EAE extracts. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMAC vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radica and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzyme from the extraction residue of sweet cherry pomace and EPPs conventional extraction from swee Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radical and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzyme from the extraction residue of sweet cherry pomace and EPPs conventional extraction from sweet cherry pomace. a,b,c Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMA vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radic and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzym from the extraction residue of sweet cherry pomace and EPPs conventional extraction from swe h b L tt h th i ifi t diff t ti th d f NEP ( Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radical and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzyme from the extraction residue of sweet cherry pomace and EPPs conventional extraction from sweet cherry pomace. a,b,c Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMA vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radic and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzym from the extraction residue of sweet cherry pomace and EPPs conventional extraction from swe h b L h h f d ff h d f NEP ( Figure 2. Experimental values of (A) TPC (Folin-Ciocalteu method), (B) total PA content (DMAC, vanillin, and butanol/HCl assays), and antioxidant capacity by (C) inhibition of hydroxyl radical and (D) TEAC assays obtained under the optimal PLE-EAE conditions and PLE without enzyme from the extraction residue of sweet cherry pomace and EPPs conventional extraction from sweet cherry pomace. 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE a,b,c Letters show the signiﬁcant differences among extraction methods of NEPs (p ≤0.05). g g (p On the other hand, TPC, PA content obtained by vanillin, DMAC, and butanol/HCl assays and antioxidant capacity measured by TEAC and percentage of inhibition of hy- droxyl radical assays showed higher values in PLE combined with EAE with PromodTM enzyme extract than PLE without EAE extracts. Despite PLE is an advanced extraction methodology that increases the extraction yields and reproducibility compared with conventional extraction techniques, this extraction methodology alone produced an incomplete extraction of NEPs. Possibly, this inefﬁcient extraction could be due to the strong interactions of NEPs with the matrix. Different compounds of the food matrix can interact with NEPs being inaccessible to pressurized liquids or conventional extraction techniques [25]. However, the combination of PLE with EAE allowed releasing higher PA content than extracts obtained with PLE without EAE. In this sense, the protease and polygalacturonase activity of PromodTM enzyme could have released NEPs from the cell wall of the residue of the extraction of sweet cherry pomace. In fact, protease activity promotes the breakage of ester linkages with carboxylic groups in proteins and polygalacturonase activity makes possible the disruption of hydroxyl groups of pectins [26,27]. The overall effect is the increase in the release of NEPs. Additionally, PLE-EAE and PLE extracts presented lower TPC values than TPC values obtained by Domínguez-Rodríguez et al. [9] from alkaline, acid and EAE with PromodTM, DepolTM and PectinaseTM enzymes extracts from the same variety of sweet cherry pomace. Nutrients 2021, 13, 3242 10 of 13 10 of 13 By contrast, PLE-EAE and PLE extracts showed higher polymeric non-extractable PA content than acid and alkaline hydrolysis [9]. These results indicated that the differences in TPC and PA contents between these studies may be due to the inﬂuence of climatic differences in each vintage. By contrast, PLE-EAE and PLE extracts showed higher polymeric non-extractable PA content than acid and alkaline hydrolysis [9]. These results indicated that the differences in TPC and PA contents between these studies may be due to the inﬂuence of climatic differences in each vintage. Additionally, antioxidant capacity results measured by the percentage of inhibition of hydroxyl radical assay showed that the EPPs presented lower antioxidant capacity than the NEPs extracts attained by PLE-EAE (see Figure 2C). 3.1. Optimization of NEPs Extraction from Cherry Pomace Extraction Residue by PLE Combined with EAE Nevertheless, TEAC values for EPPs extracts showed higher antioxidant capacity (3.27 µmol Trolox/g sample) than PLE-EAE and PLE extracts (Figure 2D). The differences between antioxidant methods may be due to the different reaction mechanisms of both antioxidant assays. In fact, in TEAC assay a radical (ABTS) is employed that is not found in food or biological systems [28–30] while in the percentage of inhibition of hydroxyl radical assay one of the most relevant radicals generated in our body is used and, thus, the percentage of inhibition of hydroxyl radical assay could provide an approximation of the antioxidant effect of the extracts on the organism. PLE-EAE and PLE extracts presented higher non-extractable PA content but lower an- tioxidant capacity than the extracts obtained by Domínguez-Rodríguez et al. [9] from EAE with PromodTM enzyme without PLE from the same variety of sweet cherry pomace [9]. However, these results cannot be strictly compared because the sweet cherry pomace from this work and the previous one correspond to different vintages and the initial composition of phenolic compounds was different in both pomaces. PLE displayed a positive effect on the extraction of NEPs since in combination with EAE showed higher antioxidant capacity than PLE alone in TEAC and inhibition of hydroxyl radical assays. As can be observed, different results were obtained depending on the extraction method and analytical assay employed due to different compounds are extracted and interact depending on the reaction mechanism of each assay. In general, results showed that NEPs remain retained in the residue of the conventional extraction of sweet cherry pomace and the combination of PLE-EAE enhances the access to release these compounds from the food matrix. 3.3. Determination of Molecular Weight of NEPs by Size Exclusion Chromatography In order to have an estimation of the molecular weight of NEPs present in PLE-EAE and PLE extracts from sweet cherry pomace residue (obtained after EPPs conventional extraction), HPLC-SEC analysis was carried out. As can be observed in Figure 3, the chromatographic analysis showed higher signal intensity in the extracts obtained only by PLE than in that from PLE-EAE. The molecular weight distribution in the main peaks of NEPs extracted with <2000 Da and 2000–6000 Da was similar in both chromatograms obtained from the PLE-EAE and PLE extracts, notwithstanding low molecular weight compounds, were more abundant in PLE than PLE-EAE extracts (Figure 3). However, PLE-EAE presented higher peak areas which corresponded to high molecular weight compounds. In fact, PLE-EAE extract exhibited a higher area of compounds with a molecular weight from 2000 Da to >8000 Da than PLE extract highlighting compounds with a molecular weight > 8000 Da that showed an area of 2145 ± 70 in PLE-EAE extract (see Table 3). By contrast, PLE extract presented a higher area of compounds with a molecular weight lower than 2000 Da than PLE-EAE extract. Compounds with this molecular weight were predominant in both extracts obtained by PLE and PLE-EAE with an area of 15,580 ± 284 and 8219 ± 49, respectively (Table 3). 11 of 13 Nutrients 2021, 13, 3242 hibited a than PLE Figure 3. HPLC-SEC chromatograms of the extracts achieved by (A) PLE-EAE with PromodTM en- zyme and (B) PLE without enzyme from sweet cherry pomace extraction residue. By contrast, PLE extract presented a higher area of compounds with a molecular weight lower than 2000 Da than PLE-EAE extract. Compounds with this molecular weight were predominant in both extracts obtained by PLE and PLE EAE with an area of 15 580 B) A) min 10 20 30 40 50 60 mAU 0 50 100 150 200 250 300 > 8000 Da 8000-6000 Da 6000-2000 Da < 2000 Da min 8 12 16 20 mAU 0 4 8 10 min 10 20 30 40 50 60 mAU 0 50 100 150 200 250 300 > 8000 Da 8000-6000 Da 6000-2000 Da < 2000 Da min 8 12 16 20 mAU 0 4 8 10 igure 3. HPLC-SEC chromatograms of the extracts achieved by (A) PLE-EAE with PromodTM enzyme and (B) PLE without nzyme from sweet cherry pomace extraction residue. Table 3. 3.3. Determination of Molecular Weight of NEPs by Size Exclusion Chromatography Estimation of the molecular weight distribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs obtained by PLE-EAE and PLE from extraction residues of sweet cherry pomace. E i M h d D D D D A) min 10 20 30 40 50 60 mAU 0 50 100 150 200 250 300 > 8000 Da 8000-6000 Da 6000-2000 Da < 2000 Da min 8 12 16 20 mAU 0 4 8 10 B) min 10 20 30 40 50 60 mAU 0 50 100 150 200 250 300 > 8000 Da 8000-6000 Da 6000-2000 Da < 2000 Da min 8 12 16 20 mAU 0 4 8 10 B) Figure 3. HPLC-SEC chromatograms of the extracts achieved by (A) PLE-EAE with PromodTM en- zyme and (B) PLE without enzyme from sweet cherry pomace extraction residue. Figure 3. HPLC-SEC chromatograms of the extracts achieved by (A) PLE-EAE with PromodTM enzyme and (B) PLE without enzyme from sweet cherry pomace extraction residue. PLE extract presented a higher area of compounds with a molecular n 2000 Da than PLE-EAE extract Compounds with this molecular weight Table 3. Estimation of the molecular weight distribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs obtained by PLE-EAE and PLE from extraction residues of sweet cherry pomace. PLE extract presented a higher area of compounds with a molecular n 2000 Da than PLE-EAE extract Compounds with this molecular weight Table 3. Estimation of the molecular weight distribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs obtained by PLE-EAE and PLE from extraction residues of sweet cherry pomace. PLE extract presented a higher area of compounds with a molecular n 2000 Da than PLE-EAE extract. Compounds with this molecular weight nt in both extracts obtained by PLE and PLE-EAE with an area of 15,580 49, respectively (Table 3). distribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs ob- esidues of sweet cherry pomace. 00 Da 8000–6000 Da 6000–2000 Da <2000 Da 5 ± 70 1604 ± 183 1760 ± 38 8219 ± 49 ± 400 1376 ± 223 569 ± 49 15,580 ± 284 Table 3. Estimation of the molecular weight distribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs obtained by PLE-EAE and PLE from extraction residues of sweet cherry pomace. 3.3. Determination of Molecular Weight of NEPs by Size Exclusion Chromatography Extraction Method >8000 Da 8000–6000 Da 6000–2000 Da <2000 Da PLE with PromodTM enzyme 2145 ± 70 1604 ± 183 1760 ± 38 8219 ± 49 PLE without enzyme 811 ± 400 1376 ± 223 569 ± 49 15,580 ± 284 In this study, PLE-EAE allowed to recover extracts with higher total peak area for NEPs than conventional extraction and acid hydrolysis from sweet cherry pomace carried out in a previous work by our research group [9], although, as it was commented above, samples analyzed proceeded from different vintages and, therefore, results are not comparable. Despite the molecular weight distribution was similar between extracts, PLE-EAE extracts showed higher total phenolic and proanthocyanidin content as well as higher antioxidant capacity than PLE extracts. For this reason, results suggested that NEPs with higher molecular weight provided higher bioactivity to the extracts. p g nt in both extracts obtained by PLE and PLE-EAE with an area of 15,580 49, respectively (Table 3). istribution by HPLC-SEC at 280 nm (expressed as peak area) of NEPs ob- Extraction Method >8000 Da 8000–6000 Da 6000–2000 Da <2000 Da PLE with PromodTM enzyme 2145 ± 70 1604 ± 183 1760 ± 38 8219 ± 49 PLE without enzyme 811 ± 400 1376 ± 223 569 ± 49 15,580 ± 284 esidues of sweet cherry pomace. 00 Da 8000–6000 Da 6000–2000 Da <2000 Da 5 ± 70 1604 ± 183 1760 ± 38 8219 ± 49 ± 400 1376 ± 223 569 ± 49 15,580 ± 284 In this study, PLE-EAE allowed to recover extracts with higher total peak area for NEPs than conventional extraction and acid hydrolysis from sweet cherry pomace carried out in a previous work by our research group [9], although, as it was commented above, samples analyzed proceeded from different vintages and, therefore, results are not comparable. Despite the molecular weight distribution was similar between extracts, PLE-EAE extracts showed higher total phenolic and proanthocyanidin content as well as higher antioxidant capacity than PLE extracts. For this reason, results suggested that NEPs with higher molecular weight provided higher bioactivity to the extracts. Nutrients 2021, 13, 3242 12 of 13 12 of 13 In general, this work proposes for the ﬁrst time an efﬁcient extraction method based on the combination of PLE with EAE using PromodTM enzyme with protease and poly- galacturonase activities to achieve the extraction of NEPs from sweet cherry pomace. 4. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. occurrence, and potential uses. Food Chem. 2006, 99, 191–203. [CrossRef] 2. Mehmet Yılmaz, F.; Mehmet, K.; Vardin, H. Optimization of extraction parameters on the isolation of phenolic compounds from sour cherry (Prunus cerasus L.) pomace. J. Food Sci. Technol. 2015, 52, 2851–2859. [CrossRef] 3. 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Conclusions In conclusion, the optimal extraction conditions to obtain NEPs using PLE-EAE with PromodTM enzyme were a temperature of 60 ◦C for 31 min at pH 10.0. In addition, PLE-EAE extracts were compared in terms of TPC, PA content, and antioxidant capacity with PLE extracts obtained under the same optimal extraction conditions but without the addition of enzymes, and with EPPs extracts. In general, PLE-EAE was the most efﬁcient extraction technique to release NEPs from sweet cherry pomace. This extract presented higher TPC, PA content, and antioxidant capacity than conventional and PLE extracts excepting polymeric PA content measured by butanol/HCl assay and antioxidant capacity measured by TEAC assay which presented higher values in the conventional EPPs extract. The estimation of the molecular weight distribution showed that PLE-EAE presented higher peak area of compounds with high molecular weight than PLE extract. For all these reasons, PLE-EAE can be recommended as an environmentally sustainable method to release antioxidant NEPs from sweet cherry pomace. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13093242/s1, Table S1: Experimental design obtained by Box-Behnken and experimental results obtained under the designed conditions by PLE combined with EAE with PromodTM enzyme from extraction residue of conventional extraction of sweet cherry pomace using Folin-Ciocalteu (mg GAE/100 g sample), DMAC (mg epicatechin/100 g sample), vanillin (mg epicatechin/100 g sample), butanol/HCl (mg epicatechin/100 g sample), TEAC (µmol Trolox/g sample) and the capacity to inhibit the formation of hydroxyl radical (% of hydroxyl radical inhibition) assays response factors. Author Contributions: Conceptualization, M.P. and M.L.M.; methodology, G.D.-R., M.P. and M.L.M.; software, G.D.-R.; validation, G.D.-R.; formal analysis, G.D.-R. and M.P.; investigation, G.D.-R.; resources, M.L.M., M.C.G. and M.P.; data curation, G.D.-R. and M.P.; writing—original draft prepara- tion, G.D.-R. and M.P.; writing—review and editing, M.P., M.L.M. and M.C.G.; visualization, G.D.-R., M.C.G., M.P. and M.L.M.; supervision, M.P. and M.L.M.; project administration, M.L.M., M.C.G. and M.P.; funding acquisition, M.L.M., M.C.G. and M.P. All authors have read and agreed to the published version of the manuscript. Funding: Authors thank ﬁnancial support from the Comunidad of Madrid (Spain) and European funding from FSE and FEDER Programs (research project S2018/BAA-4393, AVANSECAL-II-CM) and the Comunidad of Madrid and the University of Alcalá (research project CM/JIN/2019-033, SOSBIO). 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https://openalex.org/W1993814846	https://europepmc.org/articles/pmc3617006?pdf=render	English	null	The EPMA Journal introduces a new type of research article dedicated to predictive, preventive and personalised medicine	The EPMA journal	2,013	cc-by	1,103	EDITORIAL Open Access The EPMA Journal introduces a new type of research article dedicated to predictive, preventive and personalised medicine Olga Golubnitschaja1,2 What are the purpose and expected impacts of this action? complementary levels of molecular detection), advanced therapy targeting and innovative drug delivery systems, which present real progress beyond the state of the art in diagnostics, patient stratification and personalisation of treatments. 3. The manuscript should be presented to the highest didactic level. The EPMA Journal focuses on an interdisciplinary and multidisciplinary approach in educating professionals. The reason for this is that the PPPM objectives can only be achieved through a close collaboration between several professional groups. Consequently, even the most sophisticated approaches and technologies should be described in a way that is understandable to young professionals and specialists active in other biomedical fields outside of the author’s area of expertise. For this reason, it is highly recommended to make use of high-quality illustration materials, such as images and graphs, to introduce innovative technologies, new approaches, proposed molecular, subcellular and cellular mechanisms, etc. The Editorial Board and BioMed Central team have been working together to develop a journal-specific outline for research articles suitable for publication in The EPMA Journal [1]. The purpose of this action is to pro- mote scientific excellence in the area of predictive, pre- ventive and personalised medicine (PPPM), through a better understanding of the integrative approach used in PPPM, innovation in biomedical technologies and com- prehensive interpretation of the achieved results for accelerated adoption of the top sciences into daily med- ical practice and advanced health care focused on patient needs. 3. Correspondence: Olga.Golubnitschaja@ukb.uni-bonn.de 1European Association for Predictive, Preventive & Personalised Medicine, Brussels, Belgium 2Department of Radiology, Rheinische Friedrich-Wilhelms University of Bonn, Sigmund-Freud-Str. 25, Bonn 53105, Germany Golubnitschaja The EPMA Journal 2013, 4:11 http://www.epmajournal.com/content/4/1/11 Golubnitschaja The EPMA Journal 2013, 4:11 http://www.epmajournal.com/content/4/1/11 © 2013 Golubnitschaja; 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. Points to consider when writing a research manuscript When preparing a research manuscript for The EPMA Journal, authors should consider the following aspects: 1. The research topic must be in keeping with the aims and scope of the journal [2], i.e. dedicated to predictive diagnostics, targeted preventive measures and treatment algorithms tailored to the person. The authors should consider multifunctional, experimental and technological approaches and result interpretations in terms of PPPM. Why are there specific structural features required for research manuscripts in The EPMA Journal? epmajournal.com/authors/instructions/research. 2. EPMA Journal: Aims & Scope. http://www.epmajournal.com/ about#aimsscope. doi:10.1186/1878-5085-4-11 Cite this article as: Golubnitschaja: The EPMA Journal introduces a new type of research article dedicated to predictive, preventive and personalised medicine. The EPMA Journal 2013 4:11. Received: 15 March 2013 Accepted: 18 March 2013 Published: 28 March 2013 Received: 15 March 2013 Accepted: 18 March 2013 Published: 28 March 2013 Received: 15 March 2013 Accepted: 18 March 2013 Published: 28 March 2013 Why are there specific structural features required for research manuscripts in The EPMA Journal? Why are there specific structural features required for research manuscripts in The EPMA Journal? The EPMA Journal does not aim to compete with or duplicate the work of other journals dedicated to indi- vidual types of technologies, discovery of single bio- markers, drug targeting, medical imaging and so on. The journal promotes the use and amalgamation of know- ledge collected in individual scientific and technological branches in order to create integrative PPPM-related approaches that have potentials to advance health care. Therefore, any scientific and technological innovation presented in research manuscripts submitted to The EPMA Journal should consider the context of PPPM integration and the advancement of medical services. Consequently, the following article sections are asked to 2. A research manuscript should be dedicated to the development of PPPM-related technologies and demonstrate an innovation that might be presented by hybrid technologies (e.g. complementary imaging), a reasonable combination of diagnostic approaches (e.g. imaging in combination with omics, 2. A research manuscript should be dedicated to the development of PPPM-related technologies and demonstrate an innovation that might be presented by hybrid technologies (e.g. complementary imaging), a reasonable combination of diagnostic approaches (e.g. imaging in combination with omics, Page 2 of 2 Page 2 of 2 Golubnitschaja The EPMA Journal 2013, 4:11 http://www.epmajournal.com/content/4/1/11 Golubnitschaja The EPMA Journal 2013, 4:11 http://www.epmajournal.com/content/4/1/11 Golubnitschaja The EPMA Journal 2013, 4:11 http://www.epmajournal.com/content/4/1/11 be included and these points should be considered prior to submission of a manuscript: Introductory overview of the topic: this section should demonstrate the relevance of the article for the integrative field of PPPM. A detailed field overview with citations to all relevant literature is requested to inform the reader of the state of the art and demonstrate current deficits relevant to health care quality. A detailed analysis of the achieved results is an obligatory section of the manuscript to demonstrate the robustness of the discovery. An interpretation of the discovery should be provided in the context of PPPM followed by the spectrum of functions proposed for its potential utilisation in health care. Expert recommendations are requested to outline perspectives for concomitant developments in the field and related branches. The Editorial Board is confident that our current initiative will become well understood and strongly supported by the journal readers and authors. Received: 15 March 2013 Accepted: 18 March 2013 Published: 28 March 2013 References 1. EPMA Journal: Instructions for Authors: Research articles. http://www. References EPMA Journal: Instructions for Authors: Research articles. http://www epmajournal.com/authors/instructions/research. 2. EPMA Journal: Aims & Scope. http://www.epmajournal.com/ about#aimsscope. doi:10.1186/1878-5085-4-11 Cite this article as: Golubnitschaja: The EPMA Journal introduces a new type of research article dedicated to predictive, preventive and personalised medicine. The EPMA Journal 2013 4:11. 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https://openalex.org/W2943596279	https://research-information.bris.ac.uk/ws/files/200307724/e029388.full.pdf	English	null	Comprehensive ascertainment of bleeding in patients prescribed different combinations of dual antiplatelet therapy (DAPT) and triple therapy (TT) in the UK: study protocol for three population-based cohort studies emulating ‘target trials’ (the ADAPTT Study)	BMJ open	2,019	cc-by	8,226	Pufulete, M., Harris, J., Sterne, J. A. C., Johnson, T. W., Lasserson, D., Mumford, A., Doble, B., Wordsworth, S., Benedetto, U., Rogers, C. A., Loke, Y., Pithara, C., Redwood, S., & Reeves, B. C. (2019). Comprehensive ascertainment of bleeding in patients prescribed different combinations of dual antiplatelet therapy (DAPT) and triple therapy (TT) in the UK: study protocol for three population-based cohort studies emulating 'target trials' (the ADAPTT Study). BMJ Open, 9(6), Article e029388. https://doi.org/10.1136/bmjopen-2019- 029388 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1136/bmjopen-2019-029388 Link to publication record on the Bristol Research Portal PDF-document Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1136/bmjopen-2019-029388 Publisher's PDF, also known as Version of record License (if available): CC BY Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via BMJ Publishing at https://bmjopen.bmj.com/content/9/6/e029388 . Please refer to any applicable terms of use of the publisher. Strengths and limitations of this study Introduction ‘Real world’ bleeding in patients exposed to different regimens of dual antiplatelet therapy (DAPT) and triple therapy (TT, DAPT plus an anticoagulant) have a clinical and economic impact but have not been previously quantified. ► ►We designed our study using the framework recom- mended by the Cochrane Bias and Non-Randomised Studies Methods Groups for establishing appropriate patient populations, interventions and follow-up to emulate three hypothetical randomised controlled trials (target trials). Methods and analysis We will use linked Clinical Practice Research Datalink (CPRD) and Hospital Episode Statistics (HES) data to assemble populations eligible for three ‘target trials’ in patient groups: percutaneous coronary intervention (PCI); coronary artery bypass grafting (CABG); conservatively managed (medication only) acute coronary syndrome (ACS). Patients ≥18 years old will be eligible if, in CPRD records, they have: ≥1 year of data before the index event; no prescription for DAPT or anticoagulants in the preceding 3 months; a prescription for aspirin or DAPT within 2 months after discharge from the index event. The primary outcome will be any bleeding event (CPRD or HES) up to 12 months after the index event. We will estimate adjusted HR for time to first bleeding event comparing: aspirin and clopidogrel (reference) versus aspirin and prasugrel or aspirin and ticagrelor after PCI; and aspirin (reference) versus aspirin and clopidogrel after CABG and ACS. We will describe rates of bleeding in patients prescribed TT (DAPT plus an anticoagulant). Potential confounders will be identified systematically using literature review, semistructured interviews with clinicians and a short survey of clinicians. We will conduct sensitivity analyses addressing the robustness of results to the study’s main limitation—that we will not be able to identify the intervention group for patients whose bleeding event occurs before a DAPT prescription in CPRD. ► ►We will identify potential confounders systematically using literature review, semistructured interviews with clinicians (cardiologists, cardiac surgeons and general practitioners) and a short survey with an ad- ditional group of clinicians. ► ►Prepublication history and additional material for this paper are available online. To view these files, please visit the journal online (http://dx.doi. org/10.1136/bmjopen-2019- 029388 ) ► ►Because there are no medication data in Hospital Episode Statistics, we will assume that patients’ first dual antiplatelet therapy (DAPT) prescription that appears in Clinical Practice Research Datalink (CPRD) after their hospital admission is what they were prescribed at discharge. Comprehensive ascertainment of bleeding in patients prescribed different combinations of dual antiplatelet therapy (DAPT) and triple therapy (TT) in the UK: study protocol for three population-based cohort studies emulating ‘target trials’ (the ADAPTT Study) on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from Maria Pufulete, 1 Jessica Harris,1 Jonathan A C Sterne,2 Thomas W Johnson,3 Daniel Lasserson, 4 Andrew Mumford,5 Brett Doble, 6 Sarah Wordsworth,6 Umberto Benedetto,5 Chris A Rogers,1 Yoon Loke,7 Christalla Pithara,8 Sabi Redwood,8 Barnaby C Reeves1 To cite: Pufulete M, Harris J, Sterne JAC, et al. Comprehensive ascertainment of bleeding in patients prescribed different combinations of dual antiplatelet therapy (DAPT) and triple therapy (TT) in the UK: study protocol for three population-based cohort studies emulating ‘target trials’ (the ADAPTT Study). BMJ Open 2019;9:e029388. doi:10.1136/ bmjopen-2019-029388 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Protocol Open access Comprehensive ascertainment of bleeding in patients prescribed different combinations of dual antiplatelet therapy (DAPT) and triple therapy (TT) in the UK: study protocol for three population-based cohort studies emulating ‘target trials’ (the ADAPTT Study) Introduction incorporated into assessments of cost-effectiveness. For DAPT, this entails accounting for uncertainty in the abso- lute risk of bleeding, the impact of different bleeding events on health-related quality of life and treatment adherence and subsequent risk of secondary ischaemic events and the cost implications of managing these bleeding events. Dual antiplatelet therapy (DAPT), a combination of aspirin and either clopidogrel, prasugrel or ticagrelor, is recommended for secondary prevention of ischaemic events (heart attack and stroke) in people with coronary artery disease. Guidelines recommend that patients are treated with DAPT for 6 to 12 months following myocar- dial infarction (MI) and coronary interventions (percuta- neous coronary intervention [PCI] and coronary artery bypass grafting [CABG])1–3 and support the use of the more potent antiplatelet inhibitors ticagrelor and pras- ugrel.2 Antiplatelet agents reduce the risk of ischaemic events, by preventing the formation of clots in athero- sclerotic coronary arteries and within stents (following PCI) or grafts (following CABG), but increase the risk of bleeding.4 Randomised controlled trials (RCTs) have shown that adding clopidogrel to aspirin leads to 1% excess risk of major bleeding (requiring admission to hospital) compared with aspirin alone.5 6 Prasugrel and ticagrelor reduce the risk of ischaemic events further but also further increase the risk of bleeding.7 Some patients (eg, those with existing atrial fibrillation or those who develop atrial fibrillation after PCI, CABG or acute coro- nary syndrome [ACS]) are prescribed an anticoagulant (eg, warfarin, dabigatran, rivaroxaban, apixaban) in addition to DAPT (triple therapy [TT]), which further increases the risk of bleeding. We propose to use Hospital Episode Statistics (HES) and the Clinical Practice Research Datalink (CPRD) databases to estimate the incidence of all bleeding events occurring in patients prescribed different DAPT or TT regimens after undergoing coronary interven- tions (PCI and CABG) and in conservatively managed patients with ACS. Our study will also provide param- eter estimates to update existing cost-effectiveness models. We will use the framework recommended by the Cochrane Bias and Non-Randomised Studies Methods Groups for establishing appropriate patient populations, interventions and follow-up to emulate the following three hypothetical RCTs (hereafter referred to as the target trials, table 1)17. 1. In patients who have undergone PCI, estimate the ef- fect on bleeding events of assignment to aspirin and clopidogrel (reference) versus aspirin and prasugrel or aspirin and ticagrelor. 2. Open access Open access Data sources CPRD is a database of primary care electronic health record data (available online via CPRD GOLD) from participating general practices, covering 7% of the UK population.18 Patients included in CPRD are largely representative of the UK population in terms of age, sex, ethnicity and body mass index. HES covers all hospital admissions for all English patients whose treatment is funded by the UK National Health Service (NHS), whether treated by the NHS or by independent providers.19 Seventy-five per cent of English general practices included in CPRD are linked to HES data.18 We obtained data from 1 April 2009 to 31 July 2017; this period covers the introduction of the newer anti- platelet agents prasugrel and ticagrelor. This study protocol has been approved. guest. Protected by copyright. The economic impact of bleeding events associated with DAPT is also poorly characterised, in particular for minor bleeding events and their impact on health-related quality of life.8 This is not surprising given that health economic analyses often lack detailed data on adverse effects of interventions, despite consensus that such effects should be considered.15 16 To ensure appropriate decisions are made about which DAPT regimens to use in clinical practice, the health and resource use conse- quences of minor and major bleeding events should be Introduction In patients who have undergone CABG, estimate the effect on bleeding events of assignment to aspirin (ref- erence) versus aspirin and clopidogrel. ‘Real world’ bleeding events that do not require any intervention are likely to be much more frequent than those reported in RCTs, which excluded patients at high risk of bleeding and mainly reported only on major bleeding. Bleeding events that do not result in hospital- isation are largely managed in primary care and may have a significant clinical and economic impact.8 Minor and nuisance bleeding (nose and gum bleeds, bruising and prolonged bleeding from cuts) may also reduce adher- ence to DAPT9 and non-adherent patients may be at increased risk of a secondary ischaemic episode.10 Only three studies have reported the incidence and conse- quences of nuisance bleeding after DAPT11–13; these suggest that nuisance bleeding is common (affecting 29%–38% of patients) and impacts on adherence (11% of patients in one study discontinued clopidogrel12). A nested case–control study using the Health Improve- ment Network (a UK primary care database) reported an increased risk of upper gastrointestinal bleeding with clopidogrel and aspirin compared with aspirin alone (relative risk, 2.08; 95% CI 1.34 to 3.21).14 3. In patients who are conservatively managed after presenting with ACS, estimate the effect on bleeding events of assignment to aspirin (reference) versus aspi- rin and clopidogrel. Strengths and limitations of this study ► ►We will conduct sensitivity analyses to address the robustness of results to different assumptions about the unknown intervention group in patients who died or had a bleeding event before a DAPT prescription in CPRD. Received 24 January 2019 Revised 4 March 2019 Accepted 12 March 2019 For numbered affiliations see end of article. Correspondence to Dr Maria Pufulete; maria.pufulete@bristol.ac.uk © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ. Received 24 January 2019 Revised 4 March 2019 Accepted 12 March 2019 guest. Protected by copyright. UK Medicines and Healthcare Products Regulatory Agency Database Research (protocol 16_126R) and the South West Cornwall and Plymouth Research Ethics Committee (17/SW/0092). The findings will be presented in peer- reviewed journals, lay summaries and briefing papers to commissioners/other stakeholders. Trial registration number 76607611; Pre-results. Correspondence to Dr Maria Pufulete; maria.pufulete@bristol.ac.uk Ethics and dissemination This protocol was approved by the Independent Scientific Advisory Committee for the Trial registration number 76607611; Pre-results. 1 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 p g g Target trial 2 (CABG) g Consecutive patients (age ≥18 years) undergoing CABG (urgent and elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; other concomitant cardiac surgery (eg, valve surgery); major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Target trial 3 (conservatively managed ACS) Consecutive patients (age ≥18 years) hospitalised for an acute coronary syndrome (ACS): myocardial infarction (MI) with or without ST elevation or unstable angina. Exclusions: PCI or CABG performed at time of ACS diagnosis; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/ allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Relevant interventions can be identified as CPRD has information on all medications (including doses) prescribed in primary care. Relevant interventions can be identified as CPRD has information on all medications (including doses) prescribed in primary care. Clopidogrel (75 mg daily) or prasugrel (5 mg or 10 mg daily) or ticagrelor (90 mg twice daily). All patients will receive aspirin (at a dose of 75 mg daily, in line with current guidelines). g ( ) Clopidogrel (75 mg) in addition to aspirin (at a dose of 75 mg daily, in line with current guidelines) or aspirin only (any dose, reflecting variation in usual care). Target trial 3 (conservatively managed ACS) As for target trial 2. g ( ) Clopidogrel (75 mg) in addition to aspirin (at a dose of 75 mg daily, in line with current guidelines) or aspirin only (any dose, reflecting variation in usual care). Target trial 3 (conservatively managed ACS) As for target trial 2. Assignment to interventions Participants are assigned to DAPT interventions in hospital. Participants enter the study at index procedure date for PCI and CABG, and episode start date for ACS, and will be assigned to DAPT interventions using first prescription in CPRD (within 2 months of hospitalisation) as a proxy for what they were prescribed in hospital (there are no medications data in HES). This assignment will exclude a proportion of eligible patients (those who died or experienced a major bleed that caused them to stop DAPT, or patients who have no prescription for DAPT within the 2-month window); we will identify and describe the characteristics of these excluded patients. Study populations htt BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from on 1 July 2019 by guest. Protected by copyr http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from Table 1 Summary of three target trials and how observational data will be used to emulate these Study populations Target trial 3 (conservatively managed ACS) As for target trial 2. Relevant interventions can be identified as CPRD has information on all medications (including doses) prescribed in primary care. http:// BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from Table 1 Summary of three target trials and how observational data will be used to emulate these PICO component Target trial Issues in emulating the target trial using observational data Eligibility criteria Target trial 1 (PCI) Consecutive patients (age ≥18 years) undergoing PCI (emergency or elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Target trial 2 (CABG) Consecutive patients (age ≥18 years) undergoing CABG (urgent and elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; other concomitant cardiac surgery (eg, valve surgery); major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Target trial 3 (conservatively managed ACS) Consecutive patients (age ≥18 years) hospitalised for an acute coronary syndrome (ACS): myocardial infarction (MI) with or without ST elevation or unstable angina. Exclusions: PCI or CABG performed at time of ACS diagnosis; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/ allergy to aspirin, clopidogrel, prasugrel or ticagrelor. CPRD-HES linked data set contains information that allows us to identify all eligible patients for the three target trials. The study period is April 2009–July 2017. All eligible patients will have sufficient data (1 year) preceding their index event to apply the exclusion criteria and characterise the population (eg, comorbidities) and sufficient follow-up data (1 year) to identify outcomes. It is not possible to capture intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Interventions Target trial 1 (PCI) Clopidogrel (75 mg daily) or prasugrel (5 mg or 10 mg daily) or ticagrelor (90 mg twice daily). All patients will receive aspirin (at a dose of 75 mg daily, in line with current guidelines). Target trial 2 (CABG) Clopidogrel (75 mg) in addition to aspirin (at a dose of 75 mg daily, in line with current guidelines) or aspirin only (any dose, reflecting variation in usual care). Target trial 3 (conservatively managed ACS) Relevant interventions can be identified as CPRD has information on all medications (including doses) prescribed in primary care. Study populations Eligibility and exclusion criteria for the three target trials (for patients undergoing PCI, patients undergoing CABG or patients hospitalised and conservatively managed for ACS) are listed in table 1. We will identify eligible patients who are included in Clinical Practice Research Datalink (CPRD) and eligible for linkage with Hospital Episode 2 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Open access Table 1 Summary of three target trials and how observational data will be used to emulate these PICO component Target trial Issues in emulating the target trial using observational data Eligibility criteria Target trial 1 (PCI) Consecutive patients (age ≥18 years) undergoing PCI (emergency or elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Target trial 2 (CABG) Consecutive patients (age ≥18 years) undergoing CABG (urgent and elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; other concomitant cardiac surgery (eg, valve surgery); major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Target trial 3 (conservatively managed ACS) Consecutive patients (age ≥18 years) hospitalised for an acute coronary syndrome (ACS): myocardial infarction (MI) with or without ST elevation or unstable angina. Exclusions: PCI or CABG performed at time of ACS diagnosis; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/ allergy to aspirin, clopidogrel, prasugrel or ticagrelor. CPRD-HES linked data set contains information that allows us to identify all eligible patients for the three target trials. The study period is April 2009–July 2017. All eligible patients will have sufficient data (1 year) preceding their index event to apply the exclusion criteria and characterise the population (eg, comorbidities) and sufficient follow-up data (1 year) to identify outcomes. It is not possible to capture intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. Interventions Target trial 1 (PCI) Clopidogrel (75 mg daily) or prasugrel (5 mg or 10 mg daily) or ticagrelor (90 mg twice daily). All patients will receive aspirin (at a dose of 75 mg daily, in line with current guidelines). Target trial 2 (CABG) Clopidogrel (75 mg) in addition to aspirin (at a dose of 75 mg daily, in line with current guidelines) or aspirin only (any dose, reflecting variation in usual care). Issues in emulating the target trial using observational data Target trial 1 (PCI) Consecutive patients (age ≥18 years) undergoing PCI (emergency or elective). Exclusions: DAPT or anticoagulant use in the previous 3 months; major bleed requiring hospitalisation in previous 12 months; renal failure requiring dialysis; intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. CPRD-HES linked data set contains information that allows us to identify all eligible patients for the three target trials. The study period is April 2009–July 2017. All eligible patients will have sufficient data (1 year) preceding their index event to apply the exclusion criteria and characterise the population (eg, comorbidities) and sufficient follow-up data (1 year) to identify outcomes. It is not possible to capture intolerance/allergy to aspirin, clopidogrel, prasugrel or ticagrelor. p g g Target trial 2 (CABG) In sensitivity analyses, we will address the robustness of results to different assumptions about the intervention group in those patients where the DAPT medication is unknown or a major bleed occurs prior to the first DAPT medication, by using multiple imputation models for handling missing data. Prior known information regarding the likely prescription based on patient characteristics or general policies will be incorporated in these analyses. y guest. Protected by copyright. Continued 3 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Open access PICO component Target trial Issues in emulating the target trial using observational data Follow-up Starts at assignment to intervention and ends at first bleed or 12 months from assignment (whichever comes first). Starts at time of hospitalisation for PCI, CABG or ACS and ends at first bleed or 12 months from hospitalisation (whichever comes first). Primary outcome Any bleed within 12 months of the start of DAPT (DAPT is prescribed at hospitalisation for PCI, CABG or ACS). Any bleed within 12 months of hospitalisation for PCI, CABG or ACS. Analysis Intention to treat According to first prescription for DAPT in CPRD CABG, coronary artery bypass grafting; CRPD, Clinical Practice Research Datalink; DAPT, dual antiplatelet therapy; HES, Hospital Episode Statistics; PCI, percutaneous coronary intervention; PICO, Population, intervention, comparator, outcome. Table 1 Continued on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from Follow-up The start of follow-up (the index event) will be the date of the index hospital procedure (PCI, CABG) or start date of the hospital episode that contains the ACS diagnosis (ACS). Patients will be followed up until 12 months after the index event, since DAPT is prescribed for 12 months in accordance to guidelines. We will classify patients according to the first prescrip- tion recorded in CPRD during the first 2 months after hospitalisation for PCI, CABG or ACS. This 2-month window is based on variability in the amount of DAPT medication provided to patients in hospital following their PCI, CABG or ACS and hence variability in the time when they first request a repeat prescription from their general practice. A preliminary investigation showed that more than 75% of eligible patients have a prescrip- tion for one or more antiplatelet agents during this time period. If a patient only has a prescription for aspirin during the 2-month window after hospital discharge, they will be assigned to an aspirin only intervention. If patients also receive a prescription for clopidogrel, pras- ugrel or ticagrelor, they will be assigned to aspirin/clopi- dogrel, aspirin/prasugrel or aspirin/ticagrelor. If there is a prescription for more than one additional antiplatelet agent in the 2-month window, we will assign the patient to an intervention based on the agent prescribed first. For Outcomes The primary outcome will be any bleeding event, clas- sified as type 2–5 by the Bleeding Academic Research Consortium (BARC) bleeding scale.21 For each patient, we will identify all bleeding events in HES and CPRD during follow-up. We will not be able to identify BARC type 1 bleeding events (bleeding that is not actionable and does not cause the patient to seek treatment) as type 1 assumes there is no interaction with the health system or healthcare professionals; therefore, no bleed event will be recorded in HES or CPRD. We have specified a comprehensive list of bleeding codes in CPRD and HES (see online supplementary appendix 1). These will be categorised according to anatomical site for descriptive purposes. Secondary outcomes will be: any major bleeding event (leading to hospital admission, inpatient HES); any minor bleeding event (recorded within CPRD data); all-cause mortality; cardiovascular mortality; mortality from bleeding (these will be identified from linked ONS data); MI; stroke; additional coronary intervention. In the target trials, the interventions would be assigned during the hospital stay, as soon as patients are eligible for antiplatelet therapy. Our observational data set does not have information on medication given to patients at discharge, because HES does not include medications data. Therefore, the first time at which we have infor- mation on the antiplatelet regimen to which patients were assigned in hospital is when they receive their first primary care prescription/s for aspirin or DAPT, recorded in CPRD. It is reasonable to use these as a proxy for the medications that patients started in hospital, because patients’ general practitioners (GPs) are unlikely to change the prescriptions that were started in hospital. Interventions example, if a patient has an aspirin prescription and a prescription for clopidogrel before a prescription for tica- grelor, the patient will be assigned to the aspirin/clopido- grel intervention. Patients with no prescriptions in CPRD for aspirin, or aspirin and clopidogrel, prasugrel or tica- grelor within the 2-month window will be excluded from the main analysis. We will conduct sensitivity analyses of all eligible patients including those with unknown DAPT regimens or a major bleed prior to first DAPT prescrip- tion, by estimating assignment to DAPT interventions for those with no prescription data using multiple imputa- tion based on a range of assumptions.20 The interventions of interest for the three target trials are shown in table 1. Guidelines recommend low dose aspirin (75 to 100 mg/d) plus either clopidogrel (75 mg/d), prasugrel (5 mg/d or 10 mg/d) or ticagrelor (90 mg twice/d) for PCI and conservatively managed patients with ACS. For PCI patients, the interventions of interest are aspirin and clopidogrel, aspirin and prasugrel and aspirin and ticagrelor. In conservatively managed patients with ACS, clopidogrel is the most commonly prescribed second antiplatelet agent (in addition to aspirin) and a large proportion of patients are prescribed aspirin only; therefore, the interventions of interest are aspirin only (75 to 100 mg/day) and aspirin and clopidogrel. There is variation in aspirin prescription for CABG patients; some surgeons choose 75 mg/day, others 150 mg/day or 300 mg/day. Surgeons may also prescribe an additional antiplatelet agent, most commonly clopidogrel. There- fore, the comparisons of interest in CABG patients are aspirin only (any dose, reflecting variations in usual care in different hospitals) and aspirin and clopidogrel (doses as for PCI). We have specified these comparisons based on preliminary feasibility counts from CPRD, which indi- cate that few CABG and conservatively managed patients with ACS are prescribed aspirin and prasugrel or aspirin and ticagrelor. on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ blished as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from BMJ Open: first published as 10.1136/bmjopen-2019-029388 on 4 June 2019. D Open access Issues in emulating the target trial using observational data Statistics (HES) and Office for National Statistics (ONS) mortality data (because they have a valid NHS number and are registered at a practice that was participating in the linkage programme). Patients are included if they had a PCI, CABG or ACS (index event) record in HES during the study period (1 April 2010–31 January 2017), and have at least 1 year of linked CPRD-HES data before the date of their index event. They must also have been prescribed one of the treatment regimens being compared in the target trial corresponding to their index event. One year’s data preceding eligibility for the target trial is adequate to apply most of the exclusion criteria and determine comorbidities and medication history: such information would be collected at baseline in a randomised trial. The following Office of Population Censuses and Surveys (OPCS) procedure codes (PCI and CABG) and Interna- tional Classification of Diseases (ICD-10) codes (ACS no procedure) will be used to identify patients: PCI, K49, K50 & K75; CABG, K40, K41, K42, K43, K44, K45 & K46; ACS without a procedure, I20.0, I21, I22, I24.9 (with no OPCS code for PCI or CABG in the same hospital admission). Figure 1 shows full details of the inclusion and exclusion criteria. Figure 1 Study diagram describing the construction of the PCI, CABG and ACS (conservatively managed) populations. on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ Downloaded from Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 4 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Confounding and cointerventions Potential confounders (variables that predict both risk of bleeding and intervention group) will be specified a priori.22 23 We will identify confounders and cointer- ventions using literature review and clinician expertise as recommended by the Cochrane Bias and Non-Ran- domised Studies Methods Groups.17 We will carry out a comprehensive and systematic literature search to iden- tify all RCT and cohort studies of DAPT interventions, or cohort studies that identify predictors of bleeding. The literature searches for the review are included in supple- mentary appendix 2. Abstracts will be screened by one researcher and full-text papers will be obtained. Data on confounders and cointerventions will be extracted by two researchers independently using a data extraction form specifically designed for the study; variables extracted y guest. Protected by copyright. 5 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Open access data set but acknowledge that there may be missing data across patients (and time) for some confounders. will include study characteristics, population character- istics (reported in the tables of baseline characteristics), factors adjusted for in the statistical analyses and factors reported to predict risk of bleeding in our populations. We will not perform a risk of bias assessment because the aim of the review is only descriptive (ie, the output will be lists of confounders and cointerventions) and there are no established criteria for assessing the validity with which primary researchers consider potential confounders and cointerventions; therefore, it would be inappropriate to apply a risk-of-bias tool for studies estimating a treatment effect. We will use ‘saturation’ as a criterion for discontin- uing data collection, defined as review of 10 consecutive studies without identifying an additional confounder/ cointervention. Sample size Estimated rates of bleeding with the different thera- pies are 5% for aspirin, 9% for aspirin/clopidogrel and 12% f i i / l d i i / i l 5 6 24 25 Estimated rates of bleeding with the different thera- pies are 5% for aspirin, 9% for aspirin/clopidogrel and 12% for aspirin/prasugrel and aspirin/ticagrelor.5 6 24 25 Preliminary feasibility counts provided by CPRD suggest that there will at least the following numbers of patients eligible for each target trial: on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2019-029388 on 4 June 2019. Downloaded from p p g p g Preliminary feasibility counts provided by CPRD suggest that there will at least the following numbers of patients eligible for each target trial: p p g p g Preliminary feasibility counts provided by CPRD suggest that there will at least the following numbers of patients eligible for each target trial g g PCI: aspirin/clopidogrel (reference, 6738 patients) versus aspirin/prasugrel (842 patients) or aspirin/tica- grelor (770 patients) g p CABG: aspirin (reference, 2556 patients) versus aspi- rin/clopidogrel (595 patients) p g p Conservatively managed ACS: aspirin (reference, 8148 patients) versus aspirin/clopidogrel (3082 patients) In parallel, we will conduct semiquantitative interviews with six clinicians in each of three groups: cardiologists; cardiac surgeons and GPs (to determine whether DAPT prescriptions are changed in primary care). The main aim of the clinician interviews is to understand DAPT prescribing practice in the UK and identify the factors (relating to patients, centres and prescribing practices of the individual doctors) that influence the decision about which antiplatelet regimen to prescribe. All factors that influence DAPT prescribing (confounders) identi- fied from the literature review and clinician interviews will be combined in a short survey (SurveyMonkey). The survey questionnaire will be administered online to all consultant members of the British Cardiovascular Society (cardiologists) and the Society for Cardiothoracic Surgery (cardiac surgeons). The survey will be either emailed to all members individually (if the professional bodies agree) or advertised in weekly/monthly news- letters. Sample size Confounders will be grouped in confounding domains.17 We will attempt to identify each potential confounder (identified through the literature review, clinician interviews and survey) in the CPRD or HES These estimates give expected event rates of at least 700 for PCI, 180 for CABG and 680 for ACS, assuming a ratio of 8:1 (aspirin/clopidogrel:aspirin/prasugrel or aspirin/clopidogrel:aspirin/ticagrelor) for PCI, 4:1 (aspirin:aspirin/clopidogrel) for CABG and 2.5:1 (aspir- in:aspirin/clopidogrel) for ACS. The HR detectable with 90% and 80% power at the 5% statistical signifi- cance, assuming the group ratios given above, are shown in table 2. The correlation of the DAPT with other covar- iates adjusted for is unknown and we assessed the impact of a range of correlations (0, 0.3 and 0.5). Statistical analyses We will describe temporal changes in DAPT prescribing and bleeding for PCI, CABG and ACS populations. We will use descriptive statistics to summarise the characteris- tics of the different intervention groups and standardised mean differences to compare them. We will estimate rates of bleeding (number of events/person time) with 95% CIs for each group. We will separate major and minor 6 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Table 2 HR for a range of correlations for percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG) and acute coronary syndrome (ACS) Ratio of presence: absence of covariate Squared correlation with other covariates HR detectable 90% power 80% power PCI 8:1 0 (ie, unadjusted) 1.48 1.41 0.3 1.60 1.50 0.5 1.74 1.62 CABG 4:1 0 (ie, unadjusted) 1.83 1.69 0.3 2.06 1.87 0.5 2.35 2.10 Conservatively managed ACS 2.5:1 0 (ie, unadjusted) 1.32 1.27 0.3 1.39 1.33 0.5 1.48 1.40 on 1 July 2019 by guest. Protected by copyright. bmj.com/ 6 6 Open access bleeding since adverse events of each type have different health and resource use consequences. bleeding since adverse events of each type have different health and resource use consequences. those that died before receiving their first prescription, had a major bleed which caused them to stop DAPT or have no aspirin/DAPT prescription recorded in CPRD within the 2-month window). These analyses will be un- dertaken using multiple imputation methods to deal with missing information on DAPT medication and will take patient characteristics, procedure/diagnosis and general medication policies into account, using a range of assumptions. q Analyses will estimate the effects of assigned inter- vention (analogous to an intention-to-treat analysis of a randomised trial) for the antiplatelet regimens corre- sponding to the first prescription of aspirin or DAPT in CPRD (see Interventions). We will use parametric survival models to estimate adjusted HRs with 95% CIs for the time to first bleeding event, comparing intervention groups for each target trial. Exploratory analyses, including assess- ment of proportional hazards assumptions, will be used to inform the choice of survival distribution (eg, Weibull). Estimated time-dependent event probabilities will be used to update existing cost-effectiveness models.26 The confounding factors to be included in the model (which will be identified as described previously and grouped into confounding domains from our data set), the modelling strategy and the approach to handling correlated covari- ates will be documented in a data analysis plan. Statistical analyses Partici- pants free from a bleeding event will be censored at 12 months after the index event. For secondary endpoints, we will use survival models to estimate adjusted HRs with 95% CIs for time to first event. For mortality outcomes, we will take account of the competing risks of death due to other causes. 2. We will address the possibility that some minor bleeding events are not documented in CPRD but nevertheless prompt medication changes. We anticipate that most bleeding events will occur soon after the index event and before any medication change. Medication changes will be described relative to bleeding events observed (eg, before event, after event, no bleeding event observed). If a substantial proportion (>10%) of people change medication before their first bleeding event, we will per- form a sensitivity analysis excluding these patients. 2. We will address the possibility that some minor bleeding events are not documented in CPRD but nevertheless prompt medication changes. We anticipate that most bleeding events will occur soon after the index event and before any medication change. Medication changes will be described relative to bleeding events observed (eg, before event, after event, no bleeding event observed). If a substantial proportion (>10%) of people change medication before their first bleeding event, we will per- form a sensitivity analysis excluding these patients. 3. We will assess the sensitivity of our findings when the analysis is restricted to a subpopulation of patients at low risk of bleeding (ie, excluding people at high risk for bleeding) who, we hypothesise, have the lowest risk of residual confounding. on 1 July 2019 by guest. Protect http://bmjopen.bmj.com/ 88 on 4 June 2019. Downloaded from We will attempt to identify an instrumental variable (eg, clinician/GP practice prescribing practice) to control for confounding by indication. If we are successful, we will repeat the above analyses for the PCI, CABG and ACS populations. We will perform three sets of sensitivity analyses: 1. We will address the unknown intervention group of eligible patients who have no prescription data and therefore cannot be assigned to an intervention (ie, 7 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Figure 2 Study diagram describing the construction of the triple therapy (TT) populations. on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ ed from igure 2 Study diagram describing the construction of the triple therapy (TT) populations. Author affiliations 1 1Clinical Trials and Evaluation Unit, Bristol Trials Centre, University of Bristol, Bristol, UK UK 2NIHR Biomedical Research Centre, Department of Population Health Sciences, University of Bristol, Bristol, UK 3Department of Cardiology, Bristol Heart Institute, Bristol, UK 4Institute of Applied Health Research, University of Birmingham, Birmingham, UK 5Bristol Medical School, University of Bristol, Bristol, UK 6Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford UK 2NIHR Biomedical Research Centre, Department of Population Health Sciences, University of Bristol, Bristol, UK 3Department of Cardiology, Bristol Heart Institute, Bristol, UK 4Institute of Applied Health Research, University of Birmingham, Birmingham, UK 5Bristol Medical School, University of Bristol, Bristol, UK 6Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford UK 7Norwich Medical School, University of East Anglia, Norwich, UK 8Ethnography Research Team, National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care West (NIHR CLAHRC West), Bristol, UK pp In patients receiving various TT regimens (grouped by pre-index event anticoagulant prescription and post- index event anticoagulant prescription in PCI, CABG and ACS separately, see figure 2), we will describe rates of bleeding (number of events/person time) with 95% CIs for each group. We have not specified comparison groups because we know there is large variation in prescribing of anticoagulants; TT is usually prescribed for a rela- tively short period of time (1–6 months, depending on a patient’s individual risks of thrombosis and bleeding) after which an antiplatelet agent (usually aspirin) is removed and patients continue to receive an anticoagu- lant and single antiplatelet for the remainder of the 12 months after the index admission. Acknowledgements We would like to thank Taria Murray-Thomas, senior researcher at CRPD, who assisted with the data requests. We would also like to thank members of our PPI group and the PPI lead, Dr Noreen Hopewell-Kelly, for their input into the design of the study. This study was designed and delivered in collaboration with the Clinical Trials and Evaluation Unit (CTEU), a UKCRC registered clinical trials unit which, as part of the Bristol Trials Centre, is in receipt of National Institute for Health Research CTU support funding. Contributors MP: designed and will conduct the study and wrote the manuscript. JH: study statistician responsible for data linkage and analysis. JACS: provided strategic direction with respect to study design and analysis. TWJ: conceived the research question and provided cardiology expertise. Open access We will explore the consistency of treatment effect esti- mates in the following subgroups: ACS versus non-ACS (PCI and CABG populations); diabetic versus non-dia- betic; chronic kidney disease versus non-chronic kidney disease; concurrent prescription for proton pump inhib- itors versus no prescription for proton pump inhibitors. All subgroups will be defined in the data analysis plan and chosen based on the characteristics of the analysis popu- lations before carrying out any analyses by intervention group. We will report p values from tests of interaction. and patients weighing the potential benefits against the adverse side effects and practical inconvenience. Our PPI group will attend regular meetings to discuss results from the study and will also contribute and collaborate on the development of our results dissemination strategy. Author affiliations 1 DL: provided general practice expertise, clinical epidemiological expertise. AM: conceived the research question and provided haematology expertise. BD: provided health economics expertise. SW: provided health economics expertise. UB: provided cardiac surgery expertise. CAR: provided guidance with respect to the statistical analysis. YL: provided pharmacoepidemiology expertise. CP: responsible for conduct of the qualitative study with clinicians. SR: will oversee conduct and reporting of the qualitative study with clinicians. BCR: provided strategic direction with respect to study design and analysis. All authors read and approved the final version of the manuscript. Ethics and dissemination This study protocol has approval. We also obtained ethical approval for the semistructured interviews with clinicians and survey from South West Cornwall and Plymouth Research Ethics Committee, 17/SW/0092. The findings will be presented at national/international conferences, published in peer-reviewed academic journals and acces- sible formats in newsletters to patients (where available). The findings will also be reported as a briefing paper to commissioners (eg, commissioning groups, National Institute for Health and Care Excellence) and to other healthcare stakeholders with an interest in the research through the Cardiac & Stroke Networks. We will also present and discuss our results at local CPRD working groups to disseminate methods to other researchers using the CPRD database. Triple therapy We will establish additional groups of patients (PCI or CABG or conservatively managed ACS) based on TT prescription. The main reason for prescribing an anti- coagulant is to treat atrial fibrillation in a patient other- wise eligible for either of the three target trials. Atrial fibrillation can precede (long-term anticoagulation for pre-existing atrial fibrillation) or follow the index event (anticoagulation for new-onset atrial fibrillation). Figure 2 shows how the patient groups receiving TT will be constructed. on 1 July 2019 by guest. Pro http://bmjopen.bmj.com/ 29388 on 4 June 2019. Downloaded from We will identify patients on long-term anticoagula- tion by identifying prescriptions of oral anticoagulants (warfarin, dabigatran, rivaroxaban, apixaban) in the 3 months before their index event and a concomitant prescription of any anticoagulant (TT) with DAPT and in the first 2 months after the index admission. Patients who develop atrial fibrillation after the index event will be identified from any new anticoagulant prescription during follow-up. We have compiled a list of all drug codes in CPRD for antiplatelet agents (aspirin, clopidogrel, prasugrel and ticagrelor) and anticoagulants (warfarin, dabigatran, rivaroxaban, apixaban) (see supplementary appendix 3). Statistical analyses i 2 S d di d ibi h i f h i l h (TT) l i 7 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 Open access Competing interests TJ has received speaker and consultancy fees from Astra Zeneca & Daichi-Sankyo. None of the other authors have competing interests to declare. 10. Cohen M. Expanding the recognition and assessment of bleeding events associated with antiplatelet therapy in primary care. Mayo Clin Proc 2009;84:149–60. 10. Cohen M. Expanding the recognition and assessment of bleeding events associated with antiplatelet therapy in primary care. Mayo Clin Proc 2009;84:149–60. 11. Ben-Dor I, Torguson R, Scheinowitz M, et al. Incidence, correlates, and clinical impact of nuisance bleeding after antiplatelet therapy for patients with drug-eluting stents. Am Heart J 2010;159:871–5. Ethics approval Independent Scientific Advisory Committee (ISAC) of the CPRD (protocol number 16_126R). 12. Roy P, Bonello L, Torguson R, et al. Impact of "nuisance" bleeding on clopidogrel compliance in patients undergoing intracoronary drug- eluting stent implantation. Am J Cardiol 2008;102:1614–7. Provenance and peer review Not commissioned; peer reviewed for ethical and funding approval prior to submission. 13. Amin AP, Bachuwar A, Reid KJ, et al. Nuisance bleeding with prolonged dual antiplatelet therapy after acute myocardial infarction and its impact on health status. J Am Coll Cardiol 2013;61:2130–8. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 14. García Rodríguez LA, Lin KJ, Hernández-Díaz S, et al. Risk of upper gastrointestinal bleeding with low-dose acetylsalicylic acid alone and in combination with clopidogrel and other medications. Circulation 2011;123:1108–15. 15. Craig D, McDaid C, Fonseca T, et al. Are adverse effects incorporated in economic models? An initial review of current practice. Health Technol Assess 2009;13:1-71, 97-181, iii. p 16. Heather EM, Payne K, Harrison M, et al. Including adverse drug events in economic evaluations of anti-tumour necrosis factor-α drugs for adult rheumatoid arthritis: a systematic review of economic decision analytic models. Pharmacoeconomics 2014;32:109–34. References 1. NICE. Myocardial infarction: cardiac rehabilitation and prevention of further cardiovascular disease. 2013 http://www.nice.org.uk/ guidance/cg172/resources/guidance-mi-secondary-prevention-pdf. 1. NICE. Myocardial infarction: cardiac rehabilitation and prevention of further cardiovascular disease. 2013 http://www.nice.org.uk/ guidance/cg172/resources/guidance-mi-secondary-prevention-pdf. y 17. Sterne JA, Hernán MA, Reeves BC, et al. ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ 2016;355:i4919. 2. Valgimigli M, Bueno H, Byrne RA, et al. ESC focused update on dual antiplatelet therapy in coronary artery disease developed in collaboration with EACTS: The Task Force for dual antiplatelet therapy in coronary artery disease of the European Society of Cardiology (ESC) and of the European Association for Cardio- Thoracic Surgery (EACTS). European heart journal 2017;39:213–60. 2. Valgimigli M, Bueno H, Byrne RA, et al. ESC focused update on dual antiplatelet therapy in coronary artery disease developed in collaboration with EACTS: The Task Force for dual antiplatelet therapy in coronary artery disease of the European Society of Cardiology (ESC) and of the European Association for Cardio- Thoracic Surgery (EACTS). European heart journal 2017;39:213–60. 18. Herrett E, Gallagher AM, Bhaskaran K, et al. Data Resource Profile: Clinical Practice Research Datalink (CPRD). Int J Epidemiol 2015;44:827–36. 19. NHS. Hospital Episode Statistics (HES). 2018 http://content.digital. nhs.uk/hes. g y ( ) p j 3. Wijns W, Kolh P, Danchin N, et al. Guidelines on myocardial revascularization. Eur Heart J 2010;31:2501–55. 20. Sterne JA, White IR, Carlin JB, et al. Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls. BMJ 2009;338:b2393. g y ( ) p j 3. Wijns W, Kolh P, Danchin N, et al. Guidelines on myocardial revascularization. Eur Heart J 2010;31:2501–55. 4. Tada T, Natsuaki M, Morimoto T, et al. Duration of dual antiplatelet therapy and long-term clinical outcome after coronary drug-eluting stent implantation: landmark analyses from the CREDO-Kyoto PCI/ CABG Registry Cohort-2. Circ Cardiovasc Interv 2012;5:381–91.i 4. Tada T, Natsuaki M, Morimoto T, et al. Duration of dual antiplatelet therapy and long-term clinical outcome after coronary drug-eluting stent implantation: landmark analyses from the CREDO-Kyoto PCI/ CABG Registry Cohort-2. Circ Cardiovasc Interv 2012;5:381–91.i on 1 July 2019 by guest. Protected by copy http://bmjopen.bmj.com/ ne 2019. Downloaded from 21. Mehran R, Rao SV, Bhatt DL, et al. Standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the Bleeding Academic Research Consortium. Circulation 2011;123:2736–47. 5. Main C, Palmer S, Griffin S, et al. Patient and public involvement We set up a patient advisory group comprising of patients from our three cohorts (PCI, CABG and conserva- tively managed ACS). The group was consulted about several aspects of the proposed study. Group members confirmed the importance of the research topic and agreed that the choice of antiplatelet regimen should be based on shared decision making, with clinicians Funding This study is funded by the National Institute for Health Research (NIHR) Health Technology Assessment (HTA 14/192/89). The British Heart Foundation and NIHR Bristol Biomedical Research Unit for Cardiovascular Disease funded some staff time (MP, JH, BCR). Disclaimer The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 8 Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 26. Briggs A, Claxton K, Sculpher M. Decision Modelling for Health Economic Evaluation. New York: Oxford University Press Inc, 2006. References Clopidogrel used in combination with aspirin compared with aspirin alone in the treatment of non-ST- segment-elevation acute coronary syndromes: a systematic review and economic evaluation. Health Technol Assess 2004;8::iii-iv, xv-xvi, 1-141. 5. Main C, Palmer S, Griffin S, et al. Clopidogrel used in combination with aspirin compared with aspirin alone in the treatment of non-ST- segment-elevation acute coronary syndromes: a systematic review and economic evaluation. Health Technol Assess 2004;8::iii-iv, xv-xvi, 1-141. 22. Reeves BC, Deeks JJ, Higgins JPT. et al. Chapter 13: Including non-randomized studies. In: Higgins JPT, Green S, eds. Cochrane Handbook for Systematic Reviews of Interventions. Version 5.0.2: The Cochrane Collaboration, 2009. [updated September 2009]. on 1 July 2019 by guest. Protected by copyright. http://bmjopen.bmj.com/ aded from 23. Reeves BC, Higgins JP, Ramsay C, et al. An introduction to methodological issues when including non-randomised studies in systematic reviews on the effects of interventions. Res Synth Methods 2013;4:1–11. 6. Terpening C. An appraisal of dual antiplatelet therapy with clopidogrel and aspirin for prevention of cardiovascular events. J Am Board Fam Med 2009;22:51–6. 6. Terpening C. An appraisal of dual antiplatelet therapy with clopidogrel and aspirin for prevention of cardiovascular events. J Am Board Fam Med 2009;22:51–6. 7. Pilgrim T, Windecker S. Antiplatelet therapy for secondary prevention of coronary artery disease. Heart 2014;100:1750–6. 24. Wiviott SD, Braunwald E, McCabe CH, et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007;357:2001–15. 8. Doble B, Pufulete M, Harris JM, et al. Health-related quality of life impact of minor and major bleeding events during dual antiplatelet therapy: a systematic literature review and patient preference elicitation study. Health Qual Life Outcomes 2018;16:191. 25. Wallentin L, Becker RC, Budaj A, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2009;361:1045–57. y 9. Czarny MJ, Nathan AS, Yeh RW, et al. Adherence to dual antiplatelet therapy after coronary stenting: a systematic review. Clin Cardiol 2014;37:505–13. y 9. Czarny MJ, Nathan AS, Yeh RW, et al. Adherence to dual antiplatelet therapy after coronary stenting: a systematic review. Clin Cardiol 2014;37:505–13. 26. Briggs A, Claxton K, Sculpher M. Decision Modelling for Health Economic Evaluation. New York: Oxford University Press Inc, 2006. Pufulete M, et al. BMJ Open 2019;9:e029388. doi:10.1136/bmjopen-2019-029388 9
https://openalex.org/W4232854187	https://peerj.com/articles/6028v0.3/submission	English	null	Peer Review #1 of "Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study (v0.1)"	null	2,018	cc-by	10,814	Manuscript to be reviewed Manuscript to be reviewed Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study Qi Mao 1, 2 , Chengjie Chen 1 , Tao Xie 1 , Aiping Luan 3 , Chaoyang Liu 1 , Yehua He Corresp. 1 1 College of Horticultural, South China Agricultural University, Guangzhou, China 2 College of Agriculture, Guangdong Ocean University, Zhanjiang, China 3 Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, Danzhou, China Corresponding Author: Yehua He Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study Comprehensive tissue-specific transcriptome profiling pineapple (Ananas comosus) and building an eFP-brow further study Qi Mao 1, 2 , Chengjie Chen 1 , Tao Xie 1 , Aiping Luan 3 , Chaoyang Liu 1 , Yehua He Corresp. 1 1 College of Horticultural, South China Agricultural University, Guangzhou, China 2 College of Agriculture, Guangdong Ocean University, Zhanjiang, China 3 Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, Danzhou, China Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study Mao 1, 2 , Chengjie Chen 1 , Tao Xie 1 , Aiping Luan 3 , Chaoyang Liu 1 , Yehua He Corresp. Qi Mao , , Chengjie Chen , Tao Xie , Aiping Luan , Chaoyang Liu , Yehua He p 1 College of Horticultural, South China Agricultural University, Guangzhou, China 2 College of Agriculture, Guangdong Ocean University, Zhanjiang, China 3 Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, Danzhou, China Corresponding Author: Yehua He 1 College of Horticultural, South China Agricultural University, Guangzhou, China 2 College of Agriculture, Guangdong Ocean University, Zhanjiang, China 3 Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, Danzhou, China Corresponding Author: Yehua He Email address: heyehua@scau.edu.cn Corresponding Author: Yehua He Email address: heyehua@scau.edu.cn Pineapple is one of the most economically important tropical or subtropical fruit trees. However, few studies focus on the development of its unique collective fruit. In this study, we generated a genome-wide developmental transcriptomic profile of 14 different tissues of the collective fruit of the pineapple covering each of the three major fruit developmental stages. In total, 273 tissue-specific and 1,051 constitutively expressed genes were detected. We also performed gene co-expression analysis and 18 gene modules were classified. Among these, we found three interesting gene modules; one was preferentially expressed in bracts and sepals and was likely involved in plant defense; one was highly expressed at the beginning of fruit expansion and faded afterward and was probably involved in endocytosis; Another gene module increased expression level with pineapple fruit development and was involved in terpenoid and polyketide metabolism. In addition, we built a pineapple electronic fluorescent pictograph (eFP) browser to facilitate exploration of gene expression during pineapple fruit development. With this tool, users can visualize expression data in this study in an intuitive way. Together, the transcriptome profile generated in this work and the corresponding eFP browser will facilitate further study of fruit development in pineapple. PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed 1 Comprehensive tissue-specific transcriptome profiling o 2 pineapple (Ananas comosus) and building an eFP-browser for 3 further study 4 5 Qi Mao1,2,3,$, Chengjie Chen1,2,$, Tao Xie, Aiping Luan4, Chaoyang Liu1,2, Yehua He1,2,* 6 7 1. College of Horticulture, South China Agricultural University, Guangzhou 510642, Guangdong, P.R. 8 China. 9 2. Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in South China, 10 Guangzhou 510642, Guangdong, P.R. China. 11 3. College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, Guangdong, P.R. China. 12 4. Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, 13 Danzhou, Hainan, China 14 15 16 $co-first author 17 18 *Corresponding author 19 YH: heyehua@scau.edu.cn 20 21 1 Comprehensive tissue-specific transcriptome profiling of 2 pineapple (Ananas comosus) and building an eFP-browser for 3 further study 3. College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, Guangdong, P.R. China. 4. Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science, Danzhou, Hainan, China Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 22 Abstract 23 Pineapple is one of the most economically important fruit crops widely cultivated in the warm subtropical region. 24 However, few studies focus on the development of its unique collective fruit. In this study, we generated a 25 genome-wide developmental transcriptomic profile of 14 different tissues of the collective fruit of the pineapple 26 covering each of the three major fruit developmental stages. In total, 273 tissue-specific and 1,051 constitutively 27 expressed genes were detected. We also performed gene co-expression analysis and 18 gene modules were 28 classified. Among these, we found three interesting gene modules; one was preferentially expressed in bracts 29 and sepals and was likely involved in plant defense; one was highly expressed at the beginning of fruit expansion 30 and faded afterward and was probably involved in endocytosis; another gene module increased expression level 31 with pineapple fruit development and was involved in terpenoid and polyketide metabolism. In addition, we built 32 a pineapple electronic fluorescent pictograph (eFP) browser to facilitate exploration of gene expression during 33 pineapple fruit development. With this tool, users can visualize expression data in this study in an intuitive way. 34 Together, the transcriptomic profile generated in this work and the corresponding eFP browser will facilitate 35 further study of fruit development in pineapple. 36 23 Pineapple is one of the most economically important fruit crops widely cultivated in the warm subtropical region. 24 However, few studies focus on the development of its unique collective fruit. In this study, we generated a 25 genome-wide developmental transcriptomic profile of 14 different tissues of the collective fruit of the pineapple 26 covering each of the three major fruit developmental stages. In total, 273 tissue-specific and 1,051 constitutively 27 expressed genes were detected. We also performed gene co-expression analysis and 18 gene modules were 28 classified. Among these, we found three interesting gene modules; one was preferentially expressed in bracts 29 and sepals and was likely involved in plant defense; one was highly expressed at the beginning of fruit expansion 30 and faded afterward and was probably involved in endocytosis; another gene module increased expression level 31 with pineapple fruit development and was involved in terpenoid and polyketide metabolism. In addition, we built 32 a pineapple electronic fluorescent pictograph (eFP) browser to facilitate exploration of gene expression during 33 pineapple fruit development. Manuscript to be reviewed 37 Introduction 62 Since the development of the original eFP browser for Arabidopsis, eFP browsers have also been developed for 38 39 Pineapple is an economically important fruit herb widely cultivated in tropical and sub-tropical regions 40 (Bartholomew, Paull & Rohrbach, 2002). Pineapples are unique agricultural fruit not only due to their fragrance 41 and taste, but also because they form collective fruits, which differ them from other familiar fruits such as apples, 42 oranges, and mangoes. All fleshy coalesced floral parts of pineapple flowers and its inflorescence can be 43 consumed as either fresh or canned fruit (Bartholomew, Paull & Rohrbach, 2002; Botella, Ramon & Smith, 44 2008). Thus, pineapple fruit possesses both economic and research value. 45 Recently, with the popularization of high-throughput sequencing technologies, several genomic studies of 46 pineapple fruit have been performed (Moyle et al., 2005; Ong, Voo & Kumar, 2012; Redwan, Saidin & Vijay, 47 2016). Moyle et al. analyzed EST clone sequences and found that contigs from libraries of green (unripe) and 48 yellow (ripe) fruit were both dominated by metallothionein (Moyle et al., 2005). To understand the mechanisms 49 underlying fruit ripening, Ong et al. performed Illumina sequencing on the transcriptome of ripe pineapple fruit 50 flesh. Genes related to flavor, texture, appearance, and fruit sweetness were identified based on digital annotation 51 (Ong, Voo & Kumar, 2012). In addition, Redwan et al. analyzed differential gene expression in RNASeq 52 libraries from ripening pineapple fruit; this analysis revealed that ethylene-related genes may also be involved 53 in regulating non-climacteric ripening in pineapple (Redwan, Saidin & Vijay Kumar, 2016). While each of these 54 studies examined pineapple fruit, they were all concerned with fruit ripening. While understanding fruit ripening 55 is highly important, so is fruit development, which occurs immediately prior to ripening. How does each 56 pineapple fruit expand? How is the collective fruit formed? How can we breed a cultivar with smaller and 57 shallower “fruit eyes”? Answering these questions requires a better understanding of pineapple fruit 58 development, and especially a better understanding of how different tissues merge to make up collective fruits. 59 The electronic fluorescent pictograph (eFP) browser is a powerful web-based tool for exploring gene 60 expression (Winter et al., 2007). 22 Abstract With this tool, users can visualize expression data in this study in an intuitive way. 34 Together, the transcriptomic profile generated in this work and the corresponding eFP browser will facilitate 35 further study of fruit development in pineapple. 36 36 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 37 Introduction 38 39 Pineapple is an economically important fruit herb widely cultivated in tropical and sub-tropical regions 40 (Bartholomew, Paull & Rohrbach, 2002). Pineapples are unique agricultural fruit not only due to their fragrance 41 and taste, but also because they form collective fruits, which differ them from other familiar fruits such as apples, 42 oranges, and mangoes. All fleshy coalesced floral parts of pineapple flowers and its inflorescence can be 43 consumed as either fresh or canned fruit (Bartholomew, Paull & Rohrbach, 2002; Botella, Ramon & Smith, 44 2008). Thus, pineapple fruit possesses both economic and research value. 45 Recently, with the popularization of high-throughput sequencing technologies, several genomic studies of 46 pineapple fruit have been performed (Moyle et al., 2005; Ong, Voo & Kumar, 2012; Redwan, Saidin & Vijay, 47 2016). Moyle et al. analyzed EST clone sequences and found that contigs from libraries of green (unripe) and 48 yellow (ripe) fruit were both dominated by metallothionein (Moyle et al., 2005). To understand the mechanisms 49 underlying fruit ripening, Ong et al. performed Illumina sequencing on the transcriptome of ripe pineapple fruit 50 flesh. Genes related to flavor, texture, appearance, and fruit sweetness were identified based on digital annotation 51 (Ong, Voo & Kumar, 2012). In addition, Redwan et al. analyzed differential gene expression in RNASeq 52 libraries from ripening pineapple fruit; this analysis revealed that ethylene-related genes may also be involved 53 in regulating non-climacteric ripening in pineapple (Redwan, Saidin & Vijay Kumar, 2016). While each of these 54 studies examined pineapple fruit, they were all concerned with fruit ripening. While understanding fruit ripening 55 is highly important, so is fruit development, which occurs immediately prior to ripening. How does each 56 pineapple fruit expand? How is the collective fruit formed? How can we breed a cultivar with smaller and 57 shallower “fruit eyes”? Answering these questions requires a better understanding of pineapple fruit 58 development, and especially a better understanding of how different tissues merge to make up collective fruits. 59 The electronic fluorescent pictograph (eFP) browser is a powerful web-based tool for exploring gene 60 expression (Winter et al., 2007). It displays cartoon images depicting various tissue types in various colours, 61 with each hue indicating the level of expression of a queried gene at a given time point (Hawkins et al., 2017). Manuscript to be reviewed 77 of eleven tissues of the pineapple fruit at three developmental stages, as well as three vegetative tissues. We then 78 generated RNAseq libraries for all samples, resulting in 36 libraries. After cautious transcriptomic profiling, we 79 set up the first eFP browser for pineapple (available at: http://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi), aiming 80 to provide an important resource for researchers interested in pineapple breeding and crop improvement. 81 77 of eleven tissues of the pineapple fruit at three developmental stages, as well as three vegetative tissues. We then 78 generated RNAseq libraries for all samples, resulting in 36 libraries. After cautious transcriptomic profiling, we 79 set up the first eFP browser for pineapple (available at: http://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi), aiming 80 to provide an important resource for researchers interested in pineapple breeding and crop improvement. 81 110 WGCNA analysis and KEGG pathway enrichment analysis 111 112 To construct a gene co-expression network for pineapple fruit, we employed the WGCNA package (Langfelder 113 & Horvath, 2008). Genes that were not expressed or were expressed at very low levels (TPM lower than 0.1) in 114 all samples were filtered out. The filtered TPM matrix was then imported into WGCNA for analysis. All 115 parameters were kept as default except the following: networkType was set to signed, softPower to 10, 82 Methods and materials The filtered TPM matrix was then imported into WGCNA for analysis. All 115 parameters were kept as default except the following: networkType was set to signed, softPower to 10, 82 Methods and materials 83 84 Plant material preparation and RNA sequencing 85 86 All plants of pineapple cultivar “Shenwan” used for this study w 87 City, Guangdong, China. Three distinct plant stages were defined 88 when flower buds begin to bloom and the collective fruit begins to 89 ceases, and the collective fruit expands at its fastest rate. Stage 3 90 stops expanding and begins to ripen. In total, 11 tissues were manu 91 bract, core, flower disk, leaf, ovary wall, ovule, petal, placenta, r 92 samples were collected and stored immediately in liquid nitroge 93 RNA from all samples using TRIzol (Invitrogen) and determined 94 RNA samples with RIN numbers lower than eight were re-extrac 95 a sequencing provider for RNA sequencing. 96 97 Expression profiling 98 99 PF (Pre-filtered) data received from the sequencing prov 100 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All 101 were filtered using Trimmomatic (Bolger, Lohse & Usadel, 2014) 102 analysis. STAR aligner was employed to map all clean reads onto 103 2013). The resulting BAM files were then processed using String 104 et al., 2015), in which the TPM (Transcripts per Kilo base Million 105 method proposed by Yanai et al. was applied on the expression m 106 expressed or expressed in specific tissues (Yanai et al., 2005; Kr 107 with TAU scores lower than 0.2 were defined as constitutively e 108 higher than 0.99 were defined as tissue-specific genes. 109 110 WGCNA analysis and KEGG pathway enrichment an 111 112 To construct a gene co-expression network for pineapple fruit, we 113 & Horvath, 2008). Genes that were not expressed or were express 114 all samples were filtered out. The filtered TPM matrix was th 115 parameters were kept as default except the following: networ 97 Expression profiling 99 PF (Pre-filtered) data received from the sequencing provider was manually checked by FastQC 100 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All sequencing adapters and low quality reads 101 were filtered using Trimmomatic (Bolger, Lohse & Usadel, 2014). Only paired reads were kept for downstream 102 analysis. STAR aligner was employed to map all clean reads onto the published pineapple genome (Dobin et al., 103 2013). The resulting BAM files were then processed using StringTie for estimation of expression levels (Pertea 104 et al., 2015), in which the TPM (Transcripts per Kilo base Million) normalized method was employed. The TAU 105 method proposed by Yanai et al. was applied on the expression matrix to detect genes that were constitutively 106 expressed or expressed in specific tissues (Yanai et al., 2005; Kryuchkova & Robinson-Rechavi, 2015). Genes 107 with TAU scores lower than 0.2 were defined as constitutively expressed genes while those with TAU scores 108 higher than 0.99 were defined as tissue-specific genes. 82 Methods and materials 82 Methods and materials 83 84 Plant material preparation and RNA sequencing 85 86 All plants of pineapple cultivar “Shenwan” used for this study were cultivated in Shenwan Town, Zhongshan 87 City, Guangdong, China. Three distinct plant stages were defined according to fruit development. Stage 1 begins 88 when flower buds begin to bloom and the collective fruit begins to expand in size. Stage 2 begins when flowering 89 ceases, and the collective fruit expands at its fastest rate. Stage 3 corresponds to the time when collective fruit 90 stops expanding and begins to ripen. In total, 11 tissues were manually separated from pineapple. These included 91 bract, core, flower disk, leaf, ovary wall, ovule, petal, placenta, receptacle, sepal, stamen, and style tissues. All 92 samples were collected and stored immediately in liquid nitrogen before RNA extraction. We extracted total 93 RNA from all samples using TRIzol (Invitrogen) and determined RNA quality of samples using Agilent 2100. 94 RNA samples with RIN numbers lower than eight were re-extracted. All RNA samples were then forwarded to 95 a sequencing provider for RNA sequencing. 96 97 Expression profiling 98 99 PF (Pre-filtered) data received from the sequencing provider was manually checked by FastQC 100 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All sequencing adapters and low quality reads 101 were filtered using Trimmomatic (Bolger, Lohse & Usadel, 2014). Only paired reads were kept for downstream 102 analysis. STAR aligner was employed to map all clean reads onto the published pineapple genome (Dobin et al., 103 2013). The resulting BAM files were then processed using StringTie for estimation of expression levels (Pertea 104 et al., 2015), in which the TPM (Transcripts per Kilo base Million) normalized method was employed. The TAU 105 method proposed by Yanai et al. was applied on the expression matrix to detect genes that were constitutively 106 expressed or expressed in specific tissues (Yanai et al., 2005; Kryuchkova & Robinson-Rechavi, 2015). Genes 107 with TAU scores lower than 0.2 were defined as constitutively expressed genes while those with TAU scores 108 higher than 0.99 were defined as tissue-specific genes. 109 110 WGCNA analysis and KEGG pathway enrichment analysis 111 112 To construct a gene co-expression network for pineapple fruit, we employed the WGCNA package (Langfelder 113 & Horvath, 2008). Genes that were not expressed or were expressed at very low levels (TPM lower than 0.1) in 114 all samples were filtered out. 37 Introduction It displays cartoon images depicting various tissue types in various colours, 61 with each hue indicating the level of expression of a queried gene at a given time point (Hawkins et al., 2017). 62 Since the development of the original eFP browser for Arabidopsis, eFP browsers have also been developed for 63 other plants, including poplar, soybean, potato, maize, rice, and barley (http://www.bar.utoronto.ca/). In addition, 64 eFP browsers have also been developed for several fruit-bearing horticultural crops, including tomato 65 (http://bar.utoronto.ca/efp_tomato/cgi-bin/efpWeb.cgi), grape (http://bar.utoronto.ca/efp_grape/cgi- 66 bin/efpWeb.cgi), and strawberry (Hawkins et al., 2017). 67 Databases make bioinformation accessible to research community. Moyle et al. developed “PineappleDB”, 68 providing access to expressed sequences tag isolated from pineapple fruit, root and nematode infected root gall 69 vascular cylinder tissues (Moyle, Crowe & Ripi-Koia, 2005). Recently, Xu et al. have reported an integrated 70 pineapple genomics database (PGD) storing gene information resources of pineapple (Xu et al., 2018). PGD is 71 an excellent database for research community, which covering 45 RNAseq samples generated from pineapple 72 genome sequencing project that mainly focuses on crassulacean acid metabolism study (Ming et al., 2015). 73 Currently, there is no eFP browser available for pineapple. This may be because most experiments have been 74 performed treating the whole pineapple fruit as a single tissue and thus no eFP browser was needed. However, 75 to investigate the development of pineapple fruit, a scheme whereby different tissues---e.g. receptacles, ovary 76 walls, and placentas---can be examined independently is more appropriate. In this study, we obtained samples PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 84 Plant material preparation and RNA sequencing 86 All plants of pineapple cultivar “Shenwan” used for this study were cultivated in Shenwan Town, Zhongshan 87 City, Guangdong, China. Three distinct plant stages were defined according to fruit development. Stage 1 begins 88 when flower buds begin to bloom and the collective fruit begins to expand in size. Stage 2 begins when flowering 89 ceases, and the collective fruit expands at its fastest rate. Stage 3 corresponds to the time when collective fruit 90 stops expanding and begins to ripen. In total, 11 tissues were manually separated from pineapple. These included 91 bract, core, flower disk, leaf, ovary wall, ovule, petal, placenta, receptacle, sepal, stamen, and style tissues. All 92 samples were collected and stored immediately in liquid nitrogen before RNA extraction. We extracted total 93 RNA from all samples using TRIzol (Invitrogen) and determined RNA quality of samples using Agilent 2100. 94 RNA samples with RIN numbers lower than eight were re-extracted. All RNA samples were then forwarded to 95 a sequencing provider for RNA sequencing. PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed 119 eFP browser database construction 120 131 All raw data used for transcriptome analysis and database construction have been submitted to the Sequence 132 Read Archive (SRA) at NCBI accession number SRP156886. 133 131 All raw data used for transcriptome analysis and database construction have been submitted to the Sequence 132 Read Archive (SRA) at NCBI accession number SRP156886. Manuscript to be reviewed 116 minModuleSize to 100, deepSplit to 2.0, and MEDissThres to 0.1. A software package made in-house, TBtools, 117 was used for Gene Ontology Term and KEGG pathway enrichment analysis and visualization (Chen et al., 2018). 118 119 eFP browser database construction 120 121 We used a docker container to construct a pineapple fruit eFP browser that could be used over the long term. 122 Centos 7 image was pulled from docker.io, and dependent software packages for the eFP browser were installed 123 according to the manual. The eFP browser installation package was downloaded from 124 http://www.bar.utoronto.ca/efp/development/ and some scripts in the package were adapted for RNA-seq data. 125 To facilitate the data importing process, we wrote two python scripts to convert tab-delimited files into SQL 126 command records. After the browser was set up locally, we made a docker image and uploaded it to our VPS 127 (http://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi) to provide service for all users. 128 129 Accession numbers 130 131 All raw data used for transcriptome analysis and database construction have been submitted to the Sequence 132 Read Archive (SRA) at NCBI accession number SRP156886. 133 134 135 116 minModuleSize to 100, deepSplit to 2.0, and MEDissThres to 0.1. A software package made in-house, TBtools, 117 was used for Gene Ontology Term and KEGG pathway enrichment analysis and visualization (Chen et al., 2018). 118 116 minModuleSize to 100, deepSplit to 2.0, and MEDissThres to 0.1. A software package made in-house, TBtools, 117 was used for Gene Ontology Term and KEGG pathway enrichment analysis and visualization (Chen et al., 2018). 118 136 Results 140 Pineapple forms a collective fruit that is developed from approximately one hundred individual florets. This fruit 141 formation is similar to some berries (Figures 1A and 1B). Pineapple fruit development accompanies its flower 142 development. In this study we focused on fruit development, and defined the time when the first round/batch of 143 flowers started to open as the first fruit developmental stage. The stage when flowering ceases was defined as 144 the second stage. Finally, the third stage was defined as when florets reached mature status and stopped 145 expanding (Figure 1A). 146 Pineapple fruits contain several main tissue types: bracts, sepals, flower disks, receptacles, ovaries walls, 147 placentas, ovules, and cores (Figure 1B). In addition, we also examined flower tissues such as petals, styles, and 148 stamens, as well as vegetative tissues such as leaves, roots, and stems. The berry floret is enlarged as it expands. 149 Petals are coloured pale purple. Importantly, the rate of enlargement of the bract and receptacle are faster than 150 the rates of the flower disk and placenta, which results in the formation of pineapple fruit eyes. 152 The global landscape of the pineapple fruit developmental transcriptome 153 154 To characterize expression dynamics throughout pineapple fruit development, RNAseq libraries were 155 constructed and sequenced for 14 different tissues collected at three major developmental stages (Table S1 and 156 Figure S1). Using the Illumina sequencing platform, ~1.19 billion 125-bp paired-end reads (149 Gb) were 157 generated, corresponding to an average of 33 million reads per tissue sample (Table S1). Most reads were 158 mapped onto exon regions, indicating that the RNAseq data was clean and could be used in further analyses 159 (Figure S1). 160 The pineapple genome was expected to contain 27,024 genes (Ming et al., 2015). We found that 23,789 161 (88.02% of the total) annotated genes were expressed in at least one of the 14 tissue types (Figures 2A and 2B). 162 The detection of expression of such a high proportion of genes may be attributed to the diversity of tissues 163 included in the study as well as high sequencing depth and coverage. The expression of 3,235 gene models 164 (11.98 % of the total) were either not detected in any of the tissues or were detected below background levels 165 (TPM lower than 0.1). These gene models may represent pseudogenes or they may be expressed only under 166 specific biotic or abiotic stress conditions. Expression pattern correlation analysis showed that expression 167 profiles of vegetative tissues were more distinct than profiles of reproductive tissues (highlighted in purple and 168 red rectangles in Figure 2C). 170 Tissue-specific and constitutive expression dynamics 171 172 Genes can be expressed constitutively or preferentially according to their different roles in plant development. 173 To study the mechanisms underlying the development of pineapple collective fruit, we utilized TAU methods to 174 detect housekeeping (TAU scores lower than 0.2) and tissue-specific (TAU scores higher than 0.99) genes. In 175 total, 273 genes were found to be expressed constitutively with varying expression levels (Figure 3A and Tables PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed 181 Co-expression network analysis 182 183 Using the sequencing data generated earlier, we used WGCNA to detect gene co-expression during pineapple 184 fruit development. In total, 18 gene modules were classified, with the number of genes involved ranging from 185 155 (light green module) to 4,522 (turquoise module) (Figure 4A and Table S6). Among these modules, several 186 showed spatial and temporal expression patterns (Figure S3). Genes of the red module were preferentially 187 expressed in bracts and sepals. KEGG pathway enrichment analysis indicated that the functions of the genes of 188 the red module were associated with plant defense, including plant-pathogen interactions and MAPK signaling 189 (Figures 4B and S4). In reproductive tissues, such as receptacles, stamens, and styles, genes of the salmon- 190 coloured module were highly expressed at the beginning of fruit expansion and faded afterward. These genes 191 were generally not expressed in vegetative tissues such as leaves and roots. According to pathway enrichment 192 analysis, these genes were involved in endocytosis, transport and catabolism, and cutin suberine and wax 193 biosynthesis (Figures 4C and S5). Genes belonging to the light cyan module were also expressed at lower levels 194 in vegetative tissues, however, in reproductive tissues, they showed expression patterns similar to those of the 195 genes in the salmon-coloured group. As indicated by gene set enrichment analysis, genes involved in terpenoids 196 and polyketides metabolism were over-represented in this group (Figures 4D and S6). 197 Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed 176 S2-3). In contrast, we found 1,051 tissue-specific genes (Figure 3B and Tables S4-5). Of these, most were 177 expressed preferentially in stamen (286 genes), root (170 genes) and leaf (147 genes) (Figure 3B, Table 1). 178 Among these genes, most were also stage-specific, and were preferentially expressed in the same tissue at a 179 given development stage (Figure S2). 180 181 Co-expression network analysis 182 183 Using the sequencing data generated earlier, we used WGCNA to detect gene co-expression during pineapple 184 fruit development. In total, 18 gene modules were classified, with the number of genes involved ranging from 185 155 (light green module) to 4,522 (turquoise module) (Figure 4A and Table S6). Among these modules, several 186 showed spatial and temporal expression patterns (Figure S3). Genes of the red module were preferentially 187 expressed in bracts and sepals. KEGG pathway enrichment analysis indicated that the functions of the genes of 188 the red module were associated with plant defense, including plant-pathogen interactions and MAPK signaling 189 (Figures 4B and S4). In reproductive tissues, such as receptacles, stamens, and styles, genes of the salmon- 190 coloured module were highly expressed at the beginning of fruit expansion and faded afterward. These genes 191 were generally not expressed in vegetative tissues such as leaves and roots. According to pathway enrichment 192 analysis, these genes were involved in endocytosis, transport and catabolism, and cutin suberine and wax 193 biosynthesis (Figures 4C and S5). Genes belonging to the light cyan module were also expressed at lower levels 194 in vegetative tissues, however, in reproductive tissues, they showed expression patterns similar to those of the 195 genes in the salmon-coloured group. As indicated by gene set enrichment analysis, genes involved in terpenoids 196 and polyketides metabolism were over-represented in this group (Figures 4D and S6). 197 198 The Pineapple eFP browser for fruit development analysis 199 200 eFP browsers can accelerate routine gene expression analysis, especially in developmental biology. To 201 facilitate visual exploration of gene expression during pineapple fruit development, we leveraged docker 202 technology to build a pineapple eFP browser (http://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi). The browser 203 provides a user-friendly web-based interface. Users can enter a specific pineapple gene id and obtain 204 information on its expression in a short time. To ensure that the pineapple eFP browser was working correctly, 205 we manually checked the gene expression pattern graphs of three different genes. Manuscript to be reviewed Three genes (Aco031614, A 023419 d A 004566) d f hi Fi 5B d k l d hi h i 176 S2-3). In contrast, we found 1,051 tissue-specific genes (Figure 3B and Tables S4-5). Of these, most were 177 expressed preferentially in stamen (286 genes), root (170 genes) and leaf (147 genes) (Figure 3B, Table 1). 178 Among these genes, most were also stage-specific, and were preferentially expressed in the same tissue at a 179 given development stage (Figure S2). 215 We obtained a comprehensive and high-quality fruit transcriptome for pineapple fruit 216 215 We obtained a comprehensive and high-quality fruit transcriptome for pineapple fruit 216 217 Although extensive RNA sequencing data for pineapple exists on public repositories such as NCBI SRA, only a 218 few of these were produced from fruit (Ong, Voo & Kumar, 2012; Ma et al., 2015; Ming et al., 2015; Chen et 219 al., 2016; Liu & Fan, 2016; Redwan, Saidin & Vijay Kumar, 2016). No data is available for the different tissues 220 present in the pineapple collective fruit, and this fact hinders studies of pineapple fruit development. Here, we 221 present 36 libraries of pineapple, covering all distinct tissues of its fruit at three developmental stages. Almost 222 90 percent of the genes were detected in the current analysis based on gene strucutre annotation from the 223 reference pineapple genome. The difference in the number of genes detected in each tissue indicated that many 224 genes are expressed preferentially in some tissues and not at all in others. In order to generate a transcriptional 225 landscape of pineapple for further study, no replicate has been set. To ensure the data is trusted, we have applied 226 qRT-PCR to validate expression of two pineapple gene families in our previous work (Liu et al., 2017; Xie et 227 al., 2018). Manuscript to be reviewed Twenty-ni 242 genes were core-specific genes. Among them, Aco002250 coding a copper ion binding protein possessed t 243 highest expression level. Its homology, LOC_Os01g32790, in rice is preferentially expressed in inflorescen 244 according to the records in the MSU Rice database (Kawahara et al., 2013). A Chalcone synthase (CHS) gen 245 Aco008872, was also found to be core-specific. In maize, silencing CHS increases Lignin Content (Eloy et a 246 2017). Pineapple fruit core is the extension of stem, which is rich in lignin. As the fruit development, the CH 247 gene specifically expressed in core might take part in reducing lignin content, making the core more edib 248 Totally, we detected thirty-one genes preferentially expressed in flower-disk. Most of them were annotated 249 “hypothetical protein”. One auxin response factor might be useful for further study, whose homolog 250 AT1G30330, in Arabidopsis is preferentially expressed carpel according to eFP browser on TA 251 (www.arabidopsis.org). Pineapple flower-disk decides the depth of the fruit-eye. Controlling expression patte 252 of pineapple ARF6 might be a possible way to breeding pineapples for shallow fruit-eyes. Thirty-four gen 253 were found to be ovary-wall specific genes. It is interesting that, out of them, two shikimate kinase gen 254 Aco004125 and Aco029719, possessed the highest expression levels. Shikimate kinase is the key component 255 the shikimate pathway, from where a vast number of aromatic compounds originated (Bentley & Haslam, 199 213 Discussion 214 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 229 Detection of constitutively expressed and tissue-specific genes provide the basis for further 230 study of pineapple fruit development. 232 Tissue-specific genes play fundamental roles in tissue differentiation and maintenance (Pattison et al., 2015). 233 Detection of tissue-specific genes in pineapple fruit tissues can provide the basis for further study of genetic 234 mechanisms responsible for fruit phenotypes, and may also provide a basis for genetic manipulation. For 235 instance, in collaboration with a developed pineapple genetic-transformation system (Soneji & Nageswara, 236 2009), we can express a gene of interest in a specific tissue with the promoter sequence of a tissue-specific gene. 237 In this study, we identified ~1,000 tissue-specific genes. Among these, stamens possessed the most tissue- 238 specific genes; this may be expected since many pollen-specific genes have already been characterized. In 239 addition, leaf and root tissues both possessed more than 100 tissue-specific genes; this is almost twice the number 240 of genes in fruit tissues. This may be due to that these genes are involved in nutrient uptake. 241 In contrast, only a few genes were preferentially expressed in tissues of the pineapple fruit. Twenty-nine 242 genes were core-specific genes. Among them, Aco002250 coding a copper ion binding protein possessed the 243 highest expression level. Its homology, LOC_Os01g32790, in rice is preferentially expressed in inflorescence 244 according to the records in the MSU Rice database (Kawahara et al., 2013). A Chalcone synthase (CHS) gene, 245 Aco008872, was also found to be core-specific. In maize, silencing CHS increases Lignin Content (Eloy et al., 246 2017). Pineapple fruit core is the extension of stem, which is rich in lignin. As the fruit development, the CHS 247 gene specifically expressed in core might take part in reducing lignin content, making the core more edible. 248 Totally, we detected thirty-one genes preferentially expressed in flower-disk. Most of them were annotated as 249 “hypothetical protein”. One auxin response factor might be useful for further study, whose homology, 250 AT1G30330, in Arabidopsis is preferentially expressed carpel according to eFP browser on TAIR 251 (www.arabidopsis.org). Pineapple flower-disk decides the depth of the fruit-eye. Controlling expression pattern 252 of pineapple ARF6 might be a possible way to breeding pineapples for shallow fruit-eyes. Thirty-four genes 253 were found to be ovary-wall specific genes. It is interesting that, out of them, two shikimate kinase gene, 254 Aco004125 and Aco029719, possessed the highest expression levels. Manuscript to be reviewed 215 We obtained a comprehensive and high-quality fruit transcriptome for pineapple fruit 216 217 Although extensive RNA sequencing data for pineapple exists on public repositories such as NCBI SRA, only 218 few of these were produced from fruit (Ong, Voo & Kumar, 2012; Ma et al., 2015; Ming et al., 2015; Chen 219 al., 2016; Liu & Fan, 2016; Redwan, Saidin & Vijay Kumar, 2016). No data is available for the different tissu 220 present in the pineapple collective fruit, and this fact hinders studies of pineapple fruit development. Here, 221 present 36 libraries of pineapple, covering all distinct tissues of its fruit at three developmental stages. Alm 222 90 percent of the genes were detected in the current analysis based on gene strucutre annotation from t 223 reference pineapple genome. The difference in the number of genes detected in each tissue indicated that ma 224 genes are expressed preferentially in some tissues and not at all in others. In order to generate a transcription 225 landscape of pineapple for further study, no replicate has been set. To ensure the data is trusted, we have appli 226 qRT-PCR to validate expression of two pineapple gene families in our previous work (Liu et al., 2017; Xie 227 al., 2018). 228 229 Detection of constitutively expressed and tissue-specific genes provide the basis for furth 230 study of pineapple fruit development. 231 232 Tissue-specific genes play fundamental roles in tissue differentiation and maintenance (Pattison et al., 201 233 Detection of tissue-specific genes in pineapple fruit tissues can provide the basis for further study of gene 234 mechanisms responsible for fruit phenotypes, and may also provide a basis for genetic manipulation. F 235 instance, in collaboration with a developed pineapple genetic-transformation system (Soneji & Nageswa 236 2009), we can express a gene of interest in a specific tissue with the promoter sequence of a tissue-specific gen 237 In this study, we identified ~1,000 tissue-specific genes. Among these, stamens possessed the most tissu 238 specific genes; this may be expected since many pollen-specific genes have already been characterized. 239 addition, leaf and root tissues both possessed more than 100 tissue-specific genes; this is almost twice the numb 240 of genes in fruit tissues. This may be due to that these genes are involved in nutrient uptake. 241 In contrast, only a few genes were preferentially expressed in tissues of the pineapple fruit. Manuscript to be reviewed 256 Pineapple is famous for its special fragrance. The high expression level and the preferential expression pattern 257 of these two shikimate kinase genes in ovary-wall indicate that pineapple fragrance may be generated from 258 ovary-wall and these two genes are involved in fragrance production of pineapple. In total, forty-seven genes 259 are preferentially expressed in pineapple placenta. Among them, we found Aco006346 coding a homology of 260 sugar transporter SWEET17. In Arabidopsis, SWEET17 mediates fructose transport across the tonoplast (Guo 261 et al., 2014). Pineapple SWEET17 might also play a similar role during fruit development. Apart from this, we 262 also identified 273 constitutively expressed genes, and these may serve as useful reference genes for qRT-PCR 263 experiments of pineapple fruit. 265 Gene module classification can facilitate further study of pineapple fruit. In the present study, we built a pineapple eFP browser in which 289 distinct pineapple tissues are colour-coded. Compared to traditional gene expression databases, which visualized 290 gene expression matrices as tile plots (Nussbaumer et al., 2014), users can explore specific genes in a cartoon 291 image and see how expression levels differ among pineapple tissues. Moreover, the browser leverages docker 292 technology; with docker images, users can also re-build their own pineapple fruit eFP browsers locally relatively 293 easily in multiple operating systems. In this way, the pineapple eFP browser can be accessible without a network 294 connection. 295 296 229 Detection of constitutively expressed and tissue-specific genes provide the basis for further 230 study of pineapple fruit development. Shikimate kinase is the key component of 255 the shikimate pathway, from where a vast number of aromatic compounds originated (Bentley & Haslam, 1990). PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 265 Gene module classification can facilitate further study of pineapple fruit. 267 Genes function as networks in all biological processes. To uncover the mechanisms underlying different traits, 268 network-level analyses are required. Here, we classified all expressed pineapple genes into 18 gene modules. 269 Among these, genes of the red module were preferentially expressed in bracts and sepals. According to the 270 KEGG pathway enrichment result, most members were associated with plant defense, including plant-pathogen 271 interactions and MAPK signaling. As pineapple fruit develops, it faces various kinds of bio-stress like pest 272 binding. Thus, it is possible that these resistance-associated genes are expressed with higher levels in the outer 273 part of pineapple fruit to defense the adversity. Genes of salmon module might have similar functions but 274 function in a different way. These genes, functionally related to cutin, suberine and wax biosynthesis, are only 275 highly expressed at the beginning of fruit expansion. They may be important for all reproductive tissues of 276 pineapple but only functions at the specific stage. In contrast, genes in the light cyan module seems to be linked 277 to fruit quality. Most members of this group are involved in terpenoid and polyketide metabolism. It is likely 278 that this module contains key genes related to pineapple ripening and fragrance biogenesis. In addition, other 279 modules also seem to have distinct expression profiles and apparent functions. Together, these modules can be 280 used for gene screening and further experimentation. 277 to fruit quality. Most members of this group are involved in terpenoid and polyketide metabolism. It is likely 278 that this module contains key genes related to pineapple ripening and fragrance biogenesis. In addition, other 279 modules also seem to have distinct expression profiles and apparent functions. Together, these modules can be 280 used for gene screening and further experimentation. 281 282 The eFP browser is an important tool for pineapple fruit study. 283 284 Studies of fruit are important for fruit tree breeding and research, since different crops form different fruit types. 285 Tomato, the most famous fruit study model species (Kimura & Sinha, 2008), bears simple fruit developed from 286 ovaries. Strawberry, another model fruit species (Li et al., 2011), possesses aggregate accessory fruit developed 287 from receptacles (Hollender et al., 2012). Both species have eFP browsers, which provide researchers with a 288 simple method to explore gene expression data. 282 The eFP browser is an important tool for pineapple fruit study. 284 Studies of fruit are important for fruit tree breeding and research, since different crops form different fruit types. 285 Tomato, the most famous fruit study model species (Kimura & Sinha, 2008), bears simple fruit developed from 286 ovaries. Strawberry, another model fruit species (Li et al., 2011), possesses aggregate accessory fruit developed 287 from receptacles (Hollender et al., 2012). Both species have eFP browsers, which provide researchers with a 288 simple method to explore gene expression data. In the present study, we built a pineapple eFP browser in which 289 distinct pineapple tissues are colour-coded. Compared to traditional gene expression databases, which visualized 290 gene expression matrices as tile plots (Nussbaumer et al., 2014), users can explore specific genes in a cartoon 291 image and see how expression levels differ among pineapple tissues. Moreover, the browser leverages docker 292 technology; with docker images, users can also re-build their own pineapple fruit eFP browsers locally relatively 293 easily in multiple operating systems. In this way, the pineapple eFP browser can be accessible without a network 294 connection. 295 296 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 311 ACKNOWLEDGEMENTS 311 ACKNOWLEDGEMENTS 312 We thank all members of the He lab for their help in separating different pineapple fruit tissues. 313 324 Table S1. Summary statistics for transcriptome sequencing of various tissues in a genome-wide developmental 325 expression analysis of pineapple. 24 Table S1. Summary statistics for transcriptome sequencing of various tissues in a genome-wide developmental 25 expression analysis of pineapple. 299 Conclusions 300 In the present study, we provide a comprehensive and high quality pineapple fruit transcriptome. In it, we have 301 characterized thousands of tissue-specific genes as well as 18 gene co-expression modules. Moreover, a 302 pineapple fruit eFP browser has been constructed to explore gene expression in pineapple fruit and to facilitate 303 further study of pineapple fruit development. 300 In the present study, we provide a comprehensive and high quality pineapple fruit transcriptome. In it, we have 301 characterized thousands of tissue-specific genes as well as 18 gene co-expression modules. Moreover, a 302 pineapple fruit eFP browser has been constructed to explore gene expression in pineapple fruit and to facilitate 303 further study of pineapple fruit development. 304 305 306 307 308 300 In the present study, we provide a comprehensive and high quality pineapple fruit transcriptome. In it, we have 301 characterized thousands of tissue-specific genes as well as 18 gene co-expression modules. Moreover, a 302 pineapple fruit eFP browser has been constructed to explore gene expression in pineapple fruit and to facilitate 303 further study of pineapple fruit development. 304 305 306 307 308 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed 309 310 311 ACKNOWLEDGEMENTS 312 We thank all members of the He lab for their help in separating different pineapple fruit tissues. 313 314 315 SUPPORTING INFORMATION 316 317 Figure S1. Distribution of reads across gene features. 318 Figure S2. Tissue-specific gene expression patterns across all samples. 319 Figure S3. Heatmap of correlations between samples and gene modules. 320 Figure S4. Expression patterns of genes in the red module. 321 Figure S5. Expression patterns of genes in the salmon module. 322 Figure S6. Expression patterns of genes in the light cyan module 323 324 Table S1. Summary statistics for transcriptome sequencing of various tissues in a genome-wide developmental 325 expression analysis of pineapple. 326 Table S2. Expression patterns of constitutively expressed genes of pineapple. 327 Table S3. Expression patterns of constitutively expressed genes of pineapple during fruit development. 328 Table S4. Expression patterns of tissue-specific genes of pineapple. 329 Table S5. Expression patterns of tissue-specific genes of pineapple during fruit development. 330 Table S6. 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PGD: Pineapple Genomics Database. PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed 413 Horticulture Research 5. DOI: 10.1038/s41438-018-0078-2. 414 Yanai I., Benjamin H., Shmoish M., Chalifa-Caspi V., Shklar M., Ophir R., Bar-Even A., Horn-Saban S., Safran M., 415 Domany E., Lancet D., Shmueli O. 2005. Genome-wide midrange transcription profiles reveal expression 416 level relationships in human tissue specification. Bioinformatics 21:650–659. DOI: 417 10.1093/bioinformatics/bti042. 418 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Number specifically expressing genes of each tissue PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Three main stages of pineapple fruit development Three main stages of pineapple fruit development A. Three developmental stages of pineapple fruit. Stage_1 corresponds to the time when flower buds start to bloom and fruit begins to expand in size. At the same time, cell division dominates biological processes in fruit. Stage_2 corresponds to the time when flowering period ends and fruit expands its size at the fastest pace. At the same time, cell expansion dominates biological processes in fruit. Stage_3 corresponds to the time when fruit begins its ripening process and fruit expansion process stopped. B. Different parts of pineapple fruitlet. Stage_1, Stage_2 and Stage_3 denote the developmental stage of each fruitlet. Each fruitlet was detaching into bract, flower-disk, sepals, petals and others. Qi Mao took all the photos. Manuscript to be reviewed Tissue Gene Num. Bract 77 Core 29 Flower_disk 31 Leaf 147 Ovary_wall 34 Ovule 97 Petal 11 Placenta 47 Receptacle 30 Root 170 Sepal 42 Stamen 286 Stem 33 Style 17 1 Tissue Gene Num. 1 1 PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Figure 2(on next page) Figure 2(on next page) Global gene expression patterns in different tissues of pineapple. A. Number and percentage of genes expressed in each of the 14 tissue samples during pineapple fruit development. B. Heatmap illustration of spearman correlation coefficient between two of each sample. The darker the color, the higher the similarity of gene expression patterns between two samples. PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed A B Bract_1 Bract_2 Bract_3 Sepal_1 Sepal_2 Sepal_3 Flower_disk_1 Flower_disk_2 Flower_disk_3 Receptacle_1 Receptacle_2 Receptacle_3 Ovary_wall_1 Ovary_wall_2 Ovary_wall_3 Placenta_1 Placenta_2 Placenta_3 Ovule_1 Ovule_2 Ovule_3 Stamen_1 Style_1 Core_1 Core_2 Core_3 Petal_1 Stem_1 Stem_2 Stem_3 Leaf_1 Leaf_2 Leaf_3 Root_1 Root_2 Root_3 Bract_1 Bract_2 Bract_3 Sepal_1 Sepal_2 Sepal_3 Flower_disk_1 Flower_disk_2 Flower_disk_3 Receptacle_1 Receptacle_2 Receptacle_3 Ovary_wall_1 Ovary_wall_2 Ovary_wall_3 Placenta_1 Placenta_2 Placenta_3 Ovule_1 Ovule_2 Ovule_3 Stamen_1 Style_1 Core_1 Core_2 Core_3 Petal_1 Stem_1 Stem_2 Stem_3 Leaf_1 Leaf_2 Leaf_3 Root_1 Root_2 Root_3 0.75 0.8 0.85 0.9 0.95 1 B PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Manuscript to be reviewed Figure 3 Heatmap of consititutive and tissue-specific genes. A. Heatmap illustrating the expression pattern of consititutively-expressed genes across 14 tissues. TPM values are log2-tranformed for better visualization. B. Heatmap showing the expression pattern of specifically expressed genes across 14 tissues. TPM values are normalized by row for better visualization. PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) Figure 4(on next page) Figure 4(on next page) Dendrogram of co-expressing gene modules and KEGG pathways enrichment bar plot of three modules with interesting expression pattern. A. In the upper panel, a dendrogram showed all genes imported for WGCNA analysis. Each vertical line corresponds to one gene. In the bottom panel, rectangles of different color denoted the classification of all genes. B-D. KEGG pathway enrichment bar plot of red, salmon and lightcyan module. P-value were log10-transformed for better visualization. Manuscript to be reviewed Figure 4(on next page) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) 00909 Sesquiterpenoid and triterpenoid biosyn 00904 Diterpenoid biosynthesis 00940 Phenylpropanoid biosynthesis Metabolism of terpenoids and polyketides Biosynthesis of other secondary metabolites Metabolism 03440 H l bi ti KEGG Pathway 04626 Plant-pathogen interaction Environmental adaptation Organismal Systems 04016 MAPK signaling pathway - plant 04075 Plant hormone signal transduction Signal transduction Environmental Information Processing 00520 Amino sugar and nucleotide sugar metabolism 00950 Isoquinoline alkaloid biosynthesis 00960 Tropane, piperidine and pyridine alkaloid biosynthesis 00350 Tyrosine metabolism Biosynthesis of other secondary metabolites 00410 beta-Alanine metabolism 00333 Prodigiosin biosyntheses 00360 Phenylalanine metabolism 00905 Brassinosteroid biosynthesis 00260 Glycine, serine and threonine metabolism 00592 alpha-Linolenic acid metabolism 00565 Eth li id t b li 04144 Endocytosis Transport and catabolism Cellular Processes 00073 Cutin, suberine and wax biosynthesis 04130 SNARE interactions in vesicular transport 00020 Citrate cycle (TCA cycle) 00270 Cysteine and methionine metabolism 00330 Arginine and proline metabolism 00510 N-Glycan biosynthesis 00100 Steroid biosynthesis 0.0 2.0 4.0 6.0 8.0 10.0 -log10(P-value) KEGG Pathway D C Manuscript to be reviewed 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 A 00909 Sesquiterpenoid and triterpenoid biosynthesis 00904 Diterpenoid biosynthesis 00940 Phenylpropanoid biosynthesis Metabolism of terpenoids and polyketides Biosynthesis of other secondary metabolites Metabolism 03440 Homologous recombination 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 -log10(P-value) KEGG Pathway 04626 Plant-pathogen interaction Environmental adaptation Organismal Systems 04016 MAPK signaling pathway - plant 04075 Plant hormone signal transduction Signal transduction Environmental Information Processing 00520 Amino sugar and nucleotide sugar metabolism 00950 Isoquinoline alkaloid biosynthesis 00960 Tropane, piperidine and pyridine alkaloid biosynthesis 00350 Tyrosine metabolism Biosynthesis of other secondary metabolites 00410 beta-Alanine metabolism 00333 Prodigiosin biosyntheses 0360 Phenylalanine metabolism 0905 Brassinosteroid biosynthesis 260 Glycine, serine and threonine metabolism 592 alpha-Linolenic acid metabolism 565 Ether lipid metabolism 4.0 6.0 8.0 10.0 12.0 14.0 -log10(P-value) 04144 Endocytosis Transport and catabolism Cellular Processes 00073 Cutin, suberine and wax biosynthesis 04130 SNARE interactions in vesicular transport 00020 Citrate cycle (TCA cycle) 00270 Cysteine and methionine metabolism 00330 Arginine and proline metabolism 00510 N-Glycan biosynthesis 00100 Steroid biosynthesis 0.0 2.0 4.0 6.0 8.0 10.0 -log10(P-value) KEGG Pathway D C PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) B 04626 Plant-pathogen interaction Environmental adaptation Organismal Systems 04016 MAPK signaling pathway - plant 04075 Plant hormone signal transduction Signal transduction Environmental Information Processing 0520 Amino sugar and nucleotide sugar metabolism 50 Isoquinoline alkaloid biosynthesis 60 Tropane, piperidine and pyridine alkaloid biosynthesis Tyrosine metabolism 04144 Endocytosis Transport and catabolism Cellular Processes 00073 Cutin, suberine and wax biosynthesis 04130 SNARE interactions in vesicular transport 00020 Citrate cycle (TCA cycle) 00270 Cysteine and methionine metabolism 00330 Arginine and proline metabolism 00510 N-Glycan biosynthesis 00100 Steroid biosynthesis 0.0 2.0 4.0 6.0 8.0 10.0 -log10(P-value) KEGG Pathway D C C D 0.0 Figure 5 Pineapple eFP browser views of different gene query. A. Default view of pineapple eFP browser with different color denoting different tissues. B. A view of a randomly picked gene (Aco031614) with darken color denoting higher expression levels. C. A view of a stamen-specific gene (Aco023419). D. A view of a constitutively expressed gene (Aco004566). PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018) PeerJ reviewing PDF \| (2018:07:29820:2:0:NEW 25 Oct 2018)
https://openalex.org/W4229837164	https://figshare.com/articles/thesis/How_Local_People_Use_Their_Indigenous_Knowledge_to_Respond_to_Floods_and_Droughts_A_Case_Study_of_Tonle_Sap_Lake_Community_Cambodia/17145953/1/files/31704611.pdf	English	null	How Local People Use Their Indigenous Knowledge to Respond to Floods and Droughts: A Case Study of Tonle Sap Lake Community, Cambodia	null	2,021	cc-by	49,688	Sopheak Seng Sopheak Seng Abstract This thesis investigates the impacts of floods and droughts in the Tonle Sap Lake region and examines to what extent the local communities’ indigenous knowledge (IK) is used to deal with floods and droughts. The thesis begins by exploring the Tonle Sap Lake communities’ perceptions of climate change, especially floods and droughts and their impacts on local livelihoods. It then examines how the communities have used their IK to develop livelihood adaptation methods to cope with floods and droughts. To conduct this study, a qualitative methodology was adopted using semi-structured interviews, and non-participant and unstructured observation as the main methods. The semi-structured interviews were conducted with local people and local authorities from two communities, and NGO staff. The study found that the intensity of floods and droughts in the Tonle Sap Lake region has increased in the last few years. Floods and droughts have threatened local livelihoods and food security. To mitigate the effects of floods and droughts, the local communities in this region have developed various livelihood adaptation strategies to adapt to the hazards. The communities appeared to use both IK and technologies for their adaptation strategies. IK is seen as an invaluable local community asset in developing livelihood adaptation methods. Although a mixture of IK and new knowledge has been used to develop various adaptation strategies, the sufficiency of the adaptation is still limited. The current severity of climate change is seen to limit the local communities’ response capacities. To strengthen the communities’ adaptation capacity, contribution and involvement from non-governmental organisations and the government in developing climate change adaptation policy at a local level are essential. Keywords: indigenous knowledge, floods, droughts, livelihood, adaptation Keywords: indigenous knowledge, floods, droughts, livelihood, adaptation Keywords: indigenous knowledge, floods, droughts, livelihood, adaptation i Acknowledgement My deepest gratitude goes to my supervisor, Professor John Overton, who always encouraged and motivated me to work hard and complete my research. The support, comments, feedback and motivation from him was invaluable for this research thesis. His motivated guidance helped me in all the time of my thesis and writing. I could not have imagined having a better advisor for my thesis. I am also profoundly grateful to the government of New Zealand, particularly the Ministry of Foreign Affairs and Trade (MFAT) who have provided me with a scholarship to pursue my master’s degree at Victoria University of Wellington. The financial support from MFAT was crucial for both of my studies and living in New Zealand for two years. Moreover, I would like to express my sincere thanks to Victoria International, especially Tim Lawther and Clemmie Newton, who took care of my study progress and helped find me a proof-reader for my thesis. I would also like to express my great appreciation to commune councillors of Kampong Kleang commune, particularly Mr Sambo Chhang, two village heads of Mukwat and Santey villages, who helped facilitate my field research. Without their help and participation from community people in the two villages, my field research could not have been done smoothly. I really appreciate local communities’ participation in my research. They have demonstrated their willingness to participate in my research despite their hardship during a severe drought in their communities. Finally, yet importantly, my heartfelt thanks to my beloved wife, Kannitha Chan, and my daughter, SovannMalatey Hak whose love, understanding, determined support and encouragement have motivated my daily work on this thesis. Without their understanding, encouragement and support, this thesis would not be easily completed on time. ii ii Table of Contents Abstract ............................................................................................................................. i Acknowledgement ........................................................................................................... ii List of Figures ................................................................................................................. vi List of Tables ................................................................................................................. vii List of Appendices ........................................................................................................ viii Acronyms ........................................................................................................................ ix Chapter 1: Introduction ................................................................................................. 1 1.1 Research background .............................................................................................. 1 1.2 Overall objectives of the research ........................................................................... 3 1.3 Research questions .................................................................................................. 3 1.4 Significance of the research .................................................................................... 4 1.5 Research outline ...................................................................................................... 4 Chapter 2: Literature Review ........................................................................................ 6 2.1 Introduction ............................................................................................................. 6 2.2 Environment, climate change and development ..................................................... 6 2.3 Perception of climate change .................................................................................. 8 2.4 Incidence of floods and droughts .......................................................................... 10 2.4.1 Natural occurrence of floods and droughts .................................................... 10 2.4.2 Climate change and floods and droughts ....................................................... 11 2.5 Impacts of floods and droughts ............................................................................. 12 2.5.1 Environmental impacts ................................................................................... 12 2.5.2 Socioeconomic impacts .................................................................................. 13 2.6 Indigenous knowledge .......................................................................................... 16 2.6.1 Indigenous knowledge system ....................................................................... 16 2.6.2 Indigenous knowledge and development ....................................................... 17 2.7 Indigenous knowledge and climate change adaptation ......................................... 19 2.8 Conclusion ............................................................................................................. 25 Chapter 3: Research Methodology .............................................................................. 26 3.1 Introduction ........................................................................................................... 26 3.2 Epistemology ......................................................................................................... 26 3.3 Qualitative methodology ....................................................................................... 27 3.4 Research location .................................................................................................. 28 3.5 Methods ................................................................................................................. 29 3.5.1 Semi-structured interviews ............................................................................. 29 3.5.2 Observations ................................................................................................... 33 3.6 Data recording procedures, storage and analysis .................................................. 34 3.7 Ethical issues and confidentiality .......................................................................... 36 3.8 Researcher’s positionality ..................................................................................... 36 3.9 Conclusion ............................................................................................................. 37 Chapter 4: National Context of Cambodia ................................................................. 39 4.1 Introduction ........................................................................................................... 39 4.2 Geographical areas and population ....................................................................... 39 4.3 Cambodia’s climate ............................................................................................... 41 4.4 Cambodia’s agriculture and livelihoods ................................................................ 42 4.5 Climate change, floods and droughts in the context of Cambodia ....................... 45 4.6 Climate change policy in Cambodia ..................................................................... 47 4.7 Conclusion ............................................................................................................. 49 Chapter 5: Perception of Climate Change in the Tonle Sap Lake region ............... 50 5.1 Introduction ........................................................................................................... 50 5.2. Local perception of changing the weather as a result of climate change ............. 50 5.3 Local perception of droughts and high temperature ............................................. 51 5.4 Local perception of floods .................................................................................... 53 5.5 Discussion ............................................................................................................. 55 5.6 Conclusion ............................................................................................................. 57 Chapter 6: Impacts of Floods and Droughts on Local Communities and their Livelihoods ..................................................................................................................... 58 6.1 Introduction ........................................................................................................... 58 6.2 Impacts of floods ................................................................................................... 58 6.2.1 Impacts on individuals and community property ........................................... 58 6.2.2 Impacts on fishing activities and fish farming ............................................... 59 6.3 Discussion ............................................................................................................. 60 6.4 Impacts of droughts ............................................................................................... 62 6.4.1 Impact of droughts on rice farming ................................................................ 62 6.4.2. Table of Contents Impacts of droughts on fishing activities ...................................................... 63 6.4.3 Impacts of droughts on secondary livelihood activities ................................. 66 6.5 Discussion ............................................................................................................. 70 6.6 Conclusion ............................................................................................................. 72 Chapter 7: Local Practices as Adaptation Strategies to Deal with Floods and Droughts ......................................................................................................................... 73 7.1 Introduction ........................................................................................................... 73 7.2 Preparation for floods and droughts ...................................................................... 73 7.2.1 Preparation for floods ..................................................................................... 73 7.2.2 Preparation for droughts ................................................................................. 74 7.2.3 Discussion ...................................................................................................... 77 7.3 Local adaptation practices to generate income ..................................................... 78 7.3.1 Changing fishing methods .............................................................................. 78 iv 7.3.2 Changing fishing zones .................................................................................. 81 7.3.3 Crop farming .................................................................................................. 82 7.3.4 Livestock raising ............................................................................................ 83 7.3.5 Hunting wild animals and birds ..................................................................... 85 7.3.6 Wage labour ................................................................................................... 86 7.4 Local adaptation practices for survival ................................................................. 89 7.4.1 Vegetable gardening ....................................................................................... 89 7.4.2 Collecting wild edible vegetables and plants ................................................. 90 7.4.3 Eating small fish ............................................................................................. 91 7.5 Discussion ............................................................................................................. 92 7.6 Indigenous knowledge generation and its roles in developing livelihood adaptation strategies .................................................................................................... 94 7.7 Conclusion ............................................................................................................. 95 Chapter 8: Conclusion .................................................................................................. 97 8.1 Summary of the research process .......................................................................... 97 8.2 Key research findings ............................................................................................ 99 8.2.1 Local perception of climate change, floods and droughts .............................. 99 8.2.2 Impacts of floods and droughts on local livelihoods ................................... 101 8.2.3 Local practices and roles of indigenous knowledge in developing adaptation strategies to deal with floods and droughts ........................................................... 102 8.3 Final comments ................................................................................................... 105 References .................................................................................................................... 107 Appendices ................................................................................................................... 120 v v List of Figures Figure 1. Map of Tonle Sap Lake – Cambodia. ............................................................. 29 Figure 2. Map of Cambodia. ........................................................................................... 40 Figure 3. Average monthly temperature and rainfall of Cambodia (1901-2016). .......... 42 Figure 4. Total imports and exports of fish and fishery products for Cambodia ............ 44 Figure 5. Total capture and aquaculture production for Cambodia (tonnes). ................. 44 vi List of Tables Table 1: Research participants ........................................................................................ 32 Table 2: Key themes for data analysis ............................................................................ 35 vii List of Appendices Appendix 1. Ethical approval Appendix 2. Participant information sheet in English Appendix 3. Participant information sheet in Khmer Appendix 4. Participant consent form in English Appendix 5. Participant consent form in Khmer Appendix 6. Question guide for local communities Appendix 7. Question guide for local authorities Appendix 8. Question guide for NGO staff viii Chapter 1: Introduction Chapter 1: Introduction Acronyms AR5 WGII Assessment Report 5 Working Group II CCCA Cambodia Climate Change Alliance CCCO Cambodia Climate Change Office of Ministry of Environment CCCSP Cambodia Climate Change Strategic Plan DCC Department of Climate Change of Ministry of Environment FAO Food and Agriculture Organisation of United Nations GDP Gross Domestic Product GSSD General Secretariat, National Council for Sustainable Development IK Indigenous knowledge IPCC Intergovernmental Panel on Climate Change KEI Korea Environment Institute KHR Cambodian Riel (currency of Cambodia) MRC Mekong River Commission NAPA National Adaptation Programme of Action to Climate Change NCSD National Council for Sustainable Development NGO Non-governmental organisation OECD Organisation for Economic Co-operation and Development SDGs Sustainable Development Goals UN United Nations UNDP United Nations Development Programme UNDRR United Nations Office for Disaster Risk Reduction UNESCO United Nations Educational, Scientific and Cultural Organisation UNFCCC United Nations Framework Convention on Climate Change UNISDR United Nations International Strategy for Disaster Reduction USAID United States Agency for International Development USD United States Dollar AR5 WGII Assessment Report 5 Working Group II CCCA Cambodia Climate Change Alliance CCCO Cambodia Climate Change Office of Ministry of Environment CCCSP Cambodia Climate Change Strategic Plan DCC Department of Climate Change of Ministry of Environment FAO Food and Agriculture Organisation of United Nations GDP Gross Domestic Product GSSD General Secretariat, National Council for Sustainable Development IK Indigenous knowledge IPCC Intergovernmental Panel on Climate Change KEI Korea Environment Institute KHR Cambodian Riel (currency of Cambodia) MRC Mekong River Commission NAPA National Adaptation Programme of Action to Climate Change NCSD National Council for Sustainable Development NGO Non-governmental organisation OECD Organisation for Economic Co-operation and Development SDGs Sustainable Development Goals UN United Nations UNDP United Nations Development Programme UNDRR United Nations Office for Disaster Risk Reduction UNESCO United Nations Educational, Scientific and Cultural Organisation UNFCCC United Nations Framework Convention on Climate Change UNISDR United Nations International Strategy for Disaster Reduction USAID United States Agency for International Development USD United States Dollar ix 1.1 Research background Climate change is one of the main challenges for development in every country. Its impacts have been increasingly severe and apparent from year to year. Climate change has been observed to have significant impacts on people and natural systems (IPCC, 2014). According to IPCC’s 2018 report, global warming has had great impacts on the human and natural systems. Global warming has changed land and ocean ecosystems (IPCC, 2018). Wuebbles (2018) argues that people are affected by certain types of extreme weather and sea-level rise – the result of climate change. Extreme weather- related events such as floods, droughts, storms and heat waves have caused substantial damage to human society, including human health and wellbeing (Thomas, 2017; Wuebbles, 2018). In the last few decades, scientific studies have shown that climate change has significant impacts on people’s livelihoods around the globe (Carvajal, 2007). Livelihoods of the least-developed nations have been most vulnerable to climate change. The IPCC and OECD reports reveal that climate change has increased the frequency and magnitude of severe weather events and threatened local communities that rely on agricultural livelihoods and fisheries (IPCC, 2018; OECD, 2016). Local communities from developing countries have been found to be the most vulnerable to climate change since the natural resources on which they rely, such as land, water, fisheries and forest, have been devastated by climate change (Reid, 2014). Current severe floods and droughts, which are attributable, at least in part, to climate change have significant impacts on communities around the world. According to Smakhtin et al. (2014), floods and droughts are the most economically and socially damaging disasters accounting for approximately 90% of people affected by all-natural disasters. Güneralp et al. (2015) claim that, among the natural hazards, floods and droughts have caused severe damage to infrastructure and affected livelihoods of millions of people annually. In the least-developed countries, floods and droughts have been observed as major threats to local people since they have affected their livelihoods and endangered their lives. The majority of local people in the least-developed countries are dependent on 1 agricultural production for their primary livelihoods. However, agricultural production in these local communities has been threatened by severe floods and droughts. The Food and Agriculture Organization (FAO, 2016) estimated the economic losses from floods, droughts and storms to be, on average, USD250–300 billion per year. 1.1 Research background In ten years (2003–2013), crop and livestock production losses due to floods and droughts widely vary across the least-developed countries. The studies of the impact of climate- related disasters (flood, drought, and storm) on crop and livestock production in least- developed country regions such as sub-Saharan Africa, the Near East, Asia, Latin America and the Caribbean, show that floods and droughts are particularly the most harmful disasters for agriculture (FAO, 2016). In Cambodia, agriculture plays a significant role in the national economy and accounts for 35% of the country’s gross domestic product (GDP). It also employs a vast majority of the country's population, especially rural people (FAO, 2019a). Nearly 80% of the Cambodian population live in rural areas, and 65% of the rural population are dependent on agriculture, fisheries, and forestry for their main livelihoods (USAID, 2019a). In the Tonle Sap Lake region, agriculture and fishery resources are essential for rural communities in this region, and play a crucial role in shaping the country’s economy. The flood pulse of the lake is essential in retaining high aquatic productivity. It also brings water and fertile silt to the rice fields promoting rice farming in the floodplain (Nuorteva et al., 2010). Rice and fish in the region are the foundations of food security and income, and thus the local communities living in the region are strongly dependent on fishing and rice farming for their main livelihoods (Johnstone et al., 2013; Nuorteva et al., 2010). However, in the past few decades, human activities in Tonle Sap Lake and the Mekong River, as well as climate change, have caused significant changes to the lake and its ecosystems (Uk et al., 2018). Increases in water level and extent of flooding, caused by climate change and hydropower development in the Mekong River, have threatened fish production, plants and wildlife (Uk et al., 2018). Changes in flooding duration in the Tonle Sap floodplain have changed wetland habitats and therefore affected fish and other aquatic production and livestock production – the primary sources of local livelihoods (MRC, 2010). Besides flooding, droughts also occur in the region. Droughts are usually reported as more severe than floods. For instance, severe droughts that occurred in 2002 and 2012 led to crop damage, food insecurity and diseases. 1.2 Overall objectives of the research The overall objective of this thesis is to investigate the impacts of floods and droughts in the Tonle Sap Lake region, and examine to what extent the local people’s indigenous knowledge (IK) has been used to deal with floods and droughts, and its effectiveness. Since climate change has affected the local communities and their livelihoods, this research aims to study how the local communities perceive climate change, especially floods and droughts. Then, it investigates how the local communities have used their IK to formulate adaptation strategies to adapt to floods and droughts that have happened over time in this region. This study mainly focuses on livelihood adaptation methods that the communities have been practising during severe floods and droughts. 1.1 Research background The 2 droughts that happened in these two separate years affected more than two million people and destroyed hundreds of thousand hectares of farming crops and rice fields (Nguyen & Shaw, 2011; Reuy, 2012). In recent years, droughts in the region have become even more severe with more significant impacts on local communities’ livelihoods, threatening their food security. The droughts in recent years have caused the lake to become very shallow, and the tributaries in the Tonle Sap floodplain dried up. The fish catch was reported to have decreased, and thousands of hectares of crops were destroyed (Seiff, 2017; Sullivan, 2019). 1.4 Significance of the research This research is associated with development theory and the arguments about development and IK. As claimed by some researchers, IK is a very effective strategy in the ongoing struggles against poverty, hunger and underdevelopment. It also allows local people to live in harmony with nature, maintain sustainable resources and achieve balanced development (Agrawal, 1995; Briggs, 2014; Sillitoe, 1998). Furthermore, IK has been recognised for its value regarding climate change hazards and weather forecasting, and it has been a critical resource for communities in understanding the environment, and assessing and adapting to climate change in the past (Deeb et al., 2011; Green & Raygorodetsky, 2010). Therefore, this research provides academic researchers, development practitioners and policymakers with an insight into IK of the Tonle Sap Lake communities, and how it has been used to combat climate change, particularly floods and droughts. This research contributes to the existing literature of the impacts of floods and droughts in the region since it reports the real situation of current effects of floods and droughts that local communities have experienced. The research also provides final comments for local government and development practitioners so that they are able to support and strengthen local adaptation capacity to climate change effectively and sustainably. 1.3 Research questions To reach the overall objective of this study, a key question was addressed “How do local people in the Tonle Sap Lake region use their indigenous knowledge to respond to floods and droughts?” To answer the key question, I developed four sub-questions: 1. What are the local people’s perceptions of the floods and droughts? 2. How have the local people and their livelihoods been affected by floods and droughts? 3. What strategies do local people use to recover from the effects of floods and droughts? 4. How might indigenous knowledge be incorporated into adaptation policies? 3 3 2.1 Introduction This chapter reviews literature about climate change, its impacts and local responses to natural hazards. The chapter is divided into four core sections. The first section explores the relationship between environment, climate change and development. The second section examines public perceptions of climate change. The third section investigates the incidence of floods and droughts, and their impacts on environment, society and economy. The last part discusses IK, its relationship with development, and how it has been used by local communities in different countries around the globe to respond to the impacts of climate change, particularly floods and droughts. 1.5 Research outline This research thesis is divided into eight chapters. This first chapter delivers an introduction to the research by illustrating the research background, research objective, research questions and the significance of the study. The second chapter reviews literature about climate change, its impacts, IK and development, and the importance of IK in adaptation strategies to deal with climate change. The third chapter provides information about the methodology used to achieve the objectives of the research, including epistemology, research methods, research location, participant recruitment and my positionality. The fourth chapter presents a national context including geographical area and population, climate, agriculture and livelihoods, and climate change policy at international and national levels. The fifth chapter explains the Tonle Sap Lake communities’ perceptions of climate change, particularly floods and droughts. The sixth chapter describes the impacts of floods and droughts on local livelihood, while the seventh chapter explains how communities have practised their livelihood 4 adaptation strategies to deal with floods and droughts. The last chapter concludes the summary of the research process, key findings, and final comments. 5 2.2 Environment, climate change and development The environment is either a resource or a constraint for development. However, climate change is increasingly changing resource-based development, often in detrimental ways. In the twenty-first century, climate change is one of the most significant development challenges in the world. Its present threat is exacerbating the existing global problems (Morecroft & Cowan, 2010, p. 170). Climate change is affecting people's daily lives and the environment. Extreme weather-related events such as floods, droughts, storms and heat waves, which are a consequence of climate change, cause considerable damage to human society (Thomas, 2017). Over the last four decades, climate-related disasters such as floods, droughts, storms, heatwaves and temperature extremes have enormously increased (Thomas, 2017). These disasters have had great impacts on human health, agriculture, water resources, energy and transportation infrastructure, and more (Wuebbles, 2018). Furthermore, climate change has impacts on development and the environment. Since it causes death and diseases, and damages infrastructures, climate change is seen as one of the biggest obstacles to the development of every country in the world, and least- developed countries seem to have been the most severely affected by its impacts. According to IPCC and OECD reports, climate change has increased the frequency and magnitude of severe weather events and threatened local communities that rely on 6 agricultural livelihoods and fisheries (IPCC, 2018; OECD, 2016). These poor communities are most vulnerable to the changing weather conditions when the natural resources they need, such as land, water, fisheries and forests, have been ruined (Reid, 2014). Climate change and development are interconnected in complex ways. Most scientists claim that human actions are the leading cause of changes in climate. It is evident that human-induced actions are the main cause of climate change (Wuebbles, 2018, p. 15). The burning of fossil fuels and land-use changes through deforestation (Wuebbles, 2018) and the rise in industrialisation are causing the atmosphere’s increasing temperature (Morecroft & Cowan, 2010). Boyd also states that global warming is a consequence of the development of industries and the burning of fossil fuels (Boyd, 2014, p. 342). Likewise, conservationists claim that climate change and people’s reactions to climate change contribute to the severe threats to the natural environment (Morecroft & Cowan, 2010). Climate change and development are interconnected in complex ways. Most scientists claim that human actions are the leading cause of changes in climate. 2.2 Environment, climate change and development It is evident that human-induced actions are the main cause of climate change (Wuebbles, 2018, p. 15). The burning of fossil fuels and land-use changes through deforestation (Wuebbles, 2018) and the rise in industrialisation are causing the atmosphere’s increasing temperature (Morecroft & Cowan, 2010). Boyd also states that global warming is a consequence of the development of industries and the burning of fossil fuels (Boyd, 2014, p. 342). Likewise, conservationists claim that climate change and people’s reactions to climate change contribute to the severe threats to the natural environment (Morecroft & Cowan, 2010). Since the world population is expected to increase sharply in the next few decades, more food and energy sources will be inevitably needed to feed the increased population. Therefore, the food demand for the world’s burgeoning population will likely be one of the main factors contributing to the changing climate and environment. Rockström et al. (2009) argue that contemporary agriculture produces significant environmental pollution and causes environmental change. In order to meet the demand for food for the world population, increasing the farming areas and industrialised agriculture are crucial. Nevertheless, industrialised agriculture contributes much to environmental change. Within five decades, 1950–2000, the use of fertilisers rose by twenty-three times and the use of pesticide rose by fifty-three times, which has had massive impacts on the environment (Serageldin, 2009). Furthermore, the increasing world population produces more environmental stress (Rosa & Dietz, 2012). Larger population produces more greenhouse-gas emissions through people’s daily consumption of food, goods, and services, and there are more carbon dioxide emissions through congested traffic (Rosa & Dietz, 2012). Evidence shows that people’s actions have caused environmental change and global warming. In return, climate change is affecting environmental resources, people’s lives, the planet, and the development of every country. In order to mitigate the effect of 7 climate change, a sustainable development approach might be a solution. In 2015, the United Nations (UN) launched 17 Sustainable Development Goals (SDGs) that all UN members embraced. Combating climate change is one of the goals that all the UN members agreed to achieve by 2030 (UNDP, 2016). Given the unsustainable world economic growth and willing to ensure social and environmental sustainability, several international organisations have launched the initiative of "Green Economy" (Martinez- Covarrubias & Garza-Reyes, 2017). 2.2 Environment, climate change and development The green economy aims to “reduce dependence on fossil fuels, minimise impacts to ecosystems, and sustain socioeconomic wellbeing for all people by ensuring social equity and a fair distribution of wealth” (Richardson, 2013, p. 8). The green economy is developed through innovation and the development of new skills in green sectors such as energy efficiency, clean energy, alternative transportation, green design and construction, recycling, and organic agriculture (Richardson, 2013). To diminish the threat to future economic growth and stability, the global economy should not damage environmental assets and natural resources (Martinez-Covarrubias & Garza-Reyes, 2017). 2.3 Perception of climate change Perception is defined as the process by which we receive information from our nature and convert it to psychological knowledge to construct meaningful experiences of the world (Van den Ban & Hawskins, 2000, as cited in Ahmed & Atiqul, 2019). Climate change has affected every individual around the world. However, people’s perceptions of climate change vary greatly. Thus, understanding of local knowledge and perception of weather and climate is critical for developing an effective climate change policy at a given place (Gamble et al., 2010). Slovic (1987) also suggests that policy regarding hazard management needs to understand people’s perception and how they respond to hazards because failure to understand people’s perceptions could contribute to ineffective policies. Shukla et al. (2016) argue that local communities’ perceptions reflect local issues because the local people live closely with nature and have a close interactive understanding of their environment. The perceptions of these local people are developed from day-to-day connections with their nature and their dependence on climate conditions to ensure food security (Laidler, 2006). 8 8 Likewise, several studies demonstrate that local people could gain knowledge of deviations from what is perceived to be normal weather and climate conditions through their frequent observation of, and interaction with, the surrounding nature even though they have limited knowledge about the science of climate change (Gamble et al., 2010). In addition, Margaret et al. (2018) suggest that how people understand and perceive climate change is significant for developing policies that are intended to address the issues and their willingness to change behaviour. Therefore, people’s perceptions of climate change play a significant role in forming natural hazards policies (Shukla et al., 2016). Every person has a different perception of climate change. Public perceptions of the threats posed by climate change differ between regions and countries (Taylor et al., 2014). Pettenger also points out that “Ask ten people how to define climate change, its causes and effects, and you will get ten different answers” (Pettenger, 2007, p. 5). For instance, researchers have found that residents in the United Kingdom (UK) and the United States (US) have different observations and perceptions about climate change. The UK residents were likely to refer to ‘rain’ when they described climate change, while the US residents were more likely to mention ‘heat’ and ‘ice caps melting’ (Taylor et al., 2014). 2.4 Incidence of floods and droughts 2.4 Incidence of floods and droughts 2.3 Perception of climate change Even within one country, public perception about climate change could differ in accordance with the viewpoints and observations of local people living in different regions. In the UK, for instance, local people in the northern territory may have experienced hotter summers and warmer winters than those living in the southeastern territory (Palutikof et al., 2004). Perceptions of climate change differ from one individual to another depending on their knowledge, cultural worldviews, weather conditions, and geographical location (Shi et al., 2015; Shukla et al., 2016; Palutikof et al., 2004). Taylor et al. (2014) suggest that, since the perception of climate risk differs between people and within locations, the support for adaptation policies should be developed differently based on the people’s location. Geographical location, knowledge, culture and attitude of people may reflect how the individuals perceive climate change. Thus, proper adaptation policies should be conducted based on the actual people’s perception in a specific location. 9 9 Nevertheless, a number of studies that have been conducted in various developing countries across the globe have revealed that local communities with similar knowledge have perceived climate change similarly. Those local people mainly mention about rainfall pattern, flooding, droughts and extreme temperature when they describe climate change. For instance, studies conducted in southwestern Jamaica, India, Pakistan and African Sahel region demonstrated that severe droughts and extreme temperature are the major risks for local people since they have affected people’s lives and agriculture production (Abid et al., 2016; Gamble et al., 2010; Nyong et al., 2007; Shukla et al., 2016). The studies also revealed that changes in rainfall pattern have been a major risk for local people in India and Northern Thailand (Manandhar et al., 2015; Shukla et al., 2016). 2.4.1 Natural occurrence of floods and droughts The OECD 2016 report briefly describes floods as rises in the water level of a stream or water body to a peak from which the water level recedes at a slower rate. Floods happen in many ways due to an overflow of stream, rivers, lakes or oceans or as a result of excessive rain. Floods result from a number of basic factors. One of the most critical factors is meteorological in nature. Exceeding a river’s discharge of its channel’s volume causes a river to overflow onto the area surrounding the channel known as the floodplain. This phenomenon is known as flooding (Jackson, 2014, August 2). However, flooding is known as a complex phenomenon. According to (Collier & Webb, 2002), rainfall on wet ground, falling of warm rain on current snowpack, torrential rainfall throughout a whole basin or changing of basin shape, are all the causes of flooding. Rising temperature and spring melting of winter snow trigger off flooding (Collier & Webb, 2002). Lancaster et al. (2004) mention that flooding may happen from many bases: the sea (coastal and estuarine flooding), watercourses (fluvial flooding), overland flow of water that does not reach a natural drainage channel (fluvial flooding), rising groundwater, and the failure of artificial water systems. The OECD 2016 report briefly describes floods as rises in the water level of a stream or water body to a peak from which the water level recedes at a slower rate. 2.4.1 Natural occurrence of floods and droughts Floods and droughts are hydrologically life threatening. Floods are related to excessive rainfall (from tropical storms, thunderstorms, orographic rainfall, widespread extratropical cyclones, etc.), while droughts are associated with a shortage of rainfalls and frequently severe high temperatures that lead to drying (Trenberth, 2005). Collier and Webb (2002) argue that drought is not as simple as only a shortage of rainfall, but it is a lack of protracted humidity below longstanding average conditions that balance precipitation and evapotranspiration in a specific area. All droughts are not equally formed. Shortage of humidity could have diverse results depending on the time of year when they happen, previous moisture content of the soil, and other climatic features such as temperature, wind, and the amount of moisture in the air. Hydrologic drought happens when there is a shortfall of surface water supply (i.e., stream flow, reservoir and lake levels, ground water) during a dry spell (Collier & Webb, 2002). According to OECD (2016), drought is defined as a short-term loss of water in rivers or waterways, caused by lengthy deviations from average levels of rainfall. Drought is a normal and frequent feature of climate, and, under certain circumstances, becomes a disaster (OECD, 2016). 10 Floods happen in many ways due to an overflow of stream, rivers, lakes or oceans or as a result of excessive rain. Floods result from a number of basic factors. One of the most critical factors is meteorological in nature. Exceeding a river’s discharge of its channel’s volume causes a river to overflow onto the area surrounding the channel known as the floodplain. This phenomenon is known as flooding (Jackson, 2014, August 2). However, flooding is known as a complex phenomenon. According to (Collier & Webb, 2002), rainfall on wet ground, falling of warm rain on current snowpack, torrential rainfall throughout a whole basin or changing of basin shape, are all the causes of flooding. Rising temperature and spring melting of winter snow trigger off flooding (Collier & Webb, 2002). Lancaster et al. (2004) mention that flooding may happen from many bases: the sea (coastal and estuarine flooding), watercourses (fluvial flooding), overland flow of water that does not reach a natural drainage channel (fluvial flooding), rising groundwater, and the failure of artificial water systems. 2.4.2 Climate change and floods and droughts Floods and droughts occur all over the world, and they affect people’s livelihood and development of every country, especially in developing countries. In the last few decades, it is evident that global warming is changing because of natural and anthropogenic greenhouse gas emissions. It is believed that climate change or global warming has a magnifying effect on floods and droughts. According to Van Aalst (2006), the main greenhouse gas, carbon dioxide (CO2) has been emitted and dramatically increased in the atmosphere because of the industrial revolution, changes in agriculture and land use. The global warming is a result of rising greenhouse gases in the atmosphere, which leads to extreme weather events such as heatwaves, wildfires, cyclones, floods, and droughts (Van Aalst, 2006). The OECD 2016 report also describes that global warming and changing weather result in flood and drought. Excess or deficit of rainfall, together with other variables such as temperature, humidity, air and soil, and wind, are the main causes of floods and droughts (OECD, 2016). As mentioned by Schwartz (2018), the stalled weather system, caused by the changes in atmospheric circulation, can lead to extreme rainfall and the lack of it (prolonged sunny weather). Likewise, McCarthy (2001) describes that change in climate is likely to change average 11 11 temperature and precipitation values and increase the variability of precipitation events. This may lead to even more extreme and recurrent floods and droughts. temperature and precipitation values and increase the variability of precipitation events. This may lead to even more extreme and recurrent floods and droughts. 2.5 Impacts of floods and droughts 2.5 Impacts of floods and droughts In the last few decades, floods have increasingly affected our ecosystems, local people and socioeconomic activities such as fishing, farming, transportation and human health, while droughts create lengthy disruptions in forests and difficulties in river transportation, farming and fishing, which disrupts the food supply to people, disturbs hydroelectric power generation and affects human health, mainly because of smoke from forest fires (Wongchuig Correa et al., 2017). 2.5.1 Environmental impacts Floods and droughts are natural hazards that can cause harmful impacts on people and environmental resources. The freshwater system is one of the valuable natural resources for daily use of people. Availability of freshwater is essential for both human wellbeing and the economy. The supply of freshwater is used for various purposes, such as drinking, irrigation, hydropower production recreation, fisheries, etc. (Li et al., 2017). However, this resource is affected when flood or drought occur. Drought can affect water quality in many ways. Typically, decreased volume of water during a drought leads to increased salinity, longer hydraulic residence times, promoted toxic cyanobacterial blooms, and lowered dissolved oxygen concentrations (Mosley, 2015). A change in water quality because of drought also affects fish species i.e., lower fish recruitment rates, slower growth rates, and higher mortality rates (Mickle et al., 2018). In addition to freshwater, drought can also be a constraint on plant growth and limit crop yield due to the lack of precipitation and soil moisture (Li et al., 2017). Besides this, drought also causes damage to other ecosystems and environmental resources such as pastures, farms, forests and plants, and animals since they require water for survival (Devisti & Motamed, 2012). Flooding is another natural disaster that causes negative impacts on people’s lives and environmental resources. Stuyt et al. (2007) states there are very few studies that have been conducted about the impact of flooding events on the environment, instead, the 12 studies normally focus on damage to buildings, infrastructure, and economic loss. Nonetheless, flooding has an obvious negative impact on the environment. According to the study of Stuyt et al. (2007), flooding in the area of dense population may cause significant impact on the environment and agriculture because of physical damage to crops and installations and, because of toxic sediments on an agricultural field. Flood water destroys houses, industrial buildings and farms. The damage releases harmful substances such as suspended solids and chemicals that enter the flood water, affecting people and ecosystems in the flooding zone (Stuyt et al., 2007). Flash floods, on the other hand, can cause problems with drinking water. When a flash flood occurs, floodwater damages water supply systems, causing an inadequate availability of drinking-water supplies for people (Sun et al., 2016). 2.5.1 Environmental impacts The most severe effect of flooding is when drinking water is contaminated with microorganisms such as bacteria, sewage, heating oil, agricultural or industrial waste, chemicals, and other elements that can cause acute sickness (Sun et al., 2016). Furthermore, the flash flood may also cause damage to other natural vegetation such as riparian forests. According to Kozlowski (2002), riparian forest ecosystems are essential for their high-biomass productivity, which controls erosion and floods, remove nutrients from agricultural runoff, and reduces the impacts of air pollution and soil contamination. Riparian forests also provide critical habitat for various bird species and mammals. However, a sudden rise and fall of flood discharges combined with fast-flow rates during the flash flood may destroy the riparian forests or prevent them from growing (Kozlowski, 2002). 2.5.2 Socioeconomic impacts Floods and droughts are among the regular, hazardous, and pricey of all-natural disasters that cause severe damage to infrastructure and affect the people’s livelihoods of millions every year (Güneralp et al., 2015). According to Smakhtin et al. (2014), floods and droughts are the most economically and socially damaging disasters, accounting for approximately 90% of people affected by all-natural disasters. Between 1900 and 2006, the most devastating events happened 1,000 times in which almost 90% of the events were involved by water-related hazards such as floods, droughts and windstorms (Güneralp et al., 2015). Since the early 1980s, economic losses because of water-related hazards rose above 500%. In particular, floods and droughts made up 38% of the total natural disasters, 45% of the total causalities, over 84% of the total number 13 of people affected, and 30% of the total financial losses affected by all-natural disasters (Güneralp et al., 2015). According to the United Nations International Strategy for Disaster Reduction (UNISDR, 2002), in 20 years since 1992, floods and droughts also caused more than USD500 billion worth of damage, which made up 28% of the total destruction from all disasters. Two other recent examples of great financial losses caused by flooding is in Thailand (2011) and Pakistan (2010). In 2011, Thailand's floods, economically, cost USD40 billion while the cost of Pakistan's flood was estimated at USD43 billion with over 2,000 people being killed (Gopalakrishnan, 2013). Flooding has great negative impacts on people’s lives and socioeconomic activities such as fishing, farming, transportation and human health (Wongchuig Correa et al., 2017). Rapsomanikis et al. (2017) state that 20–300 million people worldwide are affected by floods each year. The vulnerability to floods has increased, and more property, assets and lives have suffered from flooding. In the floodplain of Northern Italy, severe flood events have been remarkable in the last two decades. From the year 2000 to 2014, floods in this floodplain region have caused 200,000 dead animals, more than 40,000 people to become homeless, more than 500,000 displaced people, 8 missing and/or dead people, and over a billion euros' worth of damages, including private, public, and industrial properties (Roder et al., 2017). In Pakistan, during 2010–2011 more than 2,500 people died in severe floods, and 27 million were affected. The floods also caused livestock to die and destroyed agricultural lands, resulting in billions of US dollar loss (Smakhtin et al., 2014). 2.5.2 Socioeconomic impacts Yet, droughts that have happened over several years have made the agriculture in Nepal less stable and less productive (Adhikari, 2018). According to the UNDP (2009) report, meteorological events from 1971-2007 caused, roughly, 90% of the crop or agricultural land loss in Nepal. Of the different disasters in Nepal, drought had the most severe impact on agriculture and crop loss accounting for 39% of the impact. Moreover, severe droughts that happened in Africa have caused huge losses of crop and livestock production. According to the FAO (2015) report, the absolute losses were more than USD14 billion, accounting for about 6% of the projected value of production. Severe droughts have caused most countries in Africa to experience rapid declines in yields. Such losses pose a great threat to food availability, rural livelihoods and the overall economy, especially given the important contribution of agriculture to food security and the economies of sub-Saharan Africa (FAO, 2015). Agriculture is a critical economic sector for most developing countries in the world, particularly in sub-Saharan African countries. However, in these countries where agriculture contributes to its national economy, droughts have caused major direct impacts such as reducing crop yields and forest productivity, reducing the water level, creating a fire hazard, devastating wildlife and fish habitats, and increasing livestock and wildlife mortality (UNISDR, 2015). Studies in Nepal and India also revealed that droughts have caused great damage on agricultural production of the rural communities. Agricultural production is the primary livelihood and food security of the rural people in these two countries. However, the impacts of severe droughts on agricultural production have threatened rural food security (Adhikari, 2018; Udmale et al., 2014). For example, the severe drought in one of the Indian states, Maharashtra, 2012, caused a huge impact on local livelihood. The local people in Maharashtra state experienced a shortage of water for their daily consumption and for agriculture. The severe drought in this state caused a vegetable production decrease of 11% compared to the previous year, and the inland fishery suffered a 16% production loss (Udmale et al., 2014). Likewise, in Nepal, agriculture is an important livelihood activity of rural people. Crop growing including rice, wheat, maize, millet, barley, and buckwheat, and livestock farming are major forms of food security in the rural communities. Yet, droughts that have happened over several years have made the agriculture in Nepal less stable and less productive (Adhikari, 2018). 2.5.2 Socioeconomic impacts Agriculture plays a vital role as the primary source of people’s livelihoods in all countries in the world. Natural disasters such as floods and droughts are the most significant challenges to agriculture development. Floods and droughts have caused significant damage to agricultural development, particularly in developing countries. The Food and Agriculture Organization (FAO, 2016) estimated the economic losses from natural disasters such as floods, droughts and storms at the average of USD250– 300 billion per year. During 2003–2013, the crop and livestock production losses due to climate-related disasters vary widely across developing countries. The studies of the impact of climate-related disasters (flood, drought, and storm) on crop and livestock production in developing country regions such as sub-Saharan Africa, the Near East, Asia, Latin America and the Caribbean, show that floods and droughts are the most harmful disasters for agriculture (FAO, 2016). In these four regions, the economic 14 losses of crop and livestock production due to floods and droughts account for 95% (USD67 billion), while storm accounts for only 5% (USD3 billion) (FAO, 2016). Agriculture is a critical economic sector for most developing countries in the world, particularly in sub-Saharan African countries. However, in these countries where agriculture contributes to its national economy, droughts have caused major direct impacts such as reducing crop yields and forest productivity, reducing the water level, creating a fire hazard, devastating wildlife and fish habitats, and increasing livestock and wildlife mortality (UNISDR, 2015). Studies in Nepal and India also revealed that droughts have caused great damage on agricultural production of the rural communities. Agricultural production is the primary livelihood and food security of the rural people in these two countries. However, the impacts of severe droughts on agricultural production have threatened rural food security (Adhikari, 2018; Udmale et al., 2014). For example, the severe drought in one of the Indian states, Maharashtra, 2012, caused a huge impact on local livelihood. The local people in Maharashtra state experienced a shortage of water for their daily consumption and for agriculture. The severe drought in this state caused a vegetable production decrease of 11% compared to the previous year, and the inland fishery suffered a 16% production loss (Udmale et al., 2014). Likewise, in Nepal, agriculture is an important livelihood activity of rural people. Crop growing including rice, wheat, maize, millet, barley, and buckwheat, and livestock farming are major forms of food security in the rural communities. 2.5.2 Socioeconomic impacts According to the UNDP (2009) report, meteorological events from 1971-2007 caused, roughly, 90% of the crop or agricultural land loss in Nepal. Of the different disasters in Nepal, drought had the most severe impact on agriculture and crop loss accounting for 39% of the impact. Moreover, severe droughts that happened in Africa have caused huge losses of crop and livestock production. According to the FAO (2015) report, the absolute losses were more than USD14 billion, accounting for about 6% of the projected value of production. Severe droughts have caused most countries in Africa to experience rapid declines in yields. Such losses pose a great threat to food availability, rural livelihoods and the overall economy, especially given the important contribution of agriculture to food security and the economies of sub-Saharan Africa (FAO, 2015). Agriculture is a critical economic sector for most developing countries in the world, particularly in sub-Saharan African countries. However, in these countries where agriculture contributes to its national economy, droughts have caused major direct impacts such as reducing crop yields and forest productivity, reducing the water level, creating a fire hazard, devastating wildlife and fish habitats, and increasing livestock and wildlife mortality (UNISDR, 2015). Studies in Nepal and India also revealed that droughts have caused great damage on agricultural production of the rural communities. 15 Given the affected agriculture and natural resources and decreasing incomes of local people as a consequence, together with rising food and timber rates, indirect impacts on the whole society such as malnutrition in children, increased unemployment rate, migration, reduced tax revenue, and the risk of foreclosures on bank loans to farmers (UNISDR, 2015) need to be considered. Given the affected agriculture and natural resources and decreasing incomes of local people as a consequence, together with rising food and timber rates, indirect impacts on the whole society such as malnutrition in children, increased unemployment rate, migration, reduced tax revenue, and the risk of foreclosures on bank loans to farmers (UNISDR, 2015) need to be considered. 2.6.2 Indigenous knowledge and development 2.6.2 Indigenous knowledge and development Since the second World War, the agenda for development has moved through several phases; from focusing on economic growth, equity growth, to basic needs, to participatory development, to sustainable development (Agrawal, 1995). Over recent eras, IK has an been appealing phrase in the rhetoric of development. The phrase “indigenous knowledge” has been used by development practitioners and theorists all over the world. In the 1980s, IK gained its interest due to the dissatisfaction of modernisation theory (Briggs, 2014). Through the distribution of formal scientific and technical knowledge from the North to the global South, modernisation theory was defined as a practical approach to reduce poverty and improve living standards of the poor population in the global South. As a result, the influential science discourses from Europe and North America have conceptualised development as a technical issue in the global South and IK was largely discounted. By the 1980s, it was clear that the modernisation approach could not be used successfully to transform many of the poor population, particularly in African countries (Briggs, 2014, p. 127). Likewise, post- development theorists argue that the failure of development is not only because the entire concept of development and its practice has become theoretical, illustrating the domination of the West over the rest of the world; but also because the process of development has been taken out of the hands of local people (Dada, 2016, p. 75). Accordingly, other alternatives should be pursued in order to foster local development movement. Dada (2016) mentions that development is a cultural process and people should be involved in this process since they are the objects of the development. Tradition and lived experiences of the people need to be considered in the development process. Moreover, it should be acknowledged that people have their own effective science, resources, knowledge, and management systems that are essential for development (Sillitoe, 1998). In this regard, IK has been considered one of the essential elements of the development process. Sillitoe (1998) illustrate the example that research in IK has started to make connections between local people’s understandings and practices and researchers and development practitioners, especially in the natural resources and health sectors. 2.6.1 Indigenous knowledge system The concept of indigenous knowledge (IK) has frequently been related in the Western context to the basic, the wild, and the natural (Semali & Kincheloe, 1999). Condescending Western viewpoints have meant that the insight into, and understanding of, IK have been only lightly appreciated. However, for the millions of indigenous peoples of Africa, Latin America, Asia, and Oceania, IK is a daily rationalisation that rewards local communities. To some extent, to these local communities, IK reflects the vigorous means in which the people of a given place have come to understand themselves in association with their natural environment and how they organise that local knowledge of nature, cultural beliefs, and history to improve their lives (Semali & Kincheloe, 1999). According to UNESCO (2017), local and indigenous knowledge refers to the understandings, abilities and ideologies evolved by societies with long histories of interaction with their natural environment. For over half a century, IK systems have caught the attention of anthropologists (Hunn, 2007). Recently, interest among researchers and development workers from different backgrounds such as international development, resource management, sustainability and resilience, disaster response, climate change, ethnobotany, ethnomedicine, and ethnoveterinary studies has increased both within and beyond the discipline (UNESCO, 2009). Senanayake (2006) argues that IK is the social capital of the poor communities. It is a valuable asset that the poor use to invest in the struggle for survival, to make food, to provide for shelter and to control their lives. 16 2.6.2 Indigenous knowledge and development This research seeks to obtain in-depth understanding of people’s experiences and objectives and to link them to scientific knowledge with the purpose of contributing, in the longer term, to positive change, and promoting culturally appropriate and environmentally sustainable adaptation for people who deplete their resource for profitable purposes (Sillitoe, 1998). 17 Some researchers have emphasised that IK is the latest and best strategy in the longstanding combat against poverty, hunger, and underdevelopment (Agrawal, 1995; Briggs, 2014; Sillitoe, 1998). Since IK has allowed people to live in harmony with their environment, using it sustainably is essential in discussions of sustainable resources use and balanced development (Agrawal, 1995; Briggs, 2014). This position stands in complete contrast to the viewpoints of modernisation theorists, who saw IK and institutions as inferior and barriers to development. However, modern formulations acknowledge that it is careless and naïve when the knowledge of the poor and marginalised people is disregarded (Agrawal, 1995). IK is not only an essential and appropriate method to allow poor communities to develop themselves, but it is also beneficial to the sustainable development of local communities. Breidlid (2009) suggests that the lack of respect for IK and the assumption of many Western researchers about the dominance of Western knowledge and scientific discourse is a grave problem to sustainable development in view of evident failure to meet human development needs and, at the same time, to protect environment and ecosystem. Nevertheless, some Western scientists have started to realise that they may learn something from the developing countries and that IK might depend on their scientific understanding of natural phenomena, which could be vital for sustainable development (Breidlid, 2009). Sillitoe also claims “it is increasingly recognised that development initiatives that pay attention to local perceptions and ways are more likely to be relevant to people’s needs and to generate sustainable interventions” (Sillitoe, 1998, p. 224). For centuries, indigenous communities have been practising their knowledge and adapting it to their lands. For them, their lands are extremely important; it is where social, cultural and environmental activities happen. Sustainable production and consumption practices together with resource conservation and management methods are among these activities, primarily based on their IK and routine governance structures (Magni, 2017). 2.6.2 Indigenous knowledge and development The importance of IK has been recognised and incorporated into the SDG (2030 Agenda) because IK can be used to address universal problems by explaining its meanings, natures and purposes; by understanding how it is put into practice and its consequences; and by underlining the significance for indigenous people to be able to entirely understand their rights. However, in order to ensure the effective incorporation of IK to address the primary concern of the 2030 Agenda, participatory methods are crucial to ensure the right and balanced 18 representation of different knowledge systems, especially of indigenous and scientific knowledge (Magni, 2017). In climate change research, IK has been progressively recognised for its value concerning environmental problems, particularly climate-related hazards and weather forecasting (Green & Raygorodetsky, 2010). In many recent studies, the IK role has been integrated into works that are related to climate hazards and development (Green & Raygorodetsky, 2010; Raygorodetsky, 2019). Given the importance of IK for sustainable development and adaptation to climate change, UNESCO launched its “Local and Indigenous Knowledge Systems” programme aiming to promote local and indigenous knowledge and its inclusion in global climate change science and policy processes (UNESCO, 2019). 2.7 Indigenous knowledge and climate change adaptation Adaptation has been acknowledged as a critical topic in international and local debates on climate change (Levina & Tirpak, 2006), and has been considered the most appropriate approach for vulnerable communities to combat climate change. UNFCCC (2019) defines the adaptation as an adjustment in environmental, social, or economic structures in order to respond to real or anticipated climate-related hazards and their impacts. The adaptation concerns the alterations in processes, practices, and structures to reduce serious harm or to get the advantage of the effects of climate change (UNFCCC, 2019). In recent decades, using IK to adapt to climate change has been an exciting topic for research. Yohe et al. (2007) claim that studies on indigenous environmental knowledge have been conducted in various countries in the world, usually in the context of understanding indigenous oral histories and cultural attachment of local people to their location. Since the interest in climate change and global environmental change has begun to grow, current studies have discovered how IK can be part of a shared learning effort to address climate-change effects and adaptation, and its connections with sustainability (Yohe et al., 2007). Deeb et al. (2011) also suggest "indigenous and local knowledge is a key resource for communities in understanding the environment and assessing and adapting to climate change”. 19 Indigenous people make up only 4% of the total world population (250-300 million people), but they use 22% of the world’s land surface (Raygorodetsky, 2019). However, these people have been severely affected by climate change. Indigenous people who are close to the environmental resources frequently witness the activities around them and are the first to recognise and adapt to any changes (Gyampoh et al., 2009, p. 70). With shared knowledge of the land, sky and sea, these people are outstanding in observing and interpreting the changes in ecosystems (Raygorodetsky, 2019). The resulting community-based and shared knowledge provide valued understandings, supplementing scientific information with sequential and landscape-specific accuracy and detail that is vital for validating climate models and evaluating climate change situations developed by scientists at a much fuller spatial and temporal scale. Furthermore, IK offers an essential basis for local adaptation and mitigation programmes that sustain the resilience of social-ecological systems at the interlinked local, regional and global scales (Raygorodetsky, 2019). 2.7 Indigenous knowledge and climate change adaptation The IPCC also recognises that IK is a valuable foundation for designing strategies of adaptation and natural resource management to respond to changing of the environment and other forms (Hiwasaki et al., 2015). Studies and projects on IK and its relevance to the understanding of climate change and formulating adaptation plans have been growing (Hiwasaki et al., 2015). Having seen the importance of IK in adaptation policies and its underrepresentation in previous IPCC assessments, Ford et al. (2016) suggest that IK and experience should be included in the next IPCC assessment reports. Ford et al. give an analysis on how indigenous content should be covered and framed in the Assessment Report 5 Working Group II (AR5 WGII) and propose four methods by which indigenous issues can be more thoroughly incorporated into the IPCC process. The first method is about a specific IK- focused or traditional knowledge chapter in the AR5 WGII. The significance of the chapter exposes the unique sensitivity, adaptability, resilience, and vulnerability of indigenous peoples to climate change, and other perceptions of impacts and adaptation represented in IK systems. The second method is that the Assessment Report 6 should foster more extensive involvement of authors with expertise working with indigenous communities, especially indigenous researchers, leaders, and reviewers because authorship has a critical role to shape content in the IPCC assessment reports. The third method is that the IPCC should create particular guidelines for assessing and integrating IK. The guidelines need to include invaluable information on the human dimensions of climate change that happen outside the scholarly journals; the oral histories, traditional 20 practices, and the research documents. These guidelines also need to be developed with IK owners. Lastly, Ford et al. suggest developing a special report on indigenous peoples and climate change in the next IPCC reports, merging both the efforts of WGII and WGIII, which permit global indigenous issues to be documented and examined more deeply and discussing greater flexibility for incorporating IK and the myths, stories, culture, and histories (Ford et al., 2016). It is clear from the above that IK is a potential asset and it plays a significant role in climate change adaptation. Nyong et al. (2007) have shown an example of how local people in the African Sahel have used their IK to develop a strategic plan to adapt to, and mitigate, the impacts of climate change. 2.7 Indigenous knowledge and climate change adaptation In the region of African Sahel, drought has been a serious and frequent natural hazard. The water shortage and high temperature have reduced agricultural productivity in the region. Having faced this extreme event, the local people in the Sahel region used mitigation and adaptation strategies that were developed locally and collectively to solve this problem. The adaptation approach that the local people used was the adoption of adequate environmental resources such as planting early maturing crops, adopting hardy varieties of crops and keeping livestock in areas where rainfall is lower. They also used technology to help them work appropriately in the new conditions. Having continuously adapted their farming, livestock keeping, and other income-generation activities, the local people in the Sahel region successfully improved their sustainable livelihoods level (Nyong et al., 2007). There are more examples of IK being used by local people to adapt to environmental changes in different places in the world. Below, Lebel (2013) illustrates a number of examples of how local communities in different countries, e.g., Asia-Pacific, use IK for adaptation strategies to cope with natural hazards. ● Land, water and natural resource management In Bolipara, Bangladesh, indigenous communities’ preparations for extreme rainfall events they have estimated through bioindicators and physical witnesses include harvesting crops early and covering domestic animals. When drought is expected to happen, the communities shift cultivation to earlier and save fish before ponds dry out. The local communities of the Rongelap Atoll, Marshall Islands have experienced shortage of freshwater for their daily consumption. Having faced the lack of freshwater, the local communities dig pits lined with mulch and coral rubble to minimise the use of ● Land, water and natural resource management In Bolipara, Bangladesh, indigenous communities’ preparations for extreme rainfall events they have estimated through bioindicators and physical witnesses include harvesting crops early and covering domestic animals. When drought is expected to happen, the communities shift cultivation to earlier and save fish before ponds dry out. The local communities of the Rongelap Atoll, Marshall Islands have experienced shortage of freshwater for their daily consumption. Having faced the lack of freshwater, the local communities dig pits lined with mulch and coral rubble to minimise the use of 21 freshwater. Furthermore, the communities explore fresh groundwater bodies that exist on top of saline water, and they manage them by growing shade plants to prevent water evaporation from the holes. 2.7 Indigenous knowledge and climate change adaptation • Site selection and built infrastructure In Papua New Guinea, local communities living along the Markham River build their houses on high stilts to avoid the effect of annual rainy season flooding. These people also build drains to direct water away from their cropping areas. The communities of Khok Kham, Thailand, experience coastal erosion and use two adaptation options to cope with the impact of coastal erosion. The two options are walls made from concrete and sandbags, and bamboo fences with mangrove plants. The benefit of the bamboo system is that when the fence collapses, the mangroves have developed and continue to trap sediments, while the wall option has to be rebuilt at much expense. A study of Gyampoh et al. (2009) also shows how local people living in the Offin River basin, Ghana use their IK to cope with low rainfall, drought and crop failure. Most of the indigenous people living in the Offin River are subsistence farmers who rely on the rivers, streams and rainfall for their agriculture and daily home use. However, decreasing rainfall, increasing air temperature, increasing sunshine intensity, and seasonal changes in rainfall patterns as a result of climate change have all affected these indigenous people recently (Gyampoh et al., 2009). The prolonged rainfall shortages and changes in rainfall pattern have caused crop failures in the basin, reduced water availability in the rivers and streams. High temperature has caused their vegetables to ripen too early, leading to a decrease in sale value of their produce. Heat and water- related diseases have occurred in the basin. Having become aware of this threat to their survival, the local people in the basin developed numerous strategies to adapt. One is to reuse water from washing clothes or household tools to water back gardens and nurseries. The second method is rainwater harvesting. It is traditional to collect and store rainwater in big barrels that are placed under the roofs of their houses. Later on, the local communities abandoned this method and installed wells and boreholes instead. The traditional and local authorities of communities in the Offin River realised that the clearing of riparian plants was the main factor for increased soil erosion, causing water to become dirty, and eventually reducing stream flow. 2.7 Indigenous knowledge and climate change adaptation Subsequently, the traditional and local authorities created an awareness campaign of the effects of deforestation around The traditional and local authorities of communities in the Offin River realised that the clearing of riparian plants was the main factor for increased soil erosion, causing water to become dirty, and eventually reducing stream flow. Subsequently, the traditional and local authorities created an awareness campaign of the effects of deforestation around the rivers and streams, instructing the communities on the prevention of bushfires, 22 promoting local-based management of forests and levying fines on those who set fire to the forests, and cleared riparian plants. Indigenous knowledge and livelihood adaptation strategies In rural communities, local livelihoods and food security are threatened by droughts and floods. In order to secure their livelihoods, the rural communities have used their knowledge to develop various adaptation strategies. A number of studies have revealed how rural communities in different countries such as Bangladesh, India, Nepal, Indonesia and Ghana use their knowledge to respond to natural hazards, especially floods and droughts. A study by Lebel (2013) reveals that farmers in the lower part of north-eastern Bangladesh use their livelihood adaptation strategies, including crop diversification, floating gardens and aquaculture to cope with disaster risks. Floating- gardening methods are a conventional practice that local people in this area use to recover from severe flooding and as a way to survive during future floods. Likewise, local communities in Orissa, India who have experienced severe droughts use their knowledge of indigenous varieties and biodiversity conservation to store, grow and exchange seeds of local varieties of main crops. The communities use the local varieties because they are more drought-tolerant and able to cope with abnormal rainfall. The local communities also claim that the varieties do not require expensive organic fertiliser and pesticide to grow well. Usually, the local communities in this region grow 6 to 14 crops at the same time, allowing them to cope with severe droughts (Lebel, 2013). A study by Jones and Boyd (2011) also indicates how local people in Shreenagar, Nepal use livelihood adaptation strategies to cope with droughts and abnormal rainfalls. Shreenagar is a village in midwestern Nepal. The village is located on a steep slope, with poor soil and a dry climate, limiting the capability for agriculture. Cultivation in this region happens twice in a year and relies mainly on rainfall and the natural environment. However, the changes in rainfall pattern and occurrences of drought, resulting from climate change, have affected the agricultural production in this region. In order to deal with these natural hazards, the local communities use their IK to develop livelihood adaptation strategies, such as harvesting wild food, reducing food consumption, trading livestock, seeking financial and nutritional support from others, and short-term migration (Jones & Boyd, 2011). 23 Armah et al. (2013) have conducted a study in 31 communities in Northern Ghana to explore how IK of the local communities is used to cope with climate change. Indigenous knowledge and livelihood adaptation strategies The local farmers in these communities are heavily dependent on the natural environment for their agricultural production. Climate factors such as high temperature, drought, and flooding have caused reduction in yields of farmers’ agricultural production. To cope with the production loss, farmers use their indigenous methods, such as crop management strategy, land management strategy, livestock strategy, and off-farm activities. More than half of the local farmers engage in livestock production as a method to cope with the production loss. Livestock production and sales provide work for the entire year and are one of the main sources of income for the local farmers. Livestock raising also helps farmers improve food security and the manure from livestock can be used to maintain soil fertility and increase crop productivity (Armah et al., 2013). Armah et al. (2013) have conducted a study in 31 communities in Northern Ghana to explore how IK of the local communities is used to cope with climate change. The local farmers in these communities are heavily dependent on the natural environment for their agricultural production. Climate factors such as high temperature, drought, and flooding have caused reduction in yields of farmers’ agricultural production. To cope with the production loss, farmers use their indigenous methods, such as crop management strategy, land management strategy, livestock strategy, and off-farm activities. More than half of the local farmers engage in livestock production as a method to cope with the production loss. Livestock production and sales provide work for the entire year and are one of the main sources of income for the local farmers. Livestock raising also helps farmers improve food security and the manure from livestock can be used to maintain soil fertility and increase crop productivity (Armah et al., 2013). Severe droughts, abnormal rainfall and high temperature, resulting from climate change, have posed a major threat to many local livelihoods in Indonesia. Binternagel et al. (2010) reveal that severe drought and high temperature have impacted greatly on local communities’ cacao agroforestry in an Indonesian province of Central Sulawesi. The impacts of severe drought have caused yield loss and loss of the local household incomes. Binternagel et al. (2010) also reveal how various types of adaptation strategies are used to deal with the severe drought. The most common strategies are reactive or past adaptations, which help the local people to deal with the impacts of the droughts. Indigenous knowledge and livelihood adaptation strategies Severe droughts, abnormal rainfall and high temperature, resulting from climate change, have posed a major threat to many local livelihoods in Indonesia. Binternagel et al. (2010) reveal that severe drought and high temperature have impacted greatly on local communities’ cacao agroforestry in an Indonesian province of Central Sulawesi. The impacts of severe drought have caused yield loss and loss of the local household incomes. Binternagel et al. (2010) also reveal how various types of adaptation strategies are used to deal with the severe drought. The most common strategies are reactive or past adaptations, which help the local people to deal with the impacts of the droughts. The reactive strategies comprise reducing food expenditure and changing food patterns, paid labour, raising a loan, rattan collection, hand irrigation techniques, and land-use change. The reducing food expenditure and changing food patterns strategies involve the local households reducing the number of meals by eating twice instead of three times a day, and replace rice, the main food component, with cassava, young rattan or sago. Furthermore, the local households reduce the amount of food in the meals by eating smaller portions (Binternagel et al., 2010). The reactive strategies comprise reducing food expenditure and changing food patterns, paid labour, raising a loan, rattan collection, hand irrigation techniques, and land-use change. The reducing food expenditure and changing food patterns strategies involve the local households reducing the number of meals by eating twice instead of three times a day, and replace rice, the main food component, with cassava, young rattan or sago. Furthermore, the local households reduce the amount of food in the meals by eating smaller portions (Binternagel et al., 2010). Various studies have demonstrated a variety of local adaptation strategies in different countries that have been used to cope with climate change. However, Lebel (2013) argues that the IK that used to deal with climate change may not always be sufficient. Lebel suggests that the IK needs to be strengthened and integrated with scientific knowledge in order to expand the adaptation. Likewise, Gyampoh et al. (2009) notice that the knowledge the indigenous people in the Offin River basin use to cope with 24 climate change has not always succeeded. They claim that the IK of agriculture and water management that people used in the past has become more challenging to apply in recent years because of changes in rainfall patterns. Indigenous knowledge and livelihood adaptation strategies For instance, the local people in the Offin River basin who switched to wells and boreholes instead of placing barrels under their house roofs reported that their new method did not work due to the current severe climate. Gyampoh et al. (2009) then suggest that in order to exploit, improve and mainstream IK adaptation methods into global adaptation strategies, IK should be studied more, supported and incorporated into scientific research (Gyampoh et al., 2009). Additionally, Hiwasaki et al. (2015) mention that in order to make the best use of IK for adapting to climate change, it is essential to understand how knowledge can be incorporated into science. It can be done through a process in which researchers, development workers, communities jointly undertake observation, documentation, and validation. The incorporation of IK with science could allow community people to formulate the best strategic plan to adapt to climate change and moderate the impacts of climate-related disasters (Hiwasaki et al., 2015). 3.1 Introduction This chapter outlines the methodology used to achieve the objectives of the research thesis. The chapter begins by investigating social constructivism as the epistemology that guides this research study. It then explains how this epistemology forms part of an overall qualitative methodology comprising methods such as in-depth semi-structured interview and observation. Research location, participant recruitment, data recording and analysis are also included in the chapter. 2.8 Conclusion The chapter has explored previous studies about climate change and its relationship with development. The chapter also discussed the public perception about climate change and drew examples of local perception about climate change in different countries. It then investigated the impacts of floods and droughts on the environment, socioeconomics and local livelihoods. Next, it discussed the importance of IK in development and its significant roles in developing adaptation strategies to deal with climate change. As can be seen, IK has been used in the past effectively to adapt to droughts and floods. However, climate change has been increasing in severity and frequency. Therefore, IK needs to be studied more, strengthened and integrated with scientific knowledge in order to deal with the new situation of climate change. We need to understand better how local communities not only perceive and adapt to climate change but also, we need to examine the extent to which IK and practices can be used and modified to deal effectively with the effects of climate change. 25 3.2 Epistemology This research studies how local people have constructed knowledge to solve problems happening in their community. For this study, it is the people’s perceptions and experiences of, and knowledge about, floods and droughts that I am interested in; and how they give meaning to, and use, that knowledge and those experiences. The research specifically looks at how farmers and fishers in the Tonle Sap Lake region have constructed knowledge about floods and droughts and have used their livelihood practices to respond to such hazards. In this case, social constructivism is the most suitable epistemology for this research. According to Creswell (2014), in constructivism, people seek to understand the world where they live and work, and they interpret the meaning of their life and work based on their experiences. Likewise, Ultanir (2012) states that constructivism is a study of knowledge, a theory of learning or meaning making, which offers an explanation of the nature of knowledge and how people learn. Thus, the real understanding of how local people have constructed their IK is developed based on their experiences and practice. This theory helped me to look for the complexity of people’s views rather than limiting meanings of a few sets of ideas. The goal of this research relies on the participants’ views of the studied situation (Creswell, 2014). The study of the context of local people, collaborating with multiple participants, employing ideas and values of participants is necessary for my research (Dudovskiy, 2019). As a constructivist researcher, I addressed the processes of communication with the participants of this research project. I specifically focused on 26 the context of IK with which local people live and work to understand the historical and cultural settings of my participants (Creswell, 2014). the context of IK with which local people live and work to understand the historical and cultural settings of my participants (Creswell, 2014). 3.3 Qualitative methodology The research investigates, in particular, the IK around livelihood strategies that local communities have used to prepare for floods and droughts and recover from the effects of such natural hazards. To do this, qualitative methods are appropriate and practical for my research because this methodology allows researchers to deepen the understanding of the ways people interpret and make sense of their own experiences and the setting where they live. The research employed a qualitative approach to develop a case study of the use of IK by local people in two villages in the Tonle Sap Lake region to develop livelihood strategies to deal with regular floods and droughts. Qualitative research has been employed frequently in academic and applied social studies. The qualitative approach mainly follows the exploratory analysis and is used to explain what happens at a local setting and sometimes raises or formulates new hypotheses and theories (Johnson, 2014). Researchers adopt a qualitative methodology to discover or learn more about a little-known topic or phenomenon. Commonly, it is used to understand people’s experiences and to express their perspectives (Johnson, 2014). According to Stewart- Wither et al. (2014), qualitative research is commonly used to seek understanding of complex social facts that happen in a real place, where the goal is to both understand and find meanings, and perhaps bring about change (Stewart-Withers et al., 2014, p. 60). Qualitative research is also used to collect information in natural sites, rather than unreal or created settings (Brockington & Sullivan, 2003). This approach helps my research explore the meanings of IK by interacting with, and interpreting, the actions and views of local people in a particular setting. Tracy (2013) mentions that qualitative research focuses on the thick description where researchers engage themselves in a culture, and examine a specific situation existing in that scene. This allows researchers to explore lived experiences, located in its perspective, appreciate participants’ local meanings and interpret participants’ standpoints and stories. 27 3.4 Research location The research was conducted in two villages (Mukwat and Santey) in the Tonle Sap Lake region. Mukwat village is located along a tributary and is adjacent to Tonle Sap Lake. This village is known as a land and water-based village because it is flooded for six months and dry for the other six months of the year. This village is also known as a fishing village because the primary livelihood activity of the local communities in the village is fishing. Santey village is located about 7 km from Tonle Sap Lake and is known as a land-based or farming village because the primary livelihood activity of the villagers is rice farming. The two villages are administratively located in Siem Reap province. Siem Reap province is located in northwest Cambodia and is approximately 300 km far from the Cambodian capital city, Phnom Penh. Tonle Sap Lake is the biggest freshwater lake in Southeast Asia, making a significant part of the Mekong River system (Kummu et al., 2014). The lake is very productive, driven by an annual mono-modal flood pulse (Kummu et al., 2014). The lake is a dynamic flood-pulsed ecosystem that has a connection with the Mekong River. The lake surface area rises annually from approximately 2,500 km2 to over 12,500 km2 driven by seasonal flooding from the Mekong River (Holtgrieve et al., 2013). This flooding is believed to promote many of the significant ecological developments, including primary and secondary aquatic yield. The lake is also rich in fishery sources and a productivity area for rice production that support the livelihoods of nearly two million people (Holtgrieve et al., 2013; Johnstone et al., 2013). The local communities living in the region are strongly dependent on fishing and rice farming for their main livelihood (Johnstone et al., 2013). However, in the past few decades, human activities and climate change have caused significant changes of the lake and its ecosystems (Uk et al., 2018). 28 Figure 1. Map of Tonle Sap Lake – Cambodia. Source: canbypublications com Figure 1. Map of Tonle Sap Lake – Cambodia. Source: canbypublications.com Source: canbypublications.com There are two main reasons Tonle Sap Lake was chosen for my study. The first reason is that the local people who rely on fishery resources and agricultural farming in this region have been affected by climate change, particularly floods and droughts. 3.5 Methods With a qualitative methodological approach, this research study employed semi- structured interviews and observations as the main methods. The two methods are detailed as below. With a qualitative methodological approach, this research study employed semi- structured interviews and observations as the main methods. The two methods are detailed as below. 3.4 Research location The second reason is that I worked in the region for nearly three years, and I am familiar with the location and the local people in the studied communities. More importantly, I have a network of non-governmental organisations (NGO) in the region, which helped facilitate this research. 3.5.1 Semi-structured interviews Interviewing is one of the most significant methods that qualitative researchers usually use to collect various forms of data (Creswell, 2014). According to C. L. Briggs (1986), interviews are the most popular tool used in social science studies, and they provide 29 researchers with opportunities for mutual findings, understanding, reflection, and explanation through a way that is natural, adaptive and frequently stimulating (Tracy, 2013, p. 132). The interviews help explore individually lived experiences and the respondents’ viewpoints (Tracy, 2013). Moreover, the interviews enable the researchers to access and comprehend the way the respondents perceive, give meaning to, and define settings for, the real world (Creswell, 2014). Hence, the interviews allowed me to understand the way the local communities in the Tonle Sap Lake region perceive climate change, especially floods and droughts, and how they develop adaptation strategies to respond to the natural hazards. Interviews offer the researcher different approaches, such as face-to-face, by telephone or through focus groups (Creswell, 2014). However, in my research, I used only one-on-one face-to-face interviews with the research participants. This type of interview helped me to deeply understand how the individuals of local communities perceive the impacts of floods and droughts and respond to them using their IK and experiences. A semi-structured interview is one form of qualitative interviews, allowing researchers to use open-ended questions to discover views and opinions of the participants (Creswell, 2014). The semi-structured interview allows both researchers and participants to engage in a dialogue and the participants have the space to answer on their own terms (Edwards & Holland, 2013). Therefore, the semi-structured interview is the most suitable method for my research study since it allowed me to interact with the respondents to explore their viewpoints about the impacts of floods and droughts. Because of the open-ended questions and informal protocol, the semi-structured interview allowed the respondents to have enough space to answer different questions. It also enabled me to discover how the local people in the region use their knowledge and experiences to respond to the natural hazards. During the interviews, all the participants were voice recorded. For their convenience and, as suggested by the participants, most of the interviews were conducted at the participants’ houses. However, the interviews with NGO staff and commune councillors were conducted at their offices. By conducting the interviews at their suggested places, the participants could express their viewpoints freely in a relaxed manner. 3.5.1.1 Sampling selection In this research, the participants were selected using snowball sampling. According to Walliman (2011), snowball sampling is a non-probability sampling, which is based on the selection by non-random means. The snowball sampling is a useful technique for a study or survey where it is hard to get access to the entire group of people (Walliman, 2011, p. 111). Since my research aim was to explore the livelihood strategies adaptation to floods and droughts, I wished to talk to people who were involved with different livelihood activities in the communities, and because of time constraints, I used snowball sampling for my research. As mentioned by Tracy (2013), the researcher starts to identify some participants who fit the criteria of the study and then asks those people to suggest a colleague, a friend, or a family member be involved in the study. Using the snowball sampling technique, I was introduced by the village head to talk to two participants who fit my research criteria. I then approached and asked them if they were willing to take part in my research. Once I had conducted the interview with these two participants, I approached other potential participants who were recommended by those two participants. I kept asking the participants whom I interviewed to recommend other potential participants who were appropriate for the study until I reached the number planned. However, this process was not always smooth. Sometimes, the participants who were recommended to me were not willing to participate in my research because they were busy with their fishing and farming activities. Other participants had little knowledge about the topic and could not contribute any useful information. One more important thing, some participants only recommended their close friends and family members because they wanted them to receive the small gift that I provided for participating in the interview. This did not allow me to get information from a wide range of community people. 3.5.1 Semi-structured interviews The interviews were conducted in Khmer (the official language of Cambodia) using a question guide in Khmer as well. 30 3.5.1.2 Participant and data collection procedure The research participants of this study were primary fishers and farmers in the two communities in the region. There were 19 participants in total, including fishers, farmers, commune councillors, village heads and NGO staff (see Table 1). To gain access to the local participants (farmers and fishers), I requested permission from the 31 commune councillor and village heads and had a meeting to discuss and get detailed information about the communities and people before meeting with them. For this research, the local participants (fishers and farmers) were the main research participants because they could contribute to the aim of the study, which was to understand how the communities perceive floods and droughts and respond to these natural disasters using their IK. The interviews were conducted in two different villages. In the fishing village, most of the local participants were fishers. In the farming village, most of the local participants were farmers. I spent one week in each village to conduct my interviews. Usually, I interviewed only two people in a day so that I had time to reflect on the process of the interviews and review the recordings. The local authorities (commune councillors and village heads) were included in this research because they have valuable information about the people, local livelihoods and other relevant development programmes. The research also included one NGO staff member who was working in the farming village. This NGO staff worked directly with the local farmers to provide technical support on chicken farming, which was part of the NGO’s livelihood project. Table 1: Research participants Respondents Gender Role in community 1. Participants from local authorities Respondent MW-CC09 Male Commune Councillor Respondent ST-CC09 Female Commune Councillor Respondent ST-VH08 Male Village Head Respondent MW-VH06 Male Village Head 2. Participant from NGO Respondent NGO-01 Male 3. Participants from farming village Respondent ST-FA01 Male Farmer Respondent ST-FA02 Female Farmer Respondent ST-FA03 Male Farmer Respondent ST-FA04 Male Farmer 32 Respondent ST-FA05 Male Farmer Respondent ST-FI06 Male Fisher Respondent ST-FA07 Female Farmer Respondent ST-FA08 Male Farmer 4. Participants from fishing village Respondent MW-FI01 Male Fisher Respondent MW-FI02 Male Fisher Respondent MW-FI03 Male Farmer Respondent MW-FI04 Male Fisher Respondent MW-FI05 Female Fisher Respondent MW-FI07 Male Fisher Respondent MW-FI08 Female Fisher 3.5.2 Observations According to Scheyvens and Storey (2003), observations can be used for both qualitative and quantitative research to collect data and they provide a direct and apparently accurate method of data collection. The observations are one of the most essential tools for researchers, whether they result in making ‘hard data’ or simply impressions and surprises, which help researchers shape and interpret their research (Scheyvens & Storey, 2003). In qualitative research, the researchers use the observation method to take note of the behaviour and activities of people at the research site (Creswell, 2014). The observations can be done in an unstructured or semi-structured way, which requires researchers to be engaged in various roles from a non-participant to a complete participant (Creswell, 2014). Stewart-Withers et al. (2014) suggest researchers should be involved in the place they are studying so they are able to deeply understand the way the people look at, and interpret, the world. The method also assists the researchers to check for nonverbal expressions of feeling and allows the researchers to observe events that the respondents are unable or unwilling to share during the interview (Kawulich, 2005). In this study, I employed non-participant and unstructured observation, which included transect walks and informal meetings with non-participant villagers in the two communities to observe how IK is used to develop livelihood adaptation strategies to 33 respond to floods and droughts. Non-participant and unstructured observation allowed me to study how the local communities in the region practise their daily livelihood activities. I spent half a day in each village to conduct a transect walk to explore the villages and the rice fields. The transect walk helped me to capture the essential things, such as location of communities’ farming lands, natural and human-made ponds, and other water reservoirs that the participants did not mention in their interviews. It also allowed me to see the reality of resource use and compare it with the data I collected from the interviews. In the fishing village, I also spent half a day on the lake to explore how the local people used their fishing gear to catch fish. During the interviews, the participants had mentioned some of the unfamiliar fishing gear and explained how it was used. Hence, spending a half-day at the lake enabled me to see the unfamiliar fishing gear and how it was used. 3.5.2 Observations Moreover, it allowed me to explore the reality of the lake during severe drought. In the farming village, I spent half a day in the rice fields to see how severely the rice fields had been affected by droughts, and was able to have an informal discussion with one of the farmers about the impacts of severe droughts on the local rice farming and how they prepared for and responded to the droughts. Additionally, in the afternoon after the interviews, I usually spent about 15 to 20 minutes meeting with groups of non-participant villagers in the fishing village, who gathered to fix their fishing gear and boats, to talk about their perceptions of climate change and livelihood practices. These informal meetings allowed me to capture additional interesting information that was not mentioned in the interviews. 3.7 Ethical issues and confidentiality In qualitative research, ethical issues are related to the concern of research participant protection (Merriam & Tisdell, 2016, p. 187). As a social researcher, I took the participants’ needs and concerns into account and ensured their dignity, privacy and safety (Scheyvens & Storey, 2003). I also made sure that all the participants received the same treatment and respect. There were minor ethical considerations that arose during the interview, including voice recording and my positionality. Some participants were afraid of providing detailed information for the interview when they knew that their voice would be recorded. These participants assumed that their voice would be shared with other people, such as government officials or NGO staff. Moreover, some participants were afraid of sharing, and/or not willing to share, the information with a person whom they did not know, especially an outsider. To ease this situation, I always spent at least five minutes with the participant before the interview and explained to them the purpose of voice recording and how the data would be used. I also discussed general aspects, such as the participants’ families and their communities. Spending time with the participants and having informal discussions helped me gain trust and build a good relationship with the participants so that they were not afraid of sharing information during their interview. Before starting to record their voice, I also confirmed with the participant that they were happy to provide information with voice recording. 3.6 Data recording procedures, storage and analysis All the participants were interviewed using voice recording. Before starting the interview, I explained the purpose of the study, the process of recording, and asked the participants for their consent. I used two devices, a mobile phone and voice recorder, to record the interviews in case one of them was broken or lost during the fieldwork. During the interviews, I also took notes on interesting points and keywords that required clarification from the participants and triggered further questions. As soon as the interviews had been conducted in one village, I copied all the voice recordings to my 34 laptop and uploaded them to Google drive to avoid information loss. After the completion of the data collection in the two villages, all the voice recordings were transcribed and translated into English in word-documents. I carried out both the transcription and the translation. The collected data were then analysed manually and carefully using Microsoft Word 2016. The collected data were then analysed manually and carefully using Microsoft Word 2016. After the completion of the transcription and translation, I read through all the data and searched for similarities. Then I coded the data in accordance with themes and type of responses, and classified following the format of the question guide (see Table 2): Table 2: Key themes for data analysis Key themes Sub themes Local perceptions of climate change - Changing of weather - Extreme heat and water shortage - Floods - Droughts Local livelihood activities - Primary and secondary livelihood How floods and droughts have impacted on the local livelihood - Impacts of floods on local livelihood - Impacts of droughts on local livelihood Preparation for floods and droughts - Local preparation for floods - Local preparation for droughts Livelihood strategies to respond to floods and droughts - Local livelihood strategies to respond to floods - Local livelihood strategies to respond droughts How IK was generated for the adaptation strategies - Effectiveness of adaptation strategies - What methods have been changed How IK is shared - Knowledge sharing in community - Knowledge sharing in family Local perceptions of future climate change and droughts Possible preparation/prevention of floods Table 2: Key themes for data analysis 35 Livelihood support from outsiders - Support from NGOs - Support from government Integration of IK and new technologies to respond to climate change 3.8 Researcher’s positionality Positionality refers to the insider/outsider status of researchers, including race, gender, social class background, and sexual orientation (Merriam & Tisdell, 2016). Being a researcher engaging with the community context required me to think about how my background and experience play a role in building relationships with the research 36 participants, and in how I conduct the research (Call-Cummings & Ross, 2019). I am aware that my gender, class, educational background might affect the way I interact with my research participants. Nevertheless, my position as a Cambodian and academic student, provided me with both benefits and challenges to my research. As a Cambodian citizen, I am fully aware of what requirements need to be prepared for the field research in a specific place. Being a Cambodian, I could approach my research participants with ease. Since both my participants and I shared the same language and culture, it was easy for me to interpret and understand the facial expression, gestures and behaviours used by the participants during the interview. This could help me to interact well with the participants and keep them engaged in the conversation in a safe, relaxed and friendly environment. Nonetheless, I understand that the positionality that I used to work in the studied communities might affect the participants’ opinions, which could impact my research result. The fact that I used to be a social worker in the studied communities could influence how I interpreted the participants’ views and create a power gap between them and me. To avoid this, I always clearly explained to the participants that I was there as a student to study about the community. I told the participants that I was enthusiastic to learn more from them. During the interview, I always reminded myself to pay as much attention as possible to the way the participants expressed their opinions by not interrupting them. I also pretended that I did not know anything about what the participants mentioned by letting them talk, and I listened to them carefully. I always expressed that I wished to learn from them by asking them further questions in order to clarify and explore more about the topic that the participants mentioned. 4.1 Introduction This chapter reviews the climate change impacts in the context of Cambodia and existing adaptation policies made at the national level. This chapter is divided into five sections. The first section explores the geographical area and the population of Cambodia. The second section presents the climate in Cambodia. The third section provides a brief overview of Cambodian agriculture and livelihoods. The fourth section investigates the climate change, floods and droughts in the context of Cambodia. The last part explores the existing climate change adaptation policies in Cambodia. 3.9 Conclusion This chapter explained the methods and methodology used to explore how local people in the Tonle Sap Lake use their IK to respond to floods and droughts. The study was driven by a social constructivist epistemology to understand how local communities have constructed their knowledge and understand the changes in weather. To explore this, a qualitative research consisting of semi-structured interviews and observation were used to investigate how the local communities perceive climate change and its impacts on their livelihood, and how they respond to the natural hazards. The chapter 37 also discussed my positionality as an insider and outsider, its benefits and challenges, and how I managed it for this research. 38 4.2 Geographical areas and population Cambodia is located in mainland Southeast Asia on the coast of the Gulf of Thailand and has a total area of 181,035 square kilometres (Figure 2). The international borders of Cambodia are shared with Thailand and the Lao People’s Democratic Republic on the west and the north, and Vietnam on the east and the southeast (FAO, 2012). Cambodia also shares the Mekong River basin with these three countries as well as Myanmar and China. The river flows from China and crosses the country from the north to the south. The water surfaces of the country, including Tonle Sap Lake, cover about 2.2% of the total area of the country (FAO, 2012). In the southeast, Cambodia is bordered by the Gulf of Thailand with a 435 km coastline and an exclusive economic zone of 55,600 km2. The coastal region comprises estuaries and bays and roughly 64 islands of different sizes (MoE, 2009). With regard to Cambodia’s topography, the country is divided into two parts. The first part is the central low-lying or the central plains and the flat coastal areas. The second part is the mountainous and highland areas surrounding the low-lying land. The central plains of the country mainly contain the flat landform of the Mekong River and the Tonle Sap basin. The central plains cover 75% of the country’s total area. Around the central plains, are highlands with dense forest and a low population. These highlands include the Cardamom and Elephant Mountains of the southwest and western regions; the Dangrek Mountain to the north adjacent to the Korat plateau of Thailand; and the 39 Ratanakiri plateau and Chlong highlands on the east connecting with the central highlands of Vietnam (MoE, 2009). Figure 2. Map of Cambodia. Source: freeworldmaps net Figure 2. Map of Cambodia. Source: freeworldmaps.net Source: freeworldmaps.net In 2019, the total population of Cambodia was 16.2 million (World Bank, 2019a). Nearly 80% of the Cambodian population lives in the countryside, and 65% are heavily dependent on agriculture, fisheries, and forestry for their livelihoods (USAID, 2019a). According to World Bank (2011), the majority of Cambodian people tend to live in the central plains and around the Tonle Sap Lake. Of the country's population, 52% live in the central plains, and 30% live in the surroundings of the lake. Only 11% live in the highlands and mountains, while 7% live along the shoreline. 4.2 Geographical areas and population The average population density of Cambodia is considerably low for the region, which is at only 75 people per square kilometre. With regard to poverty in Cambodia, in the last two decades, Cambodian household income has increased while the poverty rate has rapidly fallen. Between 2000 and 2015, the average annual growth in the country’s GDP was 7.8%, which boosted the GDP per capita from USD300 in 2000 (World Bank, 2019a) to USD1,225 in 2015 (OECD, 2017). Within three years, the Cambodian GDP per capita increased to USD1,380 in 2018 (World Bank, 2019a). The poverty rate drop happened at different times for rural and urban regions. In rural areas, the sharpest reduction in poverty happened between 2007 and 2009, driven by the increase in crop prices and agricultural wages. In urban 40 regions, the dramatic decline in poverty occurred between 2004 and 2007 (OECD, 2017). Although Cambodia has experienced a decrease in poverty in the last two decades, most poor rural households, who escaped poverty, have not risen far from the poverty line. About 4.5 million people are still near-poor, and vulnerable to falling back into poverty if economic and other external shocks occur (World Bank, 2019b). 4.3 Cambodia’s climate Geographically, Cambodia is situated in Southeast Asia between latitudes 10° and 15° North and longitude 102° and 108° East, with a tropical climate that is warm all year round. Cambodia has two main seasons; the dry season from November to April and the wet season from May to October (Ministry of Environment, 2009). In the wet season, the northwest monsoon brings 90% of the annual precipitation, which is usually between 1,200 and 2,000 millimetres per year throughout the country (Ministry of Environment, 2009). However, the rainfall during the wet season was higher than 2,000 millimetres in some years. In some places, the mean monthly rainfall is higher than 5,000 millimetres (World Bank, 2019c). In several regions, the rainfall fluctuates considerably from year to year, and even in June and July (the wet season), they may experience up to half a month without rain (Ministry of Environment, 2009). The average temperature of Cambodia is between 26oC and 40oC. The temperature is ordinarily cool between November and January in the dry season, and is hotter otherwise (World Bank, 2019c) (Figure 3). The warmest month of the year is April when the temperature may go above 38oC (Ministry of Environment, 2009). The humidity in the country varies depending on the time. For instance, in January and February, the humidity ranges from 65-70% while in August and September, the humidity goes up to 85-90% (Ministry of Environment, 2009). 41 41 Figure 3. Average monthly temperature and rainfall of Cambodia (1901-2016). Source: (World Bank, 2019c) Figure 3. Average monthly temperature and rainfall of Cambodia (1901-2016). Source: (World Bank, 2019c) Figure 3. Average monthly temperature and rainfall of Cambodia (1901-2016). Source: (World Bank, 2019c) 4.4 Cambodia’s agriculture and livelihoods Cambodian agriculture is not only essential for rural communities, but it also significantly contributes to the economic growth of the country. The agricultural sector of Cambodia accounts for 35% of the national GDP and creates job opportunities for many Cambodian people (FAO, 2019a). The agricultural sector has been prominently placed in the National Rectangular Strategy and highly prioritised in the development agenda of the country (Ministry of Agriculture, Forest and Fisheries, 2015). By prioritising the agricultural sector in the national development agenda, the Cambodian government anticipates that agricultural development could reduce poverty rate through food security, increasing household incomes, and job creation. The Cambodian government also expects agricultural development to contribute to economic growth and macro-economic stability (Ministry of Agriculture, Forest and Fisheries, 2015). In relation to the agriculture sector, crop production makes up the largest portion of the sector’s GDP. It accounts for 54%, while the combination of fisheries and livestock contribute 40%, and forestry and logging account for only 6%. Despite inadequate irrigation systems for rice cultivation, rice production has gradually increased. The increase in rice production has made Cambodia not only self-reliant in rice, but it has 42 also made Cambodia a significant rice exporter (FAO, 2019a). In 2017, Cambodia was one of the ten biggest rice-exporting countries in the world. Cambodia exported approximately 635,000 tons of milled rice; almost double compared to 2013 (Royal Government of Cambodia, 2018). Other important Cambodian food crops include corn, soybean, mung bean, cassava and fruits such as mango, pineapple, jackfruit, durian, rambutan and banana. Cash crops such as sugar cane, rubber, and palm oil have also significantly increased (FAO, 2019a). Most of the crops exported are raw, i.e., unprocessed. They are mainly exported to neighbouring countries such as Thailand and Vietnam, where they are processed (Netherlands Embassy, 2018). Fisheries have played an essential role in the economy of Cambodia (FAO, 2019b). A vast wetland consisting of floodplains around Tonle Sap Lake, and along the Tonle Sap and Mekong rivers, covering 30% of Cambodia’s land area delivers one of the largest, most diverse and intensive freshwater fisheries worldwide. In 2012, the freshwater fishery production value was estimated at USD1.2–1.6 billion per year, and it contributed up to 12% of the national annual GDP (Joffre et al., 2012). 4.5 Climate change, floods and droughts in the context of Cambodia Cambodia is a country highly exposed to the impacts of climate change and was ranked 13th in the Global Climate Risk Index from 1995–2015. It was then ranked 8th in the World Risk Index 2016. In 2014, the Standard and Poor’s Rating Services ranked Cambodia as the most vulnerable to the effects of climate change in the world in terms of climate-risk exposure on its economy (GSSD, 2017). In 2017, the Notre Dame Global Adaptation Index also gave Cambodia a high score in terms of its vulnerability to climate disruption, and a low score in terms of its readiness to leverage private and public sector investment for adaptive actions (University of Notre Dame, 2019). Keo (2014) states that Cambodia’s vulnerability to climate change is not only because of climate risk but also because of the lack of the capacity to adapt to it. The vast majority of the country’s population lives in rural areas with limited knowledge, inadequate infrastructure and little opportunities. More than 70% of those rural communities are dependent on agriculture, which is massively sensitive to climate disruption, placing Cambodia’s economic and social development at risk (Keo, 2014). Cambodia has abundant natural resources that are essential for livelihoods and food security. However, natural resources have been threatened by climate change (USAID, 2019b). Climate change has significantly affected Cambodian livelihoods and destroyed infrastructure, with average damage at USD235 million annually (World Bank, 2017). Changing climate poses continuing threats to Cambodia’s sustainable development goals. In 2015 alone, it was estimated that climate change impacts caused Cambodia to lose roughly USD1.5 billion, which is equivalent to 10% of the annual GDP (USAID, 2019b). In the last decade, Cambodia has experienced extreme weather events such as droughts, floods and windstorms, which placed a high social and economic cost on the country’s economy and people's livelihoods (Kim et al., 2014). Due to the geographical area, the incidence of floods and droughts in Cambodia is diverse. While floods affect lowland areas, droughts occur elsewhere across the country (GSSD, 2017). Rising temperature and changing rainfall patterns have also had high impacts on Cambodia’s economy. Since 1960, the average temperature in Cambodia has increased by 0.8oC, at a rate of approximately 0.18oC per decade (World Bank, 2019c). The increasing rate is fastest in the dry season (December to February and March to May), increasing 0.20 - 0.23°C per decade. 4.4 Cambodia’s agriculture and livelihoods In 2017, the fisheries sector provided jobs to 654,692 people directly, and more than 2 million people gained some livelihood benefit from the involvement in this sector (FAO, 2019b). The fishery sector has not only made a significant contribution to the employment and livelihoods of Cambodian people, but it has also contributed to national economic growth. In 22 years, the value of fish and fishery products exported increased from USD15,106,000 in 1995 to USD69,091,000 in 2017 (FAO, 2019b) (Figure 3). Aquaculture also plays a significant role in the total fish production of the country. Although the aquaculture sector is small compared to fish capture, the contribution of aquaculture to total fish production has gradually increased from 10.9% in 2010 to 24% in 2017 (FAO, 2019b). In 2017, the Cambodian government estimated the aquaculture production was at 205,3000 tonnes while the capture production was at 649,500 tonnes (Figure 4). 43 Figure 4. Total imports and exports of fish and fishery products for Cambodia (USD1000) Source: (FAO, 2019b) Figure 4. Total imports and exports of fish and fishery products for Cambodia (USD1000) Source: (FAO, 2019b) Figure 4. Total imports and exports of fish and fishery products for Cambodia (USD1000) Source: (FAO, 2019b) Figure 5. Total capture and aquaculture production for Cambodia (tonnes). Source: (FAO, 2019b) Figure 5. Total capture and aquaculture production for Cambodia (tonnes). Source: (FAO, 2019b) 44 4.5 Climate change, floods and droughts in the context of Cambodia In the wet seasons (June to August and September to November), the increasing In the last decade, Cambodia has experienced extreme weather events such as droughts, floods and windstorms, which placed a high social and economic cost on the country’s economy and people's livelihoods (Kim et al., 2014). Due to the geographical area, the incidence of floods and droughts in Cambodia is diverse. While floods affect lowland areas, droughts occur elsewhere across the country (GSSD, 2017). Rising temperature and changing rainfall patterns have also had high impacts on Cambodia’s economy. Since 1960, the average temperature in Cambodia has increased by 0.8oC, at a rate of approximately 0.18oC per decade (World Bank, 2019c). The increasing rate is fastest in the dry season (December to February and March to May), increasing 0.20 - 0.23°C per decade. In the wet seasons (June to August and September to November), the increasing 45 rate is slower, increasing only 0.13°-0.16°C per decade (World Bank, 2019c). Cambodia’s Climate Change Strategic Plan report predicts that the temperature in Cambodia could continue rising between 0.13°C and 0.36oC per decade in the future (NCSD & CCCA, 2018). Rising temperature is likely to damage the productivity in agriculture, fisheries and forestry, and decrease labour productivity in most sectors. Every 1oC rise is predicted to cause rice yields to decline by 10% (NCSD & CCCA, 2018). Rainfall patterns in Cambodia are not clear, with some regions experiencing increases while other regions are experiencing decreases (World Bank, 2019c). In the mountainous areas, rainfall has increased its average in the wet season and decreased in the dry season. The changing rainfall patterns has led to increased flooding, droughts and storms, which reduced productivity in agriculture and fisheries (NCSD & CCCA, 2018). In the past 20 years, floods account for 62% for the losses of agriculture, forest and fisheries productivity while droughts account for 36% (GSSD, 2017). Under the future climate conditions, floods and droughts have also been predicted to have huge impacts on most of Cambodia’s agricultural lands. Floods and droughts will reduce the growing period of farming areas to less than five months per year, which could affect the agricultural yields (GSSD, 2017). Among the natural hazards, floods and droughts have been seen as the most disastrous phenomenon in Cambodia. Floods in Cambodia occur frequently and extensively. 4.5 Climate change, floods and droughts in the context of Cambodia The Mekong River, Tonle Sap Lake, tributary flash floods, urban flooding, and failure of structures such as protection dams and water storages are the source of floods in Cambodia (KEI, 2018). Current severe floods in the Mekong River affected Cambodia in 1978, 1991, 1994, 1996, 2000, 2001, 2002, 2011, and 2013. The flood that occurred in 2000 was reported the worst in more than 70 years and the worst in Cambodia’s history (KEI, 2018) — then followed by the flood in 2011. The 2000 and 2011 floods caused thousands of people to evacuate, killed hundreds of people, and caused considerable damages to rural and urban infrastructures in the Tonle Sap and Mekong floodplains (Kim et al., 2014). In 2009, Cambodia also experienced flash floods following heavy rainfall during the wet season and tropical storms, affecting 180,000 households and causing 43 deaths and many injured (USAID, 2019b). 46 Droughts in Cambodia frequently correlate with crop productions because most of the agricultural production mainly relies on rainfall (KEI, 2018). Over the last 25 years, Cambodia has experienced a significant number of various drought events, which occurred in 1986–1987, 1994, 1997–1998, 2001, 2002, 2004, 2005, 2009, 2011, 2012, 2014, and 2015 (KEI, 2018). In 1997 and 1998, Cambodia was reported to have food shortages because of prolonged droughts (Pech, 2015) followed by consecutive prolonged droughts in 2001, 2002, 2004, 2005, 2011, and 2012 (KEI, 2018; Nguyen & Shaw, 2011; Reuy, 2012). The drought that occurred in 2002 was considered the most severe drought since it affected more than 2 million people, damaged more than 100,000 hectares of rice fields, and led to only 35% of the agricultural land being planted with rice (Nguyen & Shaw, 2011). Then, the drought in 2004 was ranked the second-worst drought in the history of Cambodia. It destroyed cultivated lands and affected food security, including food reserve and food accessibility for poor households over the year (Nguyen & Shaw, 2011). In recent years, droughts in Cambodia have become more extreme. For instance, the Cambodian Prime Minister declared the 2016 drought as the country’s worst disaster in 100 years (UNDRR, 2019). It affected all the provinces and cities in Cambodia. It was estimated that at least 2.5 million people were affected by this severe and prolonged drought (KEI, 2018). 4.5 Climate change, floods and droughts in the context of Cambodia In 2019, severe drought and a rise in high temperatures affected more than 20,000 hectares of rice fields in 13 provinces across the country (UNDRR, 2019). 4.6 Climate change policy in Cambodia The Cambodian government and other development agencies have put a lot of effort into combating the effects of climate change. The Cambodian government recognises the issues of climate change and is fully committed to the global efforts to address climate change, at both national and international levels. At the international level, Cambodia signed the United Nations Framework Convention on Climate Change (UNFCCC) on 18 December 1995. In 2002, Cambodia ratified the Kyoto Protocol, which entered into force in February 2005 (Ministry of Environment, 2006). Both treaties (UNFCCC and Kyoto Protocol) primarily aim to steady greenhouse gas concentrations in the atmosphere at a level that might prevent hazardous human 47 involvement with the climate system (GSSD, 2015). The signing and ratification of the UNFCC and the Kyoto Protocol by Cambodia was evidence of its commitment to fulfilling the responsibilities under the Convention. It also indicates that Cambodia is concerned about the impacts of climate change on its people and on their survival and development (GSSD, 2015). Cambodia also started to develop various climate change-related policies at the national level after signing the two treaties. In 2003, the Ministry of Environment founded a separate office titled “Cambodia Climate Change Office (CCCO)”, which was responsible for all activities related to climate change in Cambodia (DCC, 2019). In 2009, this office was upgraded to the Climate Change Department of the Ministry. Its mission was to contribute to sustainable development under climate change conditions and according to the policy of the Cambodian government (DCC, 2019). Having acknowledged the negative effects of climate change on social and economic development, the environment and livelihoods, especially for poor rural communities, the Ministry of Environment collaborated with other government ministries and organisations, and international donors and partners to develop the National Adaptation Programme of Action to Climate Change (NAPA). The NAPA was then sanctioned by the Council of Ministries in 2006 (Ministry of Environment, 2006). The NAPA was developed in line with Cambodia’s development objectives, and its crucial goal was to provide a framework to guide the coordination and implementation of adaptation plans through a participatory method and to build collaborations with other relevant environment and development programmes (Ministry of Environment, 2006). More specifically, the NAPA’s objectives were to understand the key features of climate hazards, and existing local communities’ adaptation strategies, then identify appropriate adaptation responses to climate change (Ministry of Environment, 2006). 4.6 Climate change policy in Cambodia Furthermore, in 2013, the Cambodian government issued a Cambodia Climate Change Strategic Plan 2014–2023 (CCCSP). The CCCSP was developed with contributions from various organisations such as national ministries and agencies, development partners, NGOs, academia, and private sectors (Royal Government of Cambodia, 2013a). This CCCSP was the first all-inclusive national policy document to respond to climate change issues. Its primary purpose was to reduce climate change vulnerability on vulnerable groups and ecosystems; to promote low-carbon development and 48 technologies through green development; and to disseminate education and participation of people in climate change adaptation activities (Royal Government of Cambodia, 2013a). In the meantime, the Cambodian government also issued another national policy named “Green Growth” and the National Strategic Plan on Green Growth 2013– 2030. The National Policy on Green Growth was created to improve people’s wellbeing and livelihoods in harmony with ecological welfare through green growth, based on a green economy, environment protection, social safety nets system and maintenance of national and cultural identity (Royal Government of Cambodia, 2013b). Besides the national policies, many climate change adaptation-related projects have been implemented by local and international NGOs in Cambodia. Nevertheless, Cambodian people, particularly rural communities, have still been the most vulnerable to the impacts of climate change. This is because there are some challenges that have rendered the implementation of climate change policies and projects in Cambodia ineffective. Those challenges include budget limitation for project implementation, lack of human capacity, limited reliable data and research related to climate change, and lack of technology transfer capacity (Un, 2018). Chapter 5: Perception of Climate Change in the Tonle Sap Lake region Chapter 5: Perception of Climate Change in the Tonle Sap Lake region 5.1 Introduction The purpose of this chapter is to explore the Tonle Sap Lake communities’ unprompted perceptions and feelings about the weather that is happening in their region. This chapter will begin by exploring the local people’s experiences of changing weather patterns in the studied area. The chapter will also investigate the other faces of climate change, such as droughts, high temperatures and floods. The investigation compares and contrasts the local people’s experiences of these natural disasters in the present day to those of past years. The data was collected from the interviews and observation during field research. 4.7 Conclusion This chapter described the geographical area, population and climate of Cambodia. It then provided a brief outline of Cambodian agriculture and livelihoods. Additionally, it investigated climate change, floods and droughts, and their impacts on people’s livelihoods, environment and county’s economy. Lastly, it discovered the existing climate change policies at both international and national level that have been developed by the Cambodian government and development partners. The current climate change policies and climate change-related projects, which have been implemented in Cambodia, still have some challenges. Rural communities, in particular, yet have been seen as vulnerable to the effects of climate change. More research regarding the impacts of climate change on rural communities and local adaptation practices need to be studied in order to contribute to climate change policy. 49 5.2. Local perception of changing the weather as a result of climate change Climate change has many faces such as high temperatures, storms, floods and droughts. People perceive it in a number of different ways. According to the data from the interviews, most participants in the region reported that the weather in their communities has changed. In general, most responses to the question about climate reflected negative feelings. The majority of the respondents said that the weather in their region has become more irregular. The irregularity of weather has been noticed since 2016 or 2017 when it became hotter and the two main seasons in Cambodia have become abnormal. The participants have also noticed that climate change reflects changes to the natural rainfall pattern in the last few years. The wet season that is supposed to have a high rainfall has been drier than normal causing a shortage of water. The dry season that used to have a short period of rainfall has become completely dry. As mentioned by a farmer in Santey, a farming village: “The weather is now changing. It is not regular. Sometimes, it rains at the beginning of the year (wet season) and dries at the end of the year. Sometimes it is dry at the beginning of the wet season and rains at the end of the season. This year, it has been dry since November 2018. Sometimes, when it rains too much, we cannot do anything. If we want to throw rice seeds in the rice field, and there is too much rainfall. It 50 damages our seeds as there is too much water in the field. Sometimes, when we need water for our rice farming, there is no rainfall. It is very different from before when the rainfall was regular, and it was easy for us to do rice farming. In the past, there was enough water in the irrigations and natural lakes, and it was so easy for dry-season rice farming as well.” (Respondent ST-FA02) Regarding irregularity of weather, some participants in Mukwat village (fishing village) have witnessed the water-rise pattern in the river and the lake has changed, particularly this year. They mentioned that the late water rise in the river was a result of the weather change. Respondent MW-FI07 remarked, “Flooding (the rise of water in the lake and tributaries) now starts to rise very late. In the past, it started to rise from mid-June. 5.2. Local perception of changing the weather as a result of climate change Before June, there was abundant rainfall, and then the water started to rise. Now (late June), the lake (Tonle Sap Lake) and tributaries are still arid.” During the interviews, participants were also asked whether they had other feelings about the weather that they have experienced. Most of them said that the weather has become hotter in the last few years. Some participants believed that the irregularity of weather pattern was a cause of rising temperature, irregular water rise in the lake and tributaries, and prolonged drought. One fisher from the fishing village mentioned that: “I noticed that the weather is changing. Sometimes, it is too hot, and the rainfall is not regular. I have also noticed that the weather has become hotter in the last five years. However, this year, the weather is extremely hot. As you can see in the lake and tributaries, there is no water. There is only mud. The flood is also irregular. Sometimes, it is low, and sometimes, it is high.” (Respondent MW-FI04) 5.3 Local perception of droughts and high temperature Based on the analysis of the interviews, all participants described that they were experiencing severe drought this year (2019). The participants noticed that drought was not something new to them, and it had already occurred for several years. However, the 2019 prolonged drought was the most critical occurrence they had encountered. As mentioned by Respondent ST-FFA02, “Drought has happened for a few years already. 51 But this year, it is the most serious problem for us. In previous years, it was not too severe, and we could still do rice farming because there was still water to do so.” The participants also said that the prolonged drought has caused the riverbeds, natural and man-made ponds, wells and other water reservoirs to become completely dry. Besides this, most participants raised a concern about the extreme temperature that was happening during the drought. Some of them noticed that the temperature in 2019 was the most extreme they had ever experienced. One individual said that the temperature during the dry season was unprecedented. Respondent MW-FA03 remarked, “I find it is extremely hot and dry this year. It is way hotter than it was before. I think the temperature was 42 degrees Celsius during Khmer New Year (April). I have never felt this extreme heat before." Remarkably, another individual mentioned that the high temperature that happened three or four years ago was even more extreme than this year (2019). Respondent MW- FI07, "I noticed that it started to become hotter and dry since a few years ago, from 2015 or 2016. That year was too dry. It was even drier than this year, and the lake was extremely shallow. Since then, there has been less rainfall and drier.” Nevertheless, the majority of the participants have witnessed the weather becoming hotter and drier since 2015. But the temperature in 2019 was the most extreme, and it has led to water scarcity for the local people in the villages. Water scarcity, as a result of prolonged drought, was seen as the most critical issue for the local people in the two villages who rely on fishing and rice farming as their main sources of income. According to most respondents, the extreme temperature in 2019 caused the most severe shortage of water, which they had never experienced before. 5.3 Local perception of droughts and high temperature They mentioned that in previous years, there was still enough water for them to use for their daily consumption, rice farming, vegetable gardening and livestock despite the drought. In the two studied villages, there were several natural and human-made ponds and reservoirs, which the local communities could use all year round. The participants explained that in the past, the water in the natural and human-made ponds could be used until April or May after the water in the lake and tributaries started to recede in December and January. Then in May and June, which is the beginning of the wet season, there was rainfall again. The people have experienced and adapted to this cycle for their entire lives. However, in 2019, the participants said that they had experienced water shortage since March or 52 April. Some of them even noticed that there had been only two rainfalls from September 2018 until late June 2019. April. Some of them even noticed that there had been only two rainfalls from September 2018 until late June 2019. “In the past, we didn’t lack water although there was drought. We used the ponds in our villages until Khmer New Year (April). Then in May, it started to rain when we had more water in the ponds and tributaries. I noticed this change of drought since 2005. Then it started to become slightly and gradually worse until now. For instance, in the past, we could use the water from the ponds for six months during the dry season. Later on, we could use it for only three months.” (Respondent MW- FA03) The water scarcity in 2019 was the most burning issue for the local people in the region. Most participants in the farming village mentioned that they had to go further into Tonle Sap Lake to carry water for their daily consumption and livestock raising. The distance from the village to the lake is approximately seven kilometres. For better-off households, they bought water from the water station located in another village. As mentioned by Respondent ST-FA02 in this farming village, “I have never seen people in this village buy water from others like today. 5.3 Local perception of droughts and high temperature This is the first time that I see people are doing it.” Likewise, the participants in the fishing village (the village which is located on a tributary and is adjacent to the lake) mentioned that all the natural and human-made ponds, wells, and tributaries were completely dry so people had to buy water from the water station for their daily consumption. Respondent MW-FA02 from this fishing village said, “It is too dry this year. All the ponds and wells in the village are completely dry. Some people have to buy water from others (water station).” 5.4 Local perception of floods Flooding is considered one of the most dangerous natural hazards since it causes damage to infrastructure, people’s livelihood and food security, and even endangers people’s lives. Flooding in the region or Tonle Sap floodplain, however, is considered as the normal rise of water level in the lake and its tributaries following the monsoon rainfall during the wet season, from May to October. 53 During the field research, the participants were asked how they perceived flooding in their areas as a result of climate change. 80% of the participants in Mukwat village (fishing village) defined flooding as a normal increase of water level in tributaries and the lake during the wet season. The participants mentioned that flooding is something that they have adapted to as it happens every year. Based on the observation, due to the high level of water in the tributaries during the wet season, the people in this village built their houses on very tall wooden stilts. These very tall stilted houses sit far above the water level during the dry season, but they are only a few centimetres above the water in the wet season. During the field research, the participants were asked how they perceived flooding in their areas as a result of climate change. 80% of the participants in Mukwat village (fishing village) defined flooding as a normal increase of water level in tributaries and the lake during the wet season. The participants mentioned that flooding is something that they have adapted to as it happens every year. Based on the observation, due to the high level of water in the tributaries during the wet season, the people in this village built their houses on very tall wooden stilts. These very tall stilted houses sit far above the water level during the dry season, but they are only a few centimetres above the water in the wet season. However, two local authority participants, said that flooding was not a normal situation in their community when there was a sudden rise of water in the tributaries. They mentioned that it was also a severe problem when it occurred. Both of them remarked that flooding had caused damage to people’s property and endangered people’s lives in the past in their community. “It (flooding) is not too normal. It affects people’s lives, as well. If it is very severe, it floods the people’s houses and affects their living. 5.4 Local perception of floods In 2011, the flood was very severe and destroyed people’s houses and we had to evacuate them to a safe place such as the pagoda (where it was built on high land). Generally, flooding is not considered as bad as drought in our area. However, it causes negative impacts as well, especially when it happens suddenly, and people have not prepared for it. For those whose houses are not tall enough, flooding affects them, and they have to move to a safe place. It causes hardship for them to make a living as well since they cannot do any livelihood activities during severe flooding. If it is just a normal rise of water in the lake and its tributaries, it is fine.” (Respondent MW-VH06) In the farming village, where flooding does not occur regularly, most participants had a similar perception of floods. They said flooding was not a critical issue for them as it did not occur frequently, and if it occurred, it did not last long. Floods usually lasted a few days or a week and then they started to recede. However, two individuals stated that people in this village used to be affected by floods in 2011 and 2017 when they were severe and sudden. They explained that the floods in these two years destroyed roads, 54 affected some people’s houses and caused damage to their vegetable gardens. Nonetheless, the 2011 and 2017 floods were not extremely severe since they lasted less than two weeks. Even though all participants recognised that floods were one of the natural hazards that could affect their livelihoods and infrastructure, they did not show serious concern about them. There were some reasons that the participants were not worried much about floods in their communities. In the farming village, according to the interview with the local authority, the 2011 flood was severe because the canals in the village were too small and the water could not flow quickly. Then, since 2017, the canals were restored and expanded in order to release water from the village if it flooded. In this village, seasonal flooding happens every year during the wet season. Typically, it only flooded rice fields, but not the village. Having experienced the seasonal flooding (rise of water level from the lake), the farmers in this village used a flood-tolerant rice variety to cope with the floods. 5.4 Local perception of floods Likewise, in the fishing village, where flooding was a normal phenomenon happening every wet season, the people built their houses on tall stilts in order to prevent the impacts of floods. 5.5 Discussion The study reveals that all respondents have noticed the changes of weather pattern in the last five to ten years. They have all experienced increasing average temperatures and decreasing average annual rainfall. Concerning the cause of weather changes, nearly half of the respondents recognised that climate change was a result of human activities. They believed that deforestation was one of the leading causes of climate change. They witnessed that the climate had changed since more people had cut down flooded forests and cleared them for farmlands. They mentioned that the destruction of flooded forests in their communities has led to severe floods and droughts. The participants stated that flooded forests in their communities provided several advantages to the community people such as fish spawning zones, fish habitats, preventing big waves when floods happen, and helping prevent severe droughts. These participants compared the droughts and floods that happened nowadays to the past when the flooded forests were well maintained in their communities. All these participants claimed the floods and droughts that occurred in the past ten years were not as severe as they are in the present day. 55 However, other respondents believed that climate change is a natural process that occurs without human intervention These participants claimed that floods and droughts have become gradually severe due to the process of nature. The study indicates that local communities had a clear perception of the weather changes, floods and droughts, although they did not comprehend the complexities of the phenomenon. Moreover, the local communities in the region had different perceptions about the phenomenon even though they shared the same culture and background. The local perceptions could be influenced by their experiences, media, and taking part in NGOs’ and local government’s development programmes. However, personal experience and observation were found as the key influences on the participants’ perception of climate change. Even though the local knowledge about climate change is limited and unscientific, the community people were well aware of the impact climate change had on their daily lives. Based on their experiences and observations, the local people could judge that the extreme temperature, floods and droughts were the result of climate change. They even noticed that the phenomenon has become more severe in recent times. This finding is aligned with the previous study by (Thomas, 2017). 5.5 Discussion Thomas, 2017 argues that extreme weather-related events such as floods, droughts, heat waves and storms, consequences of climate change, have enormously increased in the last four decades. The IPCC (2018), OECD (2016) reports and Reid (2014) also indicated that the poor communities in developing countries who rely on agricultural livelihood and fisheries were most affected by climate change. Furthermore, the local people recognised that human actions, particularly deforestation, was the cause of climate change. Based on observation, the local communities acquired the knowledge of causes of climate change through their involvement with NGOs’ development programmes, media (TV and Facebook), and peers. Since deforestation has been the main issue for both communities and the country as a whole, the knowledge about deforestation, especially the impact of deforestation, has been shared widely. In any case, this finding supports the previous research of Wuebbles (2018), which indicates that land-use change through deforestation is one of the factors that has caused the increasing temperature in the atmosphere. 56 However, due to the limitation of the local knowledge, the local communities in the region were not aware of other factors that caused extreme-weather events such as high temperature, floods and droughts. According to Van Aalst (2006), Boyd (2014), and Morecroft & Cowan (2010), besides deforestation, the industrial revolution, which contributes greenhouse gas and carbon dioxide (CO2) in the atmosphere, has caused global warming. Global warming and changing weather result in floods and droughts. According to the study, the communities were more concerned about the prolonged drought than flooding since it had severely affected their lives and livelihoods. The people were only aware that the unpredictable weather and abnormal rainfall were some of the causes of the prolonged drought. They noticed the delay of rainfall in the wet season had made the droughts worse. However, the current study about the impacts of hydropower dams in the Mekong River has been blamed for the unprecedented severe drought in the Tonle Sap Lake. Fawthrop (2019, August 26) and Sasipornkarn (2019, August 16) reveal that a serious shortage of rainfall due to the El Nino, a meteorological phenomenon in the Pacific Ocean, has affected the climate all over the Pacific basin. In the meantime, the operations of hydropower stations in China and closing of floodgates of a hydropower dam in Laos have made the drought in the Mekong River and the Tonle Sap Lake worse. 6.2 Impacts of floods 6.2 Impacts of floods 5.6 Conclusion This chapter has indicated that the local communities in the region have observed and experienced the changes of weather in the last few years. The local communities have demonstrated that they were well aware of the climate change based on their observations and perceptions. According to the participants, in the last few years, the climate has become more extreme, leading to severe floods, intense temperature and prolonged drought. Most participants have only witnessed these changes of weather in recent years when they have become increasingly more apparent and severe although climate change has happened over a considerable period. Even though flooding is one of the most devastating natural hazards, most participants in the region did not show major concerns about it because flooding is a common phenomenon in the Tonle Sap floodplain. 57 6.1 Introduction This chapter investigates the impacts of floods and droughts on communities’ daily livelihoods in the Tonle Sap Lake region. Based on the findings from my field research, the impacts of floods and droughts are categorised as damage to community property and effects on rice farming, fishing activities, fish farming, livestock raising, vegetable growing, and other alternative livelihoods. The impacts of the floods and droughts were identified by the region’s two villages’ research participants. 6.2.1 Impacts on individuals and community property According to the literature review, floods have had a great impact on people and their property. Based on the research participants’ interviews, some of them agreed that floods obviously affected them and their properties in negative ways. The participants in the region defined flooding as an issue for them when there was a sudden rise of water level in the lake and its tributaries, following heavy rainfalls and rising water level in the Mekong River. They also considered flooding as extreme (extreme flooding) when the water level in tributaries and the lake was higher than the water level of the regular seasonal flooding during the wet season. Some participants mentioned that when these two types of flooding occurred, they destroyed people’s houses and other property such as boats and fishing equipment. Some said that severe flooding caused great impacts on the community, especially poor households since their houses were not tall enough to deal with the phenomenon. These people had to evacuate from their houses to stay at a safe place such as a pagoda that was built on high land in the community. The participants added that some people had to build a temporary floor above the normal floor in their houses to deal with the severe flooding. Extreme floods destroyed not only communities’ houses but also damaged their fishing gear. According to some participants, big waves, strong winds and water hyacinth caused damage to their fishing gear. Strong winds and big waves often occur when the 58 water level is very high during the wet season. The strong winds and big waves hit and cause communities’ fishing gear such as cylindrical fish traps to collapse. Massive water hyacinth that floated from tributaries into the lake also destroyed their cylindrical fish trap and gillnets. The local authorities in the studied village said the flooding that happened in 2011 was extremely severe. It even killed some people in the village. Other participants also stated that the extreme flood caused risk to their lives when they went fishing in the lake. They explained that big waves and strong winds could collapse their boats and endanger their lives. They added that they were afraid of going fishing in the lake when the water level was too high. Instead, they fished in tributaries and flooded forests near the lakeshores. 6.2.2 Impacts on fishing activities and fish farming Extreme floods did not only affect communities’ property but also impacted on fishing activities. Poor households that relied on small-scale fishing activities as their main source of income were most affected by extreme floods. These households used only small-scale fishing gear such as cylindrical fish traps and gillnets for their fishing activities. The participants reported that when the water level was too high, they had difficulty using cylindrical fish traps and gillnets to catch fish. They added that these two types of fishing gear could only be used within three to four metres deep in the water. Respondent MW-FI05 mentioned, “When there is too much water, bushes, forests and everything is flooded. It is hard to catch fish. As I only use gillnet, I cannot catch many fish to eat.” Besides this, the participants also observed that extreme flooding also had negative impacts on fish farming. They mentioned that the water quality was very poor when extreme flooding occurred. The participants added that they noticed the water was smelly and had acid in it. This water caused diseases and killed some of their fish. The participants also said that their fish grew very slowly during the extreme flooding because of poor water quality and diseases. As mentioned by Respondent MW-FI05, “Flooding also affects my fish farming because there is acid water and the water smell badly. It caused diseases and killed my fish. I noticed strange things happened to my fish. When I cut the fish at the bottom, I saw blood balls and the meat was tender.” 59 Another individual described how the extreme flooding affected his fish farming. He mentioned that, when extreme flooding occurred, he fenced the ponds with barrier nets to prevent fish from going out of his ponds. However, big waves and strong winds that often happened during the extreme flooding caused damage to the barrier nets and the fish escaped from the ponds. Another individual described how the extreme flooding affected his fish farming. He mentioned that, when extreme flooding occurred, he fenced the ponds with barrier nets to prevent fish from going out of his ponds. However, big waves and strong winds that often happened during the extreme flooding caused damage to the barrier nets and the fish escaped from the ponds. 6.2.2 Impacts on fishing activities and fish farming Even though flooding did not often occur in the farming village, some farmers stated that extreme floods could cause damage to their rice farming as well. However, most of them said that the damage to their rice farming was not substantial since they used a flood-tolerant rice variety for their farming. Remarkably, one female farmer reported that the flood in 2017 affected her rice farming and killed her chickens. Respondent ST- FA07 mentioned that the rice productivity yield was low after the tide receded, “2017 flood affected my rice productivity. When the flood receded, the rice plants collapsed and then it rained again – too much water, and then it resulted in low productivity. The flood that year also killed heaps of my chicken. It flooded about a month.” 6.3 Discussion The findings from the study show that some local communities in the Tonle Sap Lake region have witnessed negative impacts on their lives and livelihoods from flooding. The impacts generally affected the local communities’ physical assets (i.e., houses, boats and fishing equipment), fish farming and fishing activities. Due to the geographic location, people living in the Tonle Sap floodplain adjacent to the lake, were affected by floods. The study found that extreme flooding in this region is considered a hazard phenomenon for the local people since it flooded the community rapidly without warning. The extreme flooding in the region has caused physical and socioeconomic loss of the local people. As explained by the respondents, extreme floods have destroyed the fishers’ fishing gear, boats and even houses. Poor households had been evacuated from their houses because of the extreme flooding. The extreme floods caused poor water quality for fish farming. They also destroyed the barrier net fences of the fish ponds and fish escaped, resulting in high production losses. The result of this study underpins the previous findings of other scholars that indicate floods have immensely negative impacts on people’s lives and socioeconomic activities 60 such as fishing, farming, transportation and human health (Wongchuig Correa et al., 2017). As stated by Rapsomanikis et al. (2017), 20–300 million people worldwide are affected by floods each year. The vulnerability to flooding has increased, and more property, assets and lives have faced flooding. In the floodplain of northern Italy, severe flood events have been remarkable in the last two decades. From 2000–2014, floods in this floodplain region have resulted in 200,000 dead animals, more than 40,000 people becoming homeless, more than 500,000 people being displaced, 8 people missing and/or dead, and over a billion euros’ worth of damages (including private, public, and industrial properties) (Roder et al., 2017). In Pakistan, between 2010 and 2011, more than 2,500 people died while 27 million others were affected by severe floods. The floods also killed livestock and destroyed agricultural lands, resulting in billions of US dollars loss (Smakhtin et al., 2014). Likewise, floods in South Africa caused damages to infrastructure, buildings and sewage-disposal systems. Floods in this country also damaged agricultural production and caused food insecurity for the local communities (Musyoki, Thifhulufhelwi, & Murungweni, 2016). 6.3 Discussion There are several forms of rice cultivation in the region depending on the location of the communities in relation to the huge floods of the lake (Varis et al., 2006). The rice cultivation practices in the region are deep-water rice, rain-fed lowland and/or irrigation receding rice, and dry season rice. Generally, floods are considered natural hazards that cause damages and loss to life, property, and infrastructure as well as disruption to public services. Nevertheless, floods in the Tonle Sap Lake provide several advantages for the local people such as fisheries resources and agricultural production. 6.3 Discussion Although some respondents in the floodplain community claimed that extreme floods have caused damage to their physical assets, fishing activities and fish farming, the damage was minimal for most of the local people in the region. Extreme flooding did not often occur in this region. Most importantly, the local people living in the Tonle Sap floodplain have adapted to the seasonal flooding very well. Most respondents, however, reported that flooding in their community was not a major issue for them. The respondents referred to the regular floods that happen during the wet season. Nonetheless, the periodic flooding in the region is an advantage for the local people who rely on agricultural and fishing activities as their main livelihood. The result of the study indicates that most respondents were pleased with floods since aquatic resources such as fish, plants and other aquatic animals are more abundant in the lake during flooding. According to Lamberts (2001), based on the amount of caught fish, the Tonle Sap ecosystem is one of the most productive inland waters in the world. During the wet season, the floodwater from the Mekong River flows into Tonle Sap Lake, making the lake four times bigger and increasing the water depth from 1 metre up to 10 metres (Nuorteva et al., 2010). Then fish from the Mekong River migrate into Tonle Sap Lake making fish more abundant in the lake and its tributaries (Lamberts, 2001). 61 Flooding in the Tonle Sap Lake is not only beneficial for fishing activities, but is also an advantage for agricultural activities, particularly rice cultivation. The local people in this region could do rice cultivation two to three times because of the floodwater. There are several forms of rice cultivation in the region depending on the location of the communities in relation to the huge floods of the lake (Varis et al., 2006). The rice cultivation practices in the region are deep-water rice, rain-fed lowland and/or irrigation receding rice, and dry season rice. Flooding in the Tonle Sap Lake is not only beneficial for fishing activities, but is also an advantage for agricultural activities, particularly rice cultivation. The local people in this region could do rice cultivation two to three times because of the floodwater. 6.4.1 Impact of droughts on rice farming Rice farming is the communities’ main livelihood in the farming village Santey. People in this village rely heavily on rice farming as the primary source of income and food security. However, droughts have been felt to have a huge negative impact on rice farming in the community. All participants reported that severe drought in 2019 caused serious water shortages for their rice farming, resulting in the failure of rice growth. The participants mentioned that they were facing an unusual lack of water in the community, and it caused their rice to fail to grow. The participants stated that all the natural and human-made ponds and reservoirs that they used to store water for their rice farming were completely dry. According to the participants, a large number of farmers used the IR66 rice variety, introduced by the Provincial Department of Agriculture, Forests and Fisheries, for their farming. This rice variety was known as dry-season irrigated rice and receding rice, and it was used in rain-fed and irrigation-receding conditions with a short maturity. Its growth duration was only three to four months. Farmers planted this rice variety during the dry season when the water had receded. The farmers started to grow this rice variety in April and expected to harvest in late July or early August. However, the participants reported that the severe lack of water in the rice fields had delayed their rice growth. 62 They also described that the severe drought had affected their rice productivity from the previous year, 2018, because there had been no rainfall since last September. As one farmer said (Respondent ST-FA03), “Normally, the rice productivity was three tons per hectare, but this year I got even less than one ton.” One farmer said that water scarcity and pest increase, resulting from prolonged and severe droughts, had caused damage to his rice farming. He added that his rice productivity this year was lower than the productivity in previous years. “Because of drought, we don't have enough water, and it makes rice fail to grow. I pumped water into the rice field, but it dried out because it is too hot. Another impact of the drought is increasing pests in the rice field. I tried to use pesticides, but it didn't work because there are too many pests. The more pesticides I used, the more pests increase. 6.4.1 Impact of droughts on rice farming And rice productivity is lower than before.” (Respondent MW-FA03) Due to low rice productivity, the participants were also worried about the lack of rice seed that they needed to use for rice farming in the following year. Normally, farmers keep some rice that they get from harvesting to reserve as seeds for the next season rice planting. However, since their rice productivity was not high enough, it would be a challenge for them to reserve seeds for future use. One participant complained that he had used all the seeds for rice farming this year, and he was certain that he would not have them for use in the following year because his rice productivity was very low, and there was not even enough to provide food for the entire year. The drought did not only impact on rice productivity but also reduced the duration of rice farming per year. Some participants said that they usually did rice farming twice a year. The periods of rice farming in this community were from April to August, and from August to November. Since the drought was too severe, in 2019, they could do rice farming only once per year. 6.4.2. Impacts of droughts on fishing activities Fishing is the most important livelihood option in the fishing village. People in this village rely heavily on fishing activities as the primary source of income. However, all 63 participants in this village reported that their fishing activities had negatively been impacted by severe drought. Prolonged droughts have caused the lake and its tributaries to become very shallow and muddy. High temperatures also caused water to become hot. Furthermore, the shallow lake and tributaries made it difficult for the community people to use their boats for fishing in the lake. Some of the participants said that muddy lake and tributaries also destroyed their fish traps as they got stuck in the mud and could not be pulled off. The participants also noticed that the prolonged drought had made it hard for them to catch fish when there was little water in the lake and its tributaries. They described that the high temperature and water shortage in tributaries and the lake have caused fish less abundant. As mentioned by Respondent MW-FI05, “The water is too hot, and I can catch very few fish. Sometimes, I catch nothing.” Additionally, the participants observed that this situation had happened for a few years already. They mentioned the fish had become less abundant when the water level in the lake and tributaries had become gradually lower since 2015 during the dry season. They added that in the previous years, when there was enough water during the dry season, they could catch enough fish for sale and household consumption. However, since the water level was too low, and all the tributaries, ponds and the lake were too shallow, they could not catch as many fish as before. One participant described that in the past he could catch up to 10 kg of fish per night in the dry season when there was enough water in the tributaries. But this year (2019), he could catch only 1 to 3 kg of fish per night. He added that sometimes, he caught fish less than 1 kg. Additionally, the participants observed that this situation had happened for a few years already. They mentioned the fish had become less abundant when the water level in the lake and tributaries had become gradually lower since 2015 during the dry season. 6.4.2. Impacts of droughts on fishing activities They added that in the previous years, when there was enough water during the dry season, they could catch enough fish for sale and household consumption. However, since the water level was too low, and all the tributaries, ponds and the lake were too shallow, they could not catch as many fish as before. One participant described that in the past he could catch up to 10 kg of fish per night in the dry season when there was enough water in the tributaries. But this year (2019), he could catch only 1 to 3 kg of fish per night. He added that sometimes, he caught fish less than 1 kg. Another individual consistently talked about the comparison of fish caught between the present day and in the past, before 2015. He mentioned that he could earn more in the past by catching fish with just one piece of fishing equipment, a cylindrical fish trap. As Respondent ST-FA05 remarked, “Before 2015, when there was enough water in the canals and streams, I could earn from catching fish approximately KHR500,000 within three days. I used only one cylindrical fish trap. But from 2018 until now, I could earn only about KHR300,000 within three to four days although I used more cylindrical fish traps.” Less fish abundance, resulting from prolonged drought and high temperature, has made the community people lose their profit from fishing. Since fishing is the primary source of their income, people spend more time on fishing activities than on other livelihood 64 activities. According to the interviews, some participants explained that the impact of prolonged drought had affected their income markedly. The participants described that they had to buy gasoline for their boat to fish in the lake. Their boats consume a lot of gasoline compared to motorbikes and even cars. Thus, they spend a lot of money on their boats. However, since the fish are less abundant during the drought, they could not catch many fish. Sometimes, they could not even earn enough money from catching fish to pay for gasoline. As mentioned by Respondent MW-FA03, “During the drought, there are fewer fish in tributaries and the lake because of water shortage and high temperature. When I go fishing in the lake, I hire some people to help, and I pay them KHR30,000–KHR40,000 per day. I can only earn only KHR60,000–KHR70,000 from fishing. 6.4.2. Impacts of droughts on fishing activities After paying for gasoline, I earn nothing.” Likewise, a local authority participant explained that because of the severe drought, people in the community lost their profit since they could not catch many fish for selling and household consumption. Since fishing is the most essential livelihood activity, some people had to work for others to earn money to buy gasoline for their boats to fish in the lake. He added that the people kept fishing although they could not catch many fish every day. At least, they sometimes could catch enough fish for eating. Remarkably, some people emphasised that severe drought is not the only cause of fish population decline. They mentioned that they witnessed the fish population declining for several years already. One individual said that deforestation is one of the main causes of fish decline in the lake and tributaries. He explained that flooded forests were significant for fish spawning and fish habitats. However, in the last few years, more and more people have cut down the flooded forests and cleared them for agricultural farms. Dozens of hectares of flooded forests had been cleared every year. Furthermore, other participants argued that illegal fishing activities and population growth in the Tonle Sap Lake also caused fish-population decline. They explained that the growing population in the region demanded more natural resources, especially fish because fish is the most essential food for their daily consumption and primary source of income. Since fish is the main source of income for the people in the region, people were very driven to catch more fish in the lake and its tributaries. Consequently, most fishers have used illegal and destructive fishing equipment to catch fish. Some of them even fished in fish preservation zones. However, one participant claimed that he did not 65 fish in the fish preservation zones because he was afraid of the Fisheries Administration Officers who might collect and destroy his fishing equipment. fish in the fish preservation zones because he was afraid of the Fisheries Administration Officers who might collect and destroy his fishing equipment. 6.4.3.1 Impacts on livestock raising Some people’s cows even died when they stuck there and had not been pulled out.” (Respondent ST-FA02) mud in the lake and natural ponds. So, I need to go there to pull them out of the mud. Some people’s cows even died when they stuck there and had not been pulled out.” (Respondent ST-FA02) All participants who owned cattle, buffalo and poultry claimed that loss of their animals was loss of their money and income. They claimed that they had to buy medicines to cure them. But since their animals could not survive, they lost money buying medicines and money that they could have earned from selling their animals. 6.4.3.1 Impacts on livestock raising Livestock raising in the Tonle Sap Lake region plays important roles for local communities. Local people raise cattle, buffalo and poultry for their subsistence livelihoods, source of income, as labour for rice farming, and as an asset. Nevertheless, animal raising has also been affected by severe and prolonged drought. According to the participants, the severe drought has caused diseases in their cattle and chickens. The participants noticed that high temperature has caused cattle and chickens to get sick and die. They explained that their chickens had become pale and weak when the weather was too hot. Some chickens died after being sick for one or two weeks even though they tried to cure them. They also mentioned that water scarcity had caused difficulty for them in raising their chickens. Some of them had to buy water from the water stations in another village while others had to carry water from ponds or the lake to their houses. As observed, water scarcity is one of the critical issues for their cattle and buffalo raising. The participants said that they lacked water and fresh grass to feed their cattle and buffalo since the weather was too hot and dry. They emphasised that severe and prolonged drought had caused water scarcity and fresh grass in their community. These participants had to take their cattle and buffalo to the lakeshores where there was water and fresh grass for their cattle and buffalo to eat. The participants then left their cattle and buffalo at the lakeshores for one or two months. Without proper care, some cattle and buffalo got sick and died. One participant noticed that his cow got bloody diarrhoea and died after a while. In one case, cattle walked far from the village to find water in natural ponds and streams, and even to the lakeshores. Unfortunately, they got stuck in the mud and could not move. “When it is too dry, my cows have to find water in natural ponds and reservoirs far from the village and near the lakeshore. They stuck in the 66 mud in the lake and natural ponds. So, I need to go there to pull them out of the mud. Some people’s cows even died when they stuck there and had not been pulled out.” (Respondent ST-FA02) mud in the lake and natural ponds. So, I need to go there to pull them out of the mud. 6.4.3.2 Impact on fish farming In the studied communities, fish farming is one of the income sources for local people. Most local people’s fish farming was small-scale, family operated. The severe and prolonged droughts have had negative impacts on fish farming in the communities. Some participants who did fish farming as their secondary livelihood noticed that during the prolonged drought, the water volume in their fish ponds become very low and the water became very hot. The lack of adequate water in the ponds caused an issue for fish farming. The participants had difficulty raising fish during the drought. They said that their ponds needed to have enough water so that the fish could grow healthily. Shallow fish ponds and hot water had caused diseases in the fish. The participants also claimed that the water quality in their fish ponds was very poor. They believed that the poor quality of the water also caused diseases in their fish. Moreover, the participants said that they spent a lot of money buying medicines to cure their fish. Some participants claimed that they could not save all their fish as some of the fish still died even though they had cured them. These participants believed that the severe droughts and high temperature occurring this year, 2019, had caused their fish to die. As mentioned by Respondent MW-FI04, “The weather is too hot. It makes the ponds shallow and the water hot. This causes disease and kills my fish. I spend a lot of money to buy medicines to cure it, but it could not help.” Because of the severe drought, one participant said that she had to quit doing fish farming. She said she used to do cage fish farming. She raised fish in cages in the tributary behind her house. Since the drought had been too severe and there was not enough water in the tributary, raising fish was too difficult. She said the tributary was 67 too dry to raise fish in the cage. She also wanted to switch to raising fish in ponds, but she could not afford it. She did not even have land for ponds. Eventually, she quit raising fish. 6.4.3.3 Impact on vegetables growing Severe and prolonged droughts have also affected vegetable growing in negative ways. All participants were experiencing water scarcity for their agricultural crops and vegetable gardening. The participants indicated that the impact of severe droughts on their vegetable growing was considerable. All participants observed that the phenomenon had delayed growth in crops such as corn, mung beans, eggplants and other garden vegetables. Some stated that the water shortage and high temperature had prevented their crops and vegetables from growing well. As a result, the productivity of crops was very low. “The extreme heat and water shortage this year have affected my crops badly such as mung beans and eggplants. The productivity is very low. For instance, usually I could get 500 kg of mung beans per year. But this year, I got only 200–300 kg. One of my friends who grows beans used eight sacks of seeds over three to four hectares, but he got only seven sacks of total productivity. We did not only lose money, but we also lost our labour and time that we spent on our farms. This was because there had been no rain since we started to grow the crops until the time that we harvested them. The quality of the harvested productivity is also poor. Every farmer who grew beans this year suffered a significant loss. In previous years, they used to get three to four tons of productivity, but now they got only one ton of the total productivity.” (Respondent MW-FI02) Furthermore, the participants complained about the growth failure of their garden vegetables because of severe drought. Vegetable gardens consisting of a variety of vegetables and fruits are one of the subsistence livelihoods for the local people in the farming village. The participants claimed that their vegetable gardens helped them increase the diversity of food available to their households. Nevertheless, their gardens had failed to grow well this year, (2019). The participants indicated that water shortage Furthermore, the participants complained about the growth failure of their garden vegetables because of severe drought. Vegetable gardens consisting of a variety of vegetables and fruits are one of the subsistence livelihoods for the local people in the farming village. The participants claimed that their vegetable gardens helped them increase the diversity of food available to their households. Nevertheless, their gardens had failed to grow well this year, (2019). 6.4.3.3 Impact on vegetables growing The participants indicated that water shortage 68 and extreme heat are the leading causes of dry soil in the community, which made their vegetables and fruits failed to grow. The participants also complained about spending more money on vegetables during the severe droughts. They said that they had to buy everything from the markets, including vegetables that they would not have bought if droughts had not destroyed their gardens. As Respondent ST-FA04 mentioned, “All small herbaceous plants in my garden such as lemon grass, chillies, and other vegetables died because of water shortage. So, we need to buy everything from the market. More daily expense for my family.” 6.4.3.4 Impact on other alternative livelihoods One farmer describes the serious negative effect on his family that the severe 2019 drought had: “All my children work in others’ farms and construction work to earn money for the family. The money that they earn is used for family’s expense and pay back the loan to Acleda Bank. We got a loan from the bank to buy a two-wheel tractor with a cart. We use the tractor for our rice farming and harvesting cassava in others’ farms in a distant village, near Kulen Mountain. We harvest the cassava then we carry it with our tractor to the storehouse, then we can get money from the farm’s owner. Without this tractor, it is hard for us to do so. The more cassava we can harvest and carry, the more money we can get. However, we do not have this work at the moment. Because there is no rainfall for nearly a year, the cassava is not grown well enough, so the farm’s owner stopped hiring us (his children) for a while because they do not harvest and plant it at the moment. So, my children have to go to Siem Reap town to work in construction to earn money since we need to spend every day and pay back the loan to the bank.” (Respondent ST-FA04) 6.4.3.4 Impact on other alternative livelihoods Besides the impacts described above, droughts have also affected other alternative livelihoods of the two studied villages. The alternative livelihood activities of the local people include wage labour and owning grocery stores. Wage labour in the studied communities is defined as selling labour power to other people either inside or outside the village with informal contracts such as working in farms, construction workers, house building, fixing fishing equipment, and working for other fishers who do large- scale fishing. The participants mentioned that severe droughts also have an impact on their alternative livelihood activities such as working for large-scale fishers, selling groceries, and working in agricultural farms. Some participants who worked for large- scale fishers claimed that they were not hired as often as before when the drought was too severe. They explained that the shallow lake and less fish have reduced the time they work for large-scale fishers. The large-scale fishers did not hire them when they could not catch as many fish because they could not make any profit from fishing after paying the hire. Some large-scale fishers reduced the number of working days of the participants. As mentioned by Respondent MW-FI01, “The severe drought obviously affected my other livelihood activity as well. I also work for other fishers who do large- scale fishing. Although they still hire me during the drought, but not every day. But when there is enough water in the lake, I am hired more often.” Additionally, one individual claimed that severe drought also caused an impact on his grocery business. He said that fewer people bought his goods during the drought. Since fishing is the main source of income in this community, people could not earn much money when fish were less abundant. Hence, the people in the community tended to 69 buy fewer goods such as food, drinks, cooking ingredients for their daily use. Respondent MW-FI07 mentioned, “I notice when it is too dry and people cannot make good money from fishing, they don’t come to buy stuff or buy less stuff from my store.” Some participants who worked on others’ agricultural farms also indicated that the prolonged and severe drought had impacted on their work. They consistently mentioned that they were hired less often or stopped being hired because the drought had delayed the growing crops such as corns, mung beans and cassava. 6.5 Discussion The results of this study suggest that severe and prolonged drought has been a critical issue for the local communities in the Tonle Sap Lake region. It has had great negative impacts on local livelihoods such as rice cultivation, fishing activities, fish farming, livestock raising, vegetable growing and other alternative livelihood activities. Rice farming, which is the main local livelihood activity in the land-based community, has been damaged by severe and prolonged drought. Serious water shortage, a result of prolonged drought, delayed rice growth in 2019. All local farmers reported that their 70 rice failed to grow although they used a dry-season rice variety that is believed to be drought tolerant. It was also reported that the rice productivity in the previous year (2018) was very low due to the drought. Likewise, fishing, which is the primary livelihood in the land and water-based community, had also been severely affected by the prolonged drought. Severe and prolonged droughts have caused the lake and its tributaries become very shallow and muddy, and therefore in less abundant in fish. Consequently, the amount of fish catch per household was very low. The study additionally indicates that due to its long period, the 2019 drought was the most severe that the local communities have ever encountered. The drought lasted for almost a year with low rainfall. This prolonged drought has severely affected other livelihood activities in the two communities. High temperature and water scarcity were the biggest impacts on livestock raising, fish farming and vegetable growing in the communities. Regarding livestock raising, high temperature caused diseases in cattle, buffalo and chickens, and some of these animals eventually died. For fish farming, poor water quality and warm water because of the high temperature have caused diseases and killed farmed fish. Besides, high temperature and water scarcity have caused vegetables and crops to fail. Other alternative livelihood activities, in particular, wage labour, was also affected by severe drought. The local people who worked on cassava farms and for large-scale farmers explained how their working days had been reduced because of the severe drought. Those who worked to harvest cassava reported that they were asked to postpone their work since drought had delayed the plants growth. Likewise, the local people who worked for other large-scale fishers were not hired as often to work in fishing activities in the lake. 6.5 Discussion Given the fact that the livelihood activities have been affected by the severe drought, the local people reported that their food security has been threatened and their income has decreased. This result reinforces the findings of previous studies that explored the impacts of droughts on local livelihoods in India, Nepal, and other African countries. The studies in these countries showed that droughts have negative impacts on agricultural production, the main rural livelihood, and threatened their food security. For instance, severe drought in Maharashtra State of India in 2012 impacted greatly on local livelihood (Udmale et al., 2014). The local people were reported to have a 71 shortage of water for their daily consumption and agriculture. The severe drought caused vegetable production to decrease by 11% compared to the previous year, and the inland fishery suffered 16% production loss. In Nepal, agriculture is an important livelihood activity for rural people. Crop farming, including rice, wheat, maize, millet, barley, and buckwheat, and livestock raising form a major way of food security for the rural communities. However, droughts that have happened for several years have made the agriculture in Nepal less stable and less productive (Adhikari, 2018). As stated by the UNDP (2009) report, roughly 90% of the crop or agricultural land loss in Nepal was caused by meteorological events from 1971–2007. Among the different disasters, drought had the most severe impact on agriculture with crop loss accounting for 39% of the impact. Likewise, in Africa, droughts have caused huge losses of crop and livestock production. Severe droughts have caused most countries in Africa to experience rapid declines in yields. Such losses pose a great threat to food availability, rural livelihoods and the overall economy, especially given the important contribution of agriculture to food security and the economies of sub-Saharan Africa (FAO, 2015). 7.1 Introduction Various ongoing changes of weather, particularly floods and droughts discussed in previous chapters, have greatly impacted on local people in the Tonle Sap Lake region. The people in the studied villages have significantly suffered from the extreme events, particularly prolonged and severe droughts that threaten local daily livelihoods. The focus of this chapter is to provide an insight into the everyday adaptation practices used by local people in the region to deal with the phenomenon. The chapter starts by examining the preparation practices that the local people use before floods and droughts happens. Then it looks at how local people use their indigenous knowledge (IK) in adaptation strategies to deal with floods and droughts in terms of preventing and mitigating the impacts of the phenomenon on their daily livelihoods. The local practices as adaptation strategies are divided into two main parts; the first part is the practices that people use to generate income for their households, and the second part is the practices that they use to support their daily survival. Finally, the discussion on IK generation and practices demonstrates how effectively IK is used by local people to deal with floods and droughts. 6.6 Conclusion This chapter has demonstrated that climate change in the past few years has had negative impacts on local livelihoods in the Tonle Sap Lake region. Severe droughts and floods, resulting from climate change, have affected rice farming and fish population, the main sources of the local livelihood and income. Prolonged droughts have also affected the local communities’ secondary livelihood activities, such as vegetable growing, livestock raising, fish farming, and wage labour. These effects have led to food insecurity and income loss of the communities. Hence, climate change has contributed to the changes in rural society and affected the rural economy. 72 7.2 Preparation for floods and droughts This section explains the local practices that are used in preparation of the floods and droughts occurring in their local communities. The practices were developed locally in order to prevent impacts that would cause damage to property and livelihood activities. 7.2.1 Preparation for floods According to the interviews, most participants did not prepare much for floods. The participants in fishing village indicated that flooding was just a normal phenomenon that happens every wet season, and they got used to it. However, the local authorities claimed that although flooding was not a huge issue for the local people in their communities, they did prepare for this phenomenon. They mentioned that the local 73 people always prepared their houses, boats and other fishing equipment by getting them ready to withstand the floods, and how, in the last ten years when the flooding became more severe, people started to prepare and maintain their houses to make them strong enough to deal with strong winds and big waves that occurred during flooding: “If we talk about ten years ago and before that, when the weather was not a problem for them, people tended not to prepare anything for floods, or they prepared their houses a little bit because the forests still existed, and they did not worry much about strong winds and waves. But floods nowadays cause damage to their houses. Therefore, before the water volumes increase (floods), they buy wood to maintain their houses and boats. Secondly, they buy medicines and foods to reserve for family members. They maintain their boats. In case their houses are flooded, they can live on the boats. They also prepare some woods in case the flood is too high. They can use the wood to build another floor, just above the normal flood in their houses to stay for a while during the flood.” (Respondent MW-CC09) Regarding mitigating the impact of floods on their daily livelihoods, some participants claimed that they prepared their fishing equipment such as horizontal cylinder traps and gillnets in advance of the rise in the tributaries’ and lake’s water levels. These two types of fishing gear were claimed to be suitable for catching fish in the wet season. The participants described that before flooding, they bought netting to build more and/or expand the size and length of fishing gear such as cylindrical fish traps and gillnets. As mentioned by one participant (Respondent – MW-FI03), “I prepare a netting and repair the gillnets and other equipment. When there is enough water in tributaries and the lake, we are ready to use the gear to catch fish.” 7.2.2.1 Reserving water in ponds and reservoirs There are two main seasons (dry season and wet season) in Cambodia. All Cambodian people in the country are well aware that there is little or no rainfall in the dry season. Having experienced the scarcity of rainfall in the dry season, all participants claimed 74 that they always prepared for the drought. Most participants stated that having enough water, both for household use and agricultural use, was the most important thing they needed to prepare for. They all claimed that, to avoid water shortage, they dug and restored their wells, ponds and other reservoirs to store water. Some participants dug ponds in their rice fields to store water for rice farming while others restored natural ponds and reservoirs to store water for other daily use such as household use, cattle and poultry raising, and vegetable gardening. In the farming village, most participants who farmed rice twice a year, dug their own ponds in their rice fields so they would have enough water for their rice cultivation. They mentioned that before the dry season, they pumped water from the streams into their ponds to store for use when there was no rainfall and not enough water in their rice fields in the dry season. One participant who had been practising this method for several years believed that there was a cycle of water that he pumped from the pond to his rice field. Respondent STVH06 “I dug ponds in my rice field to store water for use when it is too dry and there is no rainfall. When it is very hot and dry, I pump water from the ponds into my rice field. Then the water dries out from the field and goes under the earth into the pond. Thus, I have water for entire year.” Likewise, the commune councillor in the fishing village indicated that the local people had been practising this method for more than ten years. In the past, people had relied only on the water in tributary and one commune-funded pond. Then the local people started to dig ponds near their houses for easy access after they had experienced a severe drought that happened before 2010. Now there are several ponds in the community. 7.2.2.1 Reserving water in ponds and reservoirs One pond was shared between three or four households, but other households dug their own ponds because of the multipurpose water usage such as household use, growing vegetables, fish farming and chicken raising. It was observed during the transect walk in the fishing village that several households living on the other side of the road (not at the tributary shore) had their own ponds just behind their houses while other households shared the ponds. Some households who did fish farming had at least two ponds. One pond was for fish farming and the other was for household use. Although their ponds were close to their houses, most local communities needed to use pumping machines to pump water from the ponds up to their 75 houses. Carrying water from the ponds up to their houses was not an easy task for the local people in this fishing village because their houses were built on very tall stilts. 7.2.2.2 Selling fish before droughts occur As discussed in the previous chapter, droughts had serious impacts on small-scale fish farming in the studied communities. Having experienced the negative effects of drought on fish farming, the community people conceived a solution that prevented them from losing profit because of fish dying. The local people simply sold their fish before the drought happened. Based on their experience, the local people acknowledged that the drought usually occurred in the dry season between April and June. Normally, the high temperature during April and May could kill farmed fish. Hence, the people started to raise their fish in late June or early July. According to the participants, they had to be cautious with the time they started to raise their fish because they needed to buy small wild fish to feed their farmed fish. They calculated and identified which month the small wild fish were affordable. If the small wild fish were too expensive, they could not make any profit from their fish farming. Generally, these local fish farmers started to raise their fish just before the water level in tributaries and the lake rose. As claimed by the participants, when the water level rose, small wild fish became more abundant in tributaries and the lake. Then, the price of small wild fish was affordable for the fish farmers to buy to feed their farmed fish. The participants also stated that fish raising took approximately eight months for fish to mature. Typically, the farmed fish were fully grown and ready for harvesting in February and March. “To avoid the benefit loss, I have to sell my fish before the drought happens in April, May or June. I start to raise fish from July until March, and I have to sell them in early April before the Khmer New Year (Mid- April). I know about the method based on my experience as the drought usually happens between April and June.” (Respondent MW-FI07) “For fish farming, we know that in March or April, it is very hot. I normally prepare medicines in case the fish have diseases. If the condition of the fish is too severe, I sell my fish before it is too late 76 (before they die) to avoid benefit loss. I know that I don’t have enough money to buy medicines to prevent fish from diseases. So, I need to sell them. 7.2.2.2 Selling fish before droughts occur Some well-off people spend lots of money to cure their fish, but the fish still keep dying. Then, they sell them out.” (Respondent MW-FI04) 7.2.3 Discussion Regarding the preparation for the disasters, the local people have learned how to adapt to changes in weather patterns from the past experiences. The traditional way of rice farming in the studied community relied very much on the availability of rainfall. Hence, the local prediction about the rainfall was significant for the local people in preparing for their rice farming. The participants claimed that they prepared themselves in accordance with the rainfall. The local people observed the rainfall pattern from the late dry season or early wet season and predicted whether there would be enough rainfall for their rice farming. This method has been developed and used since the ancient past. The local people learned about this method from their ancestors and their own experiences. It was reported that there was a religious event during the Khmer New Year in April when local people celebrated and predicted rainfall pattern in the upcoming wet season. The method used in the studied community was similar to the method that the local people in one community in Tanzania used to prepare for their agricultural production. The local people in Tanzania used their IK systems to predict the rainfall pattern. The IK systems of the Tanzanian community people were developed using the combination of culture and belief systems, production systems and connections with the surrounding nature (Mwiturubani, 2017). The local people in Cambodia, however, appeared to rely more on new technology for the weather. As observed, most people in the communities had smartphones, televisions and radios, on which they could easily access the information about rainfall pattern. Concerning the effectiveness of the preparation for the droughts, the local people reported that the method of digging and restoring ponds and reservoirs previously helped them to have enough water for agriculture, livestock raising and household consumption. Nevertheless, the severe drought in 2019 exacerbated the water scarcity in 77 their community. The method of digging and restoring ponds and reservoirs could not provide them enough water for their agriculture, livestock raising and daily household use. This finding aligns with Gyampoh et als’ (2009) previous study on the Offin River basin, Ghana, where they explored how IK was used to cope with climate change. The study reveals that severe droughts and severe water scarcity in the Offin River basin caused a great negative impact on local agriculture and daily lives. 7.2.3 Discussion In order to get water for their daily consumption, the local people had used a traditional way of harvesting rainwater by placing barrels under the roofs of their houses. Then this method was abandoned when the local communities installed wells and boreholes. However, most community people reported that they were not able to harvest enough rainwater due to severe droughts (Gyampoh et al., 2009). Conversely, the method used by local fish farmers in the land- and water-based communities in the Tonle Sap Lake region of selling fish before the drought happened was efficient and practical. The local fish farmers used this method to avoid economic loss caused by drought on their fish farming. 7.3.1.1 Switching fishing equipment during floods As mentioned in the previous chapter, fishing is the most important livelihood activity in Mukwat fishing village. The participants in this village mentioned that they switched their fishing equipment when the water was very high during the wet season because some of the fishing equipment could only be used in certain water conditions. To be able to catch fish, the local fishers switched from using Lob (cylindrical fish trap) to Leu (horizontal cylinder trap for gourami) and gillnets when the water level was high. Some participants said that they still kept using Lob (cylindrical fish trap) during floods, but they needed to expand its size in order to catch fish. However, some participants reported that Lob could not catch fish as much as Leu. Leu (horizontal cylinder trap for gourami and other small fish) was a very popular fishing gear used during flooding. The gourami fish were usually sold to other fishers 78 who used it as bait for their Sai Yern (small vertical cylinder trap for shrimp). Some people used gourami fish and other small fish caught by Leu to make salted and fermented fish paste. This fishing gear could be used for six months from August to January when their village was flooded. The participants claimed that this fishing gear was easy to use and could even catch more fish compared to gillnets. As mentioned by one participant (Respondent MW- FI01), “I switched to Leu (horizontal cylinder trap for gourami) when it floods. I use this gear (Leu) for more than 10 years now. I have also been using gillnets since I grew up. However, Leu is easier to use than gillnet. Using gillnet, you need to collect fish one by one from the net. For Leu, you just need to pour fish into the basket. It can also catch more fish than gillnet.” In this fishing community, shrimp was also one of the most popular aquatic resources that people caught daily. The local people could catch shrimp for the entire year, but they had to change their gear to catch it when the tributaries and the lake were flooded. The local communities reported that they switched to using a Sai Yern in order to catch shrimp during the flood. However, this fishing gear was not affordable for everyone in the community. 7.3.1.2 Switching fishing equipment during droughts Since the impacts of severe drought on the local communities’ fishing activities were too great, most of the common and traditional fishing gear that they used in the dry season was no longer practical. The local communities reported that they could not use their traditional gear because the water was too shallow and muddy and switched to using a Proyong (seine net with lead sinkers) in order to catch fish during severe droughts. According to the commune councillor, the local people made this type of fishing gear from a net with a maximum size of 60–70 metres long and 2.5–4 metres high. Lead sinkers were tied at the bottom of the net and floaters were tied at the top of the net. However, the local communities claimed that they made this fishing gear up to 80–100 metres long. The Proyong is used with at least two fishers holding the end of each side and pulling and walking for about one kilometre in order to collect fish if the lake is very shallow. However, if the lake was not too shallow, the fishers used their boats to pull the Proyong instead. Most participants claimed that they started to use this Pryong when the water was very shallow during the drought. It had been used in the community for the last few years since the drought became too severe. The village head noticed that the local people in his village had started to use the Proyong since 2015 when the weather had become more severe. He said that, before 2015, the local communities could still catch fish during the dry season since there was enough water in the lake. Likewise, the participants said that they used to use a Lob and/or gillnet in the dry season, when the water level was low. However, these two types of fishing gear were no longer practical when the lake and its tributaries were too dry and muddy because of severe droughts. Hence, the local people switched to use the Proyong instead. The participants reported that they learned how to make and use the Proyong from people living in another commune. They said the people there used a smaller size of Proyong (about 30 metres long) to catch fish in small streams. 7.3.1.1 Switching fishing equipment during floods One participant claimed that a Sai Yern was very expensive and only the better-off households could afford it. In order to catch shrimps during the drought, the local communities used a different type of fishing gear locally known as a Kansom (brush bundle trap). According to the participants, a Kansom was made by tying branches, sticks, or brush into bundles for either placing on the bottom of the tributary or lake or suspending in the water. Once it was placed in the water, shrimps and small fish sheltered under the bundle. Then the fishers used a long-handled scoop net to collect the shrimp or fish from underneath the bundle. The research study was conducted in June, which was the time when the local people in this fishing community prepared their fishing equipment before the water rise in the lake and its tributaries. As observed during the transect walk, the people were active fixing and making their fishing gear so that they had them ready for use when the water level in tributaries and the lake was high enough. As observed, each household had made and hung their fishing gear such as a Lob and/or Sai Yern on their balconies. 79 7.3.1.2 Switching fishing equipment during droughts The participants added that they had to make it in a larger size (80–100 metres) in order to catch fish in an open space in the lake. To use this fishing gear, they needed to work collectively with three to four households and fish together in the lake. They then shared the profit after selling fish. This method was generally used among relatives and friends only. However, most 80 participants mentioned that this method could only help them get enough fish for household consumption and generate supplemental income for the household. “We have used this fishing gear for about three to four years when the water became too shallow in the lake. We learnt about this fishing gear from the people who live in another commune, upper streams. They used it in small streams. Then we followed them, but we made the gear bigger and used it in the lake. Normally, we work with 3 to 4 households to pull this fishing gear in the lake. It is big and heavy. So, we need more people to pull it in the lake. After fishing we share the money that we earn from selling fish. Sometimes, we can earn about KHR30,000–40,000 per day. But sometimes, we cannot earn anything after paying for gasoline.” (Respondent MW-FI05) According to the participants, all the above methods they used could only help them get enough for eating and living during floods and droughts. Although these methods could not help them improve their livelihood, it was claimed that they were more comfortable to do than other wage labour, particularly construction work where they had to go away from their home and work in the town. Moreover, the money that they earned from the above methods and wage labour was comparable. It was estimated that the money they could earn from construction was KHR25,000–30,000 per day. However, the income that local people could earn from fishing during severe floods and droughts was between KHR10,000–40,000 per day depending on water level in the lake. 7.3.2 Changing fishing zones Another fishing-related method that the local communities in the studied villages used during severe drought was changing the fishing zones. The participants claimed that the lake near their villages was too shallow to fish. They had to go further in the lake and explored other places where there was enough water to fish. Some of them mentioned that they had to cross the lake, which was approximately 30 km away from their villages, to catch fish. They claimed that the water on the other side of the lake was not as shallow as the water near their villages, and fish were also more abundant. Hence, 81 they could catch more fish there. In the last few years, the participants had started to go further afield for fishing when the lake became very shallow. they could catch more fish there. In the last few years, the participants had started to go further afield for fishing when the lake became very shallow. “We have to cross the lake to the other side of the lake, in Raing Til village, Pusat province to catch fish using Proyong. Over there, the water is deeper, and the fish is more abundant. We could not catch fish at our side because of shallow water.” Respondent MW-FI05 “In the last two years, my husband goes further to the other side of the lake (in Pursat province) to catch fish. The water in the lake near the community has become too shallow and muddy and we could not catch many fish. So, he has to go there to catch more fish. Over there, there are more places to catch fish and the lake is not muddy and no water hyacinth. Sometimes, when there is enough rainfall in that area, he can catch many fish and earn about KHR100,000–200,000 per day. But this income is not regular because the rainfall is not regular either.” (Respondent ST-FA02) 7.3.3 Crop farming According to some participants, besides fishing and rice farming, they grew crops and vegetables for supplemental income. The local people had practised this method for decades, but some participants emphasised that they started to do crop farming when they realised that they could not earn as much money as before from their main livelihoods such as fishing and rice farming. It was observed that only some households in the studied villages had been doing crop farming for supplemental income. Crop farming was divided into two categories; large-scale farming and small-scale farming. Large-scale crop farming was done on large land sizes of several hectares of land owned by well-off households who could afford to buy land, a two-wheel tractor, and fertiliser. Small-scale crop farming was done on small land sizes of about one hectare or less. Every household in the studied villages that had land could do this small-scale farming. Typically, this small-scale farm was located in the village. One participant claimed that he started to buy farmland and grew crops once he realised that rice farming could not help him earn as much money as he wanted to support the 82 family. He also stated that the productivity of rice was not as regular as before. He noticed that the changing of weather and water scarcity caused the irregularity of rice productivity. The local people usually spent their time growing vegetables after they had finished their rice farming and fishing. Some well-off households in the farming village had their farmlands approximately 17 km far from their village. The participants reported that they grew soybeans and sesame on their farms. Since their farmlands were located in the land-based area, far from the Tonle Sap floodplain, the farmers could do their crop farming all year round. However, they claimed that their crop farming could only help them gain enough money to support their daily expenses in the family. They blamed the prolonged 2019 drought for the low productivity of their crops. On the other hand, some households in the fishing village had their farmlands either in the village behind their houses or near the lakeshore. The farmland near the lakeshore was located approximately 3 km from the village. Commonly, the local people in this village grew corn and mung beans. 7.3.3 Crop farming It was observed that the people in this village could only grow their crops in the dry season after the water in the lake had receded. The period of farming was from February to April or May. The community people did their crop farming when they were free from fishing. They have practised this method for a long time. The participants reported that this method only helped gain supplemental income besides fishing. Similar to what farmers in the farming village mentioned, the local people in this village also complained about the impact of severe drought causing low productivity of their crops. 7.3.4 Livestock raising But this year, I can do it only one time, and my rice has not grown maturely yet. Since my rice production failed, I decided to raise pigs to gain more income for the family” (Respondent ST-FA04) 7.3.4 Livestock raising Most participants recognised that livestock raising was one of the most important livelihoods that helped generate income when their main sources of income were affected by severe droughts. The people in the farming village commonly raised pigs and chickens for their supplemental income. Although livestock raising had been practised for decades, the participants claimed that they could earn extra income from this method. In this village, the local people also raised cattle (buffalo and cows). However, for most people in the community, cattle were considered an asset rather than an income. On the other hand, there was a small number of households raising pigs and chicken as their supplemental income in the fishing village. 83 Nevertheless, the households who raised chickens reported that they only did it on a small scale for household use only. The participants said that they normally raised less than ten chickens in each household, and that they only used traditional knowledge learned from the elderly people to raise their chickens. As mentioned by a participant (Respondent MW-FI04), “Besides fishing, I also raise some chickens, but they are for household use only. I use traditional techniques to raise chickens. I never learn from outsiders.” In contrast, most people in Santey, the farming village, raised chickens on a large scale for their income. It was reported that the local communities in this village had been provided several NGO trainings on chicken raising. The NGOs also provided baby chicks to some households who could not afford to buy them. Besides chicken raising, some people in this village also raised pigs in order to generate additional income. Some participants in the farming village claimed that they started to raise pigs and chickens to generate income when they could not earn as much from fishing as before due to the decline in the fish population in the lake and its tributaries. These people noticed that climate change was the cause of the fish population decline. One farmer emphasised that his rice production had also been destroyed by droughts: “Six years ago, I quit fishing because I could not catch as many fish in the streams because fish became less abundant. Then I started to do vegetable farming and raised chickens instead of fishing. And this year, 2019, my rice production has been affected severely by drought. I usually did rice farming two times a year. The use of indigenous medicines to cure cattle and chickens Regarding cattle and chicken raising, a village head in the farming village claimed that some people in his community still used traditional medicines to cure their livestock's diseases although the majority switched to use modern medicines. The village head explained that when the people’s cows and chickens got sick because of the extreme heat, the local people used their IK that they had learned from the elderly people to 84 make traditional medicines to cure their animals. He mentioned that high temperature during the dry season often caused cows and chickens to become sick. Cows usually had bloody diarrhoea while chickens became sick and died. The village head described that to cure a cow when it got diarrhoea, people fed it small insects such as crickets and other flying insects mixed with liquor. They did this for two to three days in order to make their cow recovered. To cure sick chickens, they fed them lemongrass, and the peel of kapok tree immersed in water mixed with some modern medicine. 7.3.5 Hunting wild animals and birds Some participants were reported to catch wild animals and birds when their primary sources of income had been affected by droughts. The participants in the studied villages said that in order to find supplemental income for their family, they caught wild rats and snakes to sell. In the studied communities, wild rat meat was one of the most popular diets. As observed, rat meat was sold throughout the communities. Rat meat was not only sold in the communities but also to middlemen who sold it to Vietnamese people. This type of rat is known as a rice-field rat, which the local communities catch in their rice fields using rat traps. Some participants stated that they hunted rats to sell while others hunted them for household consumption only. The participants mentioned that they caught rats to sell when they could not make an income from their fishing and rice farming. Rat hunting could help them earn some extra money that they needed to spend on their daily foods and other household expenses. One participant in the farming village claimed that he started to hunt rats this year (2019) when the severe drought had affected his fishing greatly. I buy some rat traps to catch rice-field rats when I go fishing. I catch them only for eating. Sometimes, I bring them home for my family as well. I started to do it this year because I could not earn much money from fishing and my rice production because of the severe drought. (Respondent ST-FA05) The local authorities stated that the local people in the two studied villages sometimes hunted snakes and sold them to the middlemen. However, the participants stated that 85 there were not many people hunting snakes for business in their communities. The people who hunted snakes were skilful in doing it. Ordinary people in the communities were not able to catch snakes. It was reported that the local people started to catch snakes, especially aquatic snakes, once they noticed the fish population in their communities had declined. Aquatic snakes were sold to food sellers in the communities who sold the snake meat to locals and middlemen. Besides hunting rats and snakes, the local people in these two communities were also reported to hunt birds for their supplemental income. 7.3.5 Hunting wild animals and birds The commune councillor from the fishing village stated that some people in his communities used traps to catch birds in the forest near the lake and at the lakeshore. The birds that the local people caught included egrets, waterhens and herons. The caught birds were sold to the people in the communities who bought them for food. The local communities not only caught birds to sell, but they also for food. According to the participants, the above methods have been practised for several years. Some participants claimed that they learned how to make traps and catch wild animals and birds from their parents and elderly people in their communities. The local people have been using these methods only to gain supplemental income to their farming and fishing. However, not many people practised these methods in the two communities because they could not use these methods to earn as much money as other livelihood activities. 7.3.6 Wage labour Because of income loss from the damaging impacts of severe droughts, the local people in the Tonle Sap Lake region developed several adaptation methods to maintain food security and generate additional income. Wage labour was one of the adaptation strategies that the local communities used to generate income when their main source of income became negatively affected by severe droughts. The wage labour that the communities used included working in agricultural farms, working with fishing-related activities, working as a housebuilder and/or a construction worker in town. 86 7.3.6.1 Working in agricultural farms Most participants in the farming village reported that they worked on other farmers’ agricultural farms to earn money to support their family. Some participants said that they decided to work for other farmers because droughts had negatively affected their main and secondary livelihood activities. As mentioned in the previous chapters, severe droughts had affected the communities’ rice farming and fishing activities. The failure of rice growth and not being able to catch as many fish in the lake and its tributaries during the drought, had led to a shortage of food security and income loss for the local people. Therefore, the local people in this farming village decided to work for others’ farms in order to gain additional income to support their family. As mentioned by Respondent ST-FA03, “Fishing is my secondary livelihood, but droughts had affected it. I noticed that I could not catch as many fish since 2018. This year the drought has been too severe, then I decided to work for other farmers to get wage instead of fishing.” However, some others stated that they have already been practising this method for several years even though their rice farming was not affected by droughts. They added that they had to work for other farmers since their rice farmland was small and could not help them earn good money. They stated that rice farming could only help them to have enough rice for household consumption. Consequently, they needed to do something else to generate income for their daily living. The participants were reported to work in cassava farms, which were about 17 km far from their village, where they were hired to plant and harvest cassava. 7.3.6.2 Working in fishing-related activities Most participants in the fishing village reported that they worked for other fishers in their community to earn more income when they could not catch fish. The fishing- related activities consist of fixing fishing gear, making fishing equipment, and being hired to work for large-scale fishing. Most participants, especially women, claimed that they worked for other fishers fixing torn nets of fishing gear when they could not catch fish in the lake during the drought. Typically, the fishing gear needed to be fixed every year after it was used. The participants were hired to fix fishing gear such as Lob (cylindrical fish trap) and Sai Yern (vertical cylinder trap). 87 Besides fixing fishing gear, the participants were also hired to make fishing equipment such as small bamboo sticks used to attach Lob to the bottom of the lake. As claimed by the participants, it was just a casual job that they had to do to earn extra money. One fisher hired them to work for only a few days. Then they had to work for other fishers. Some were hired to work for only a few days for the entire year due to the severe drought. Respondent MW-FI01mentioned, “Besides growing vegetables and raising pigs, my wife works for other fishers to fix their Lobs (cylindrical fish traps). She earns KHR10,000 per day from fixing the fishing gear. We use this money to buy food and rice.” Moreover, some participants in this fishing community were hired to work with other large-scale fishers. These participants reported that they worked in fishing-related activities such as pulling Proyong (seine nets), collecting freshwater clams (using boats) and installing large fish traps with fences in the lake. They also emphasised that this method could help them gain good money. They worked for a full day and could earn between KHR20,000 and KHR35,000. Nevertheless, this work was not regular, and the fishers did not hire them to work long term. They could work for only a few days. 7.3.6.3 Working as a housebuilder and a construction worker Besides working in fishing-related activities, some male participants mentioned that they worked as a housebuilder in their communities and as a construction worker in Siem Reap town. According to the participants, working as housebuilder was a casual job that they did during the drought. The participants stated that they were not professional housebuilders, and they could only work on what they were told to do. Mostly, the participants could work for a few days only when they were needed. They were needed when there was a lack of labour or when extra labour was needed for just a while. As mentioned by Respondent ST-FA04, “Working as a house builder is a casual and irregular job. They called me to help them when there is a lack of labour. Sometimes, I could work for a few days, then there is nothing for me to do. After a while, they called me to help out again.” However, one participant claimed that he was a full-time housebuilder during the dry season. He explained that he quit fishing in the dry season from March–June and became a housebuilder since he had noticed the decline of fish populations in the lake. 88 Nonetheless, he went back to fishing when the water level rose in the lake and its tributaries in the wet season. Some participants, particularly in the farming village also stated that they went to Siem Reap town to work in construction. Detecting income loss from the impacts of the severe drought, the local communities worked as construction workers to earn money for their daily household expenses. Since their rice production had been devastated by the severe drought, the participants claimed that they did not have enough rice to eat. They did not even have enough rice seeds for the next cultivation. Hence, they had to do anything to get income to maintain their food security. The participants stated that they went to Siem Reap town to work as construction workers. These participants received a daily wage, which enabled them to buy food and rice for their household consumption. One of the participants said that she sent one of her children to work in construction when her chickens had died because of high temperature and her rice production had been destroyed by severe drought. As Respondent ST-FA07 mentioned, “I could not imagine that the drought this year is too severe. 7.3.6.3 Working as a housebuilder and a construction worker It destroyed my rice production. I used all the seeds and could not get them back. Most of my chickens also died. What I can do is sending my elder son, who just quit school to work in construction to get money to buy medicines for chickens and support the family.” Regarding knowledge of building houses and construction work, all participants emphasised that they had learned from the people who were skilful at house building and construction. They were told how to do it and then they started to learn through experiences. According to participants, the practice of these adaptation strategies could only help get enough income to support their daily expenses for a short period of time. Therefore, they had to practise other methods in order to gain more income while they could not depend on their main livelihoods (rice production and fishing). 7.4.1 Vegetable gardening The participants stated that their vegetable garden was essential for them to have enough food to eat while they could not rely much on the income from their rice 89 production and fishing. As observed during the transect walk, most households, particularly in the farming village, had their own small vegetable garden. The local people in this community normally grow herbaceous plants such as chillies, lemongrass, spring onions and galangal roots in their small garden. Some people had a bigger garden in which they planted water spinach, potato, cucumbers, long sponge gourd, wax gourd and eggplants. These vegetables were grown for both selling and household consumption. Some participants claimed that they could earn a little money from selling the vegetables to their neighbours. This method has been used for decades in the community. As mentioned by Respondent ST-FI04, “I have a small vegetable garden. I grow a few types of vegetable such long sponge gourd and water spinach. I could earn a little money from it, KHR1,000 to KHR2,000 a day. And this money is for my children which I give them when they go to school.” Even though there was a water scarcity during the drought, the local communities tried to retain the vegetable growth by buying water or carrying water from a distance to water them. However, some participants reported that most of their vegetables failed to grow due to the high temperature. The participants stated that their small garden could at least help them reduce their daily expenses while they could not earn money from their rice production. 7.4.2 Collecting wild edible vegetables and plants Some local people in the studied communities were reported to have been collecting wild vegetables and plants for their daily consumption when they could not rely on their main sources of income. It was noted that most poor households practised this method for their daily survival. According to the interviews, most participants from poor households reported that they picked edible vegetables and plants for their daily consumption when their income from fishing and rice production was lost. These participants said they picked vegetable and plant species that grew with minimal water, such as drumstick leaves, ivy gourd leaves, and edible amaranth. These species could be found in the village. Other plant species such as water spinach and sesbania flowers could be found at natural ponds and on the lakeshore, and freshwater mangrove leaves could be found at the lakeshore. As mentioned by Respondent ST-FA03 in the farming village, “We could not make income from our rice production this year. Sometimes, I 90 just collect drumstick leaves and ivy gourd leaves in the village, and water spinach at the natural ponds for our food.” In the fishing village, where most households did not have a vegetable garden, the local people collected wild vegetables and plants, such as water spinach, sesbania flowers, freshwater mangrove leaves and other edible plants for their daily food. The participants reported that these vegetables and plants could be found in tributaries, ponds and the lakeshore. As mentioned by a village head, “In this village, most people do not grow vegetables for consumption. For those well-off households, they buy vegetables from the market. But for poor households, they go and collect wild vegetables and plants such as water spinach, sesbania flowers and mangrove leaves to eat. People can find these vegetables in the tributary, ponds and the lakeshore.” (Respondent VH-MW06) 7.5 Discussion Climate change, particularly extreme floods and droughts have hugely affected the local livelihood in the Tonle Sap Lake region. In response to the effects of the floods and droughts on their livelihood, the local communities developed various livelihood adaptation approaches in order to survive. The adaptation strategies were developed based on their IK and experiences. Since the droughts have been the most devastating disasters to affect the local people, most of their adaptation strategies were used to cope with the severe droughts. Two main livelihood adaptation strategies have been adopted by the local people. The first and most important livelihood adaptation strategy was for income generation. The second strategy was for their daily food consumption. The adaptation strategies adopted for income generation included switching fishing gear and changing fishing zones, vegetable and crop farming, livestock raising, wage labour and hunting wild animals and birds. The local fishers mainly used the methods of changing fishing zones and switching fishing gear in the land and water-based village (fishing village). The method of changing fishing zones has been practised since the local fishers noticed the lake had become too shallow in the last few years. The local fishers had to travel across the lake to fish in deeper fishing zones where they claimed that fish were more abundant than in fishing zones near their villages. For the local fishers, the method of switching fishing gear has been used since the past even though there were no threats from floods and droughts. They had switched the fishing gear in accordance with the level of water in the lake of the two seasons. However, the current severe climate has meant the dry season is too dry and the lake is too shallow, which has led to a decline in the fish population. Since the fish were less abundant, the fishers switched to using fishing gear that had never been used in the past. They also changed the material of the fishing gear from bamboo to netting. It was reported that the fishing gear made from netting was lighter and more comfortable to carry. Most importantly, the netting gear could catch more fish than the bamboo gear when fish were less abundant. 7.4.3 Eating small fish Fish is both a source of income and vital food security for people in rural areas. The local people in the studied village relied on fish as their main source of income and daily food security. Fish that could be sold for a good amount of money must be in the right size. After fishing, the local fishers in the studied villages divided their fish into two groups of size, big and small. The big fish was for sale, and small fish was for household consumption. Some participants in the fishing village reported that they did not eat big caught fish during the drought because it was hardly caught and expensive. Instead, they had to eat small fish. The participants said that they had been using this method for several years after they noticed that they could not catch as many fish during the drought. They also mentioned that, in the past, the small fish were usually sold to other fishers who used it as bait to catch shrimps and big fish. As mentioned by Respondent MW-FI02, “We try to eat less or eat only small fish when we cannot catch as many fish during the drought. Mostly we can catch small fish. Once I catch big fish, I keep it for selling.” Likewise, the commune councillor mentioned that the people in his commune tended to eat small fish in the dry season when they could not catch as many fish. Then they started to eat big fish as normal when fish was more abundant in the wet season. 91 91 7.5 Discussion Besides switching fishing methods, the local communities in the region also engaged in livestock raising, vegetable and crop farming, wage labour and hunting wild animals and birds as a mean of generating income while their primary livelihood has been affected by the severe drought. The methods of livestock raising and wage labour that 92 the communities have adopted are similar with the adaptation strategies practised by local people in Northern Ghana, Nepal and Indonesia (Armah et al., 2013; Jones and Boyd, 2011; Binternagel, 2010). The studies in these three countries showed that the local people used various adaptation strategies to respond to floods and droughts. The studies in these countries revealed that the local communities adopted livestock farming and wage labour as their livelihood strategies to adapt to severe droughts that caused damage to their agricultural crops. Concerning wage labour, the study by Jones and Boyd (2011) showed that the local people in Nepal migrated for six months to find jobs further afield while the study in Indonesia by Binternagel (2010) indicated that the local communities mostly worked on other villagers’ farms. The adaptation strategies that the local communities adopted for daily food consumption consisted of vegetable gardening, collecting wild edible vegetables and plants, and eating small fish. These strategies are similar to the adaptation methods that local communities in Nepal, Indonesia and Bangladesh have used to deal with severe droughts. To adapt to severe droughts, the local people in Nepal harvested wild vegetables and plants for their food, while the local people in Indonesia reduced the food consumption (Jones & Boyd, 2011; Binternagel 2010). The local people in Indonesia were reported to reduce the number of meals by eating twice instead of three times a day. Nonetheless, the communities kept eating three meals a day, but they ate less than usual during the severe drought. Most communities, especially in the land- based village, also had their own vegetable garden that they used for their household consumption. The community people claimed that this method was among other livelihood strategies for their survival while their income was reduced because of the drought. Regarding vegetable gardening, the rural communities in northeastern Bangladesh also used this method for their livelihood adaptation strategy to cope with the disaster risks. However, the local communities in this region used a different technique for their vegetable garden because they adopted this method during the severe flooding. 7.5 Discussion Instead of growing vegetables on the ground, the local communities used bamboo rafts for their vegetable gardens; these are known as floating gardens (Lebel 2013). The results of my research also reinforce the previous study of Nuorteva et al. (2010) who conducted a study in the Tonle Sap Lake region to explore the traditional 93 adaptation of the local people used to cope with unusual environmental changes. Nuorteva et al. (2010) reveal that the local communities take on short-term employment and collect edible aquatic plants for additional income when their agriculture and fishing activities are affected by floods and droughts. Nuorteva et al. also argue that the local people used savings and other kinds of physical assets to support and broaden their livelihood base. The well-off households commonly practised the saving method while the poor households had to borrow money or rice from their neighbours, relatives or middlemen and moneylenders. However, the method for saving was not mentioned by the local people in my study. Concerning the competence of the livelihood adaptation strategies, the communities claimed that the methods they used to cope with droughts and floods could only help them have enough for household consumption. The money earned from the alternative livelihood activities was supplemental income while their main sources income had been lost. For most households in the studied communities, the adaptation strategies could not help them improve their livelihood. adaptation strategies The role of indigenous knowledge (IK) of the local communities is significant in developing those adaptation methods. The communities have developed their livelihood adaptation strategies based on their knowledge and experiences with the changes in climate/weather. The knowledge that the local people used to develop the adaptation strategies is classified into indigenous and western knowledge. The IK of the local communities in the two studied villages is the knowledge that they learned from elderly people, family members and their ancestors. The western knowledge of the community people is defined as the knowledge they gained from being involved with the local governments’ and NGOs’ development programmes and from other community people who shared this knowledge with them. Although the local communities have adopted western knowledge, the IK is still the primary source of knowledge that the local communities used to develop their livelihood adaptation practices to deal with floods and droughts. Most of the adaptation strategies have been developed based on their IK and experiences. Those strategies include fishing-related activities such as switching fishing gear and changing fishing zones, repairing fishing equipment, vegetable 94 gardening, hunting wild animals and birds, collecting wild vegetables and plants, and eating small fish. The local communities claimed that they gained the knowledge from older people in their community and their parents and ancestors, and from the experiences they have encountered daily with the surrounding environment. This IK is typically shared among family members, relatives and best friends. The local people tended not to share their knowledge with other communities. Hence, most of the livelihood adaptation strategies were adopted individually, not collectively. The western knowledge used by the local people comprises making fishing equipment, fish farming, rice farming and livestock raising. This knowledge was introduced by NGOs and local government officials through their development programmes in the Tonle Sap Lake region. The western knowledge has also been shared among communities in the region. Most western knowledge was about the techniques for rice farming, livestock raising and fish farming. These techniques were about how to improve productivity and prevent animals and fish from diseases and death. Although western knowledge was introduced to the local communities, IK still has its roles in the livelihood strategies. Instead of using IK alone, some local people integrated it with western knowledge in doing their rice farming, fish farming and livestock raising. adaptation strategies For example, some communities in the region still used indigenous medicine and treatment for their livestock. Chapter 8: Conclusion This chapter presents a summary of the thesis comprising research objectives, research location, conceptual framework, core research questions, methodology, participants, key research findings and final comments. The chapter is divided into three sections. The first section summarises the research process. The second section presents the key research findings, and the last part presents some final comments regarding the future of the topic. 7.7 Conclusion This chapter has indicated that the Tonle Sap Lake communities have adopted a number of adaptation strategies to deal with severe floods and droughts. The chapter began with an explanation of how the communities used their IK to mitigate the impacts of floods and droughts. Then, it demonstrated how various adaptation strategies had been developed with a mixture of IK and new knowledge. The local adaptation strategies were classified into two categories. The two categories were household income generation and daily survival. The communities were aware that using IK alone was not sufficient to deal with severe floods and droughts. The communities, therefore, integrated new knowledge that they gained from the outsiders and by being involved in NGOs’ and local government’s development programmes, with IK to create a new opportunity for their daily livelihood. Nevertheless, the adaptation strategies with a mixture of IK and new knowledge could only help them survive during the severe 95 floods and droughts. These adaptation methods were unable to improve the local livelihoods of the local people. floods and droughts. These adaptation methods were unable to improve the local livelihoods of the local people. 96 8.1 Summary of the research process Tonle Sap Lake is the biggest freshwater lake in Southeast Asia. It is rich in fishery resources and is a highly productive area for rice production that supports the livelihoods of nearly two million people (Holtgrieve et al., 2013; Johnstone et al., 2013; Kummu et al., 2014). However, in the last few decades, the local people who rely on fishery resources and agriculture in the region have been affected by climate change, particularly floods and droughts. The main objective of this research was to find out how local people in the region use their IK to adapt to floods and droughts. The study specifically focused on livelihood adaptation strategies that the local communities used to respond to floods and droughts. The research was conducted in two villages in the Tonle Sap Lake region. One village is located along the tributary of the lake and is adjacent to Tonle Sap Lake. This village is known as a fishing village because the primary livelihood activity of the local communities in the village is fishing. The other village is located about 7 km from the lake and is predominantly a farming village because the primary livelihood activity of the villagers is rice cultivation. The research firstly investigated how local communities in the region perceived climate change, especially floods and droughts. In this part, how the patterns of floods and droughts have evolved over time was also explored. Then, the effects of severe floods and droughts on local communities were examined. In this section, I addressed the impacts of severe floods and droughts on local livelihood activities, then turned to how the communities developed preparation methods for floods and droughts. Lastly, I 97 examined how the communities used their IK to develop livelihood adaptation strategies to respond to severe floods and droughts. In this part, the question of how the adaptation strategies have changed over time following the patterns of floods and droughts was addressed. Finally, I studied the effectiveness of using adaptation strategies to adapt to floods and droughts. To fulfil the main objective of this research, I employed four sub-core questions: To fulfil the main objective of this research, I employed four sub-core questions: 1. What are the local people’s perceptions of the floods and droughts? (see 8.2.1) 2. How have the local people and their livelihoods been affected by floods and droughts? (see 8.2.2) 3. What strategies do local people use to recover from the effects of floods and droughts? (see 8.2.3) 4. How might indigenous knowledge be incorporated into adaptation policies? (see 8.3) 4. How might indigenous knowledge be incorporated into adaptation policies? (see 8.3) 8.3) To answer the above questions, I adopted a qualitative methodological approach using semi-structured interviews and observations as the primary methods. The semi- structured interview was suitable for my research study as it allowed me to interact with the participants to explore their viewpoints about the impacts of floods and droughts. Because of the open-ended questions and informal approach, the semi-structured interview allowed the participants to have enough space to answer different questions. It also enabled me to discover how the local people in the region used their knowledge and experiences to develop livelihood adaptation strategies to respond to floods and droughts. To answer the above questions, I adopted a qualitative methodological approach using semi-structured interviews and observations as the primary methods. The semi- structured interview was suitable for my research study as it allowed me to interact with the participants to explore their viewpoints about the impacts of floods and droughts. Because of the open-ended questions and informal approach, the semi-structured interview allowed the participants to have enough space to answer different questions. It also enabled me to discover how the local people in the region used their knowledge and experiences to develop livelihood adaptation strategies to respond to floods and droughts. To conduct the interviews, I approached the participants individually. I used a snowball sampling technique to approach farmers and fishers in the two villages. There were 19 participants in total, including fishers, farmers, commune councillors, village heads and NGO staff. Besides the semi-structured interviews, I also employed non-participant and unstructured observation, which included transect walks and informal meetings with other non-participant villagers in the studied communities to observe how IK was used to develop livelihood adaptation strategies to adapt to severe floods and droughts. Non- 98 participant and unstructured observation allowed me to study how the local communities practise their daily livelihood activities. 8.2 Key research findings The findings of this thesis are divided into three major themes. The first theme presents the perceptions of local communities in the region about climate change, floods and droughts. The second theme reveals the impacts of floods and droughts on local communities and their livelihoods. The last theme indicates various livelihood adaptation methods that the local communities have adopted to prepare for, and respond to, floods and droughts. To fulfil the main objective of this research, I employed four sub-core questions: The transect walk helped me to capture the essential things that the participants did not mention in the interviews. I was also able to see the reality of resource use and compare it with the information that I collected from the interviews. 8.2.1 Local perception of climate change, floods and droughts The first core question of this thesis sought to understand the perceptions of the local communities about climate change, and floods and droughts. The research specifically explored how the local farmers and fishers perceived floods and droughts, which occur regularly in the region. The study found that the communities had clear perceptions of climate change, floods and droughts although they had little knowledge about the complexities of the phenomenon. They all said they had experienced increasing average temperatures and decreasing average annual rainfall. All participants claimed that they had experienced severe drought since 2015. The communities observed that the 2019 drought was the most extreme one they had ever encountered. Water scarcity, which was a result of prolonged drought, was seen as the most critical issue for the local people in the region who rely on fishing and rice farming as their main sources of income. All participants recognised that prolonged drought and high temperature that occurred in 2019 were the most critical issues for them, causing damage to their agricultural production, especially rice cultivation, and affecting their fishing and other livelihood activities. In the last few years, they suggested, climate has become more extreme, which has led to severe floods, intense temperature and prolonged drought. Most participants have just noticed the change of weather in recent years when it has 99 become increasingly evident and severe, although climate change has apparently happened over a considerable period. In terms of flooding, most participants did not show much concern. Although they recognised that flooding was one of the natural hazards that can affect their livelihoods, properties and endanger their lives, the participants claimed that it was not a significant issue for them. There were some reasons that the participants thought that flooding was not a big issue for the communities. In the farming village, extreme flooding did not happen often. Even though the seasonal flooding happened every wet season, it flooded only the farmers’ rice fields, not the village. Having experienced seasonal flooding, the farmers in this village adopted flood-tolerant rice varieties to cope with the flood. Likewise, in the fishing village, where flooding is a normal phenomenon happening every wet season, the local people built their houses on tall stilts to avoid the impacts of floods. Regarding the cause of weather change, nearly half of the respondents recognised that climate change is a result of human activities. 8.2.2 Impacts of floods and droughts on local livelihoods The study revealed that flooding was one of the natural hazards that affected local communities and their livelihood. For some participants in the fishing village, extreme flooding was considered as a hazard since it flooded the villages rapidly without warning. The extreme flooding caused damage to local assets such as houses, boats and fishing equipment, and affected fish farming, and fishing activities of the local communities. The local authorities claimed that the extreme flooding in the past years had caused damage to low-income families’ houses and forced people to evacuate from their houses to a safe place. For some communities who did fish farming, the extreme flooding caused poor water quality for fish farming. It also destroyed the barrier net fences of the fish ponds and the farmed fish escaped from the ponds, resulting in high production losses. However, for most communities, the impact of extreme floods was not a critical issue since the extreme flooding did not often occur in their region. Moreover, the local communities were well prepared for regular floods that happened every wet season. In terms of the impacts of droughts, the study indicated that severe and prolonged droughts had caused significant negative impacts on local communities and their livelihoods. The severe and prolonged droughts affected local livelihoods such as rice cultivation, fishing activities, fish farming, livestock raising, vegetable growing and other alternative livelihood activities. Rice cultivation, which was the main livelihood and source of income of the communities in the farming village, was severely affected by prolonged drought. Severe water scarcity, which was a result of prolonged drought, delayed rice growth. All farmers reported that their rice failed to grow even though they used a flood-tolerant variety of rice. Due to the prolonged drought, the local farmers’ rice productivity was very low. In the fishing village, fishing activities were the main livelihood and source of income. However, the prolonged drought caused rivers, and the lake to become very shallow and muddy, and therefore less abundant in fish. The amount of fish catch per household was meagre. The prolonged drought has also severely affected other livelihood activities in the two communities. High temperatures and water shortages were the most significant issues for livestock raising, fish farming and vegetable growing in the communities. Regarding livestock raising, high temperatures have caused diseases in cattle, buffalo and chickens, and some of these animals died eventually. 8.2.1 Local perception of climate change, floods and droughts However, they looked to more local human activities as a cause, rather than seeing it as a global phenomenon caused by increasing CO2 emissions worldwide. They believed that deforestation is one of the leading causes of climate change. They noticed that the climate had changed since more people cut down flooded forests and cleared them for farmlands. They believed the destruction of flooded forests in their communities has led to severe floods and droughts. The participants indicated that flooded forests in their communities provided several advantages to the community people such as fish spawning zones, fish habitats, preventing big waves when floods happened, and helping to prevent severe droughts. These participants compared the droughts and floods that had happened recently to those in the past when the flooded forests were well maintained in their communities. All these participants claimed floods and droughts that occurred ten or more years ago were not as severe as those of the present day. However, other respondents believed that climate change was a natural process that occurred beyond the control of humankind. These participants claimed that floods and droughts have become gradually severe due to the process of nature. 100 8.2.2 Impacts of floods and droughts on local livelihoods For fish farming, poor water 101 quality and warm water because of high temperatures have caused diseases and killed farmed fish. Likewise, high temperatures and water shortages have caused vegetables and crops to fail. Wage labour was also affected by the prolonged drought. The working day of local communities who worked in cassava farms was reduced because of the drought. The local communities who worked to harvest cassava were asked to postpone their work because the severe drought had delayed the plants’ growth. Likewise, the local fishers who worked for other large-scale fishers were not hired as often. Given the impacts of droughts on livelihood, the food security of the communities has been threatened, and their income has decreased. Droughts have also contributed to the changes in rural society and affected the rural economy as a whole. adaptation strategies to deal with floods and droughts Different challenges within climate change, specifically floods, and droughts, had high impacts on local people in the region. To cope with these natural hazards, the communities have practised various preparation and adaptation methods. Regarding the preparation for floods and droughts, the local people have learned how to adapt to changes in weather patterns from past experiences. The traditional way of rice farming in the studied community relies very much on the availability of rainfall. Hence, local predictions regarding rainfall is significant for the local people when preparing their rice cultivation. The communities prepared themselves in accordance with the rainfall. The local people observed the rainfall pattern from the late dry season or early wet season and predicted if there would be enough rainfall for their rice cultivation. This method has been developed and used since ancient times. The local people learned this method from their ancestors and their own experiences. It was reported that there was a religious event during the Khmer New Year in April when the local communities celebrated and predicted the rainfall for the upcoming wet season. The Tonle Sap Lake communities were found to be practising two main preparation methods for droughts. The first and most important method was to reserve water in ponds and reservoirs. Having experienced the scarcity of rainfall in the dry season, the local communities dug and restored their wells, ponds and other reservoirs to store water. The stored water was used for different purposes, including household use, vegetable gardens, livestock raising, rice cultivation and crop growing. The second 102 method, commonly used by fish farmers in the fishing village, is based on having experienced the adverse effects of drought on fish farming. The local communities found a solution that could prevent them from losing profit because of fish dying. The local communities sold their fish before the drought happened. Based on their experience, the fish farmers acknowledged that the drought usually occurred in the dry season between April and June. Normally, high temperatures during April and May could kill farmed fish. Hence, the fish farmers started to raise their fish in late June or early July and sold it in February or March. In terms of adaptation strategies, the communities have developed various livelihood adaptation methods to respond to floods and droughts. These adaptation methods were developed based on their indigenous knowledge (IK) and lived experiences. adaptation strategies to deal with floods and droughts The IK has played a significant role in local communities, which allows the local people to use it to adapt to severe floods and droughts. The knowledge that the local people used to develop the adaptation strategies is classified into indigenous and western or new knowledge. Although the communities have adopted western or new knowledge, the IK is still the primary source of knowledge that the communities used to develop their livelihood adaptation practices dealing with floods and droughts. The adaptation strategies were classified into two main categories. The first and most crucial livelihood adaptation strategy was for income generation. The second strategy was for their daily food consumption. The adaptation strategies for income generation comprised changing fishing zones and fishing gear, vegetables and crops farming, livestock raising, wage labour, and hunting wild animals and birds. The local fishers mainly used the methods of changing fishing zones and switching fishing gear in the fishing village. The method of changing fishing zones has been practised since the local fishers noticed the lake becoming very shallow in the last few years. The fishers in the studied communities had to travel across the lake to fish in deeper zones where they claimed that fish were more abundant than fishing zones near their villages. For the local fishers, the method of switching fishing gear has been used in the past even though there were no threats from floods and droughts. The local fishers switched their fishing gear in accordance with the level of water in the lake of the two seasons. However, the current severe climate has meant the dry season is too dry, and the lake is 103 too shallow, which has led to a decline in the fish population. Since the fish were less abundant, the fishers changed to use some fishing gear that they had never used before. The local fishers also changed the material of making the fishing gear from bamboo to netting. The gear made from netting was lighter and more comfortable to carry. Most importantly, the netting gear could catch more fish than the bamboo gear while fish was less abundant. The adaptation strategies for daily food consumption comprised vegetable gardening, collecting wild edible vegetables and plants, and eating small fish. Typically, the local communities had their small vegetable garden in which they commonly grow herbaceous plants such as chillies, lemongrass, spring onions and galangal roots. 8.3 Final comments This study has shown that the local people in the Tonle Sap Lake region see climate change as a result of local causes rather than global. The local people referred to local deforestation when they were asked about the cause of climate change. They assumed that the destruction of flooded forests in their communities had led to severe floods and droughts. These communities believed that if the flooded forest were well maintained, the effects of floods and droughts would not have been as severe. This local perception and knowledge could be useful to inform local environmental practices. Although it does not link to global action, it may help mitigate some effects of climate change. As mentioned by Shukla et al. (2016), local knowledge and perception reflect local issues because they live closely with nature and have a close interactive understanding of their environment. Hence, local knowledge plays a significant role in adaptation strategies. This study has shown that the local people in the Tonle Sap Lake region see climate change as a result of local causes rather than global. The local people referred to local deforestation when they were asked about the cause of climate change. They assumed that the destruction of flooded forests in their communities had led to severe floods and droughts. These communities believed that if the flooded forest were well maintained, the effects of floods and droughts would not have been as severe. This local perception and knowledge could be useful to inform local environmental practices. Although it does not link to global action, it may help mitigate some effects of climate change. As mentioned by Shukla et al. (2016), local knowledge and perception reflect local issues because they live closely with nature and have a close interactive understanding of their environment. Hence, local knowledge plays a significant role in adaptation strategies. Likewise, other scholars suggest that local or indigenous knowledge is a key resource for local communities that can be used for designing local adaptation and mitigation policy to not only mitigate the effects of climate change, but also to manage natural resources in an effective and sustainable way (Agrawal, 1995; Briggs, 2014; Deeb et al., 2011; Hiwasaki et al., 2015; Raygorodetsky, 2019). adaptation strategies to deal with floods and droughts Some households had bigger gardens where they grew a greater variety of vegetables. The vegetable garden could provide enough food for their daily consumption. The poor households that did not have a vegetable garden were found to collect wild vegetables, which they could either find inside or outside the villages. Moreover, these community people were found to catch wild animals (such as rice-field rats and snakes) and birds for both consumption and supplemental income. The local communities were also found to eat only small fish when fish were not abundant during the drought. On the whole, the Tonle Sap Lake communities have just noticed the growing severity of climate change in the past few years when it has become more apparent and extreme. Nonetheless, instead of mitigating the effects of climate change, the communities developed various adaptation strategies to adapt to it. Some methods have been practised for some time while others have been adopted recently, providing the local communities more opportunities to deal with climate change, especially severe droughts. However, due to the current severe climate, particularly severe and prolonged drought in 2019, the adaptation methods that the communities adopted were only sufficient for their daily survival. Although a mixture of IK and new knowledge has been used to develop the strategies, the sufficiency of the adaptation was still limited. Most of the adaptation strategies, particularly for income generation, could only help the local communities gain enough supplemental income for their daily consumption. These methods could not help them improve their livelihoods. IK has been considered a basic and undervalued knowledge practised by indigenous people. However, IK should be seen not so much as a fixed set of practices and skills 104 developed from the past. IK should be seen or considered more as a dynamic set of ideas and practices used to read the environment and adapt accordingly. To adapt to the current environmental changes, IK can also absorb new technologies and resources from the outside world. What is important is the way these technologies and resources are used in wider environmental knowledge. 8.3 Final comments Therefore, the IK and perceptions of the communities should be taken into consideration and incorporated into local environmental practices in this region so that the local environmental practices could be used more effectively to reduce negative impacts of climate change at the local level. Tonle Sap Lake is a region of climatic variation where floods and droughts occur naturally. However, the intensity of the floods and droughts has increased in the last few years. Since the effects of floods and droughts have threatened their livelihoods and food security, the local communities in this region have been found to adopt various established practices so that they are able to respond to the hazards. The Tonle Sap Lake people are not passive. They have actively adopted various livelihood adaptation 105 practices to deal with such natural hazards. This finding underpins the previous study of Nuorteva et al. (2010), which was also conducted in the region. Nuorteva et al. (2010) reveal that the local communities in both fishing and farming villages in the region actively increase their adaptive capacity to respond to climate change. Having faced extreme environmental phenomena, the communities have formed different kinds of response aiming to increase the local communities’ possibilities to cope with current and future events. Various climate change adaptation methods have been developed in local communities in the region. However, there appears to be little official involvement in climate change adaptation from the government and NGOs. The communities have been left to themselves to deal with climate change. Most local communities claimed that their climate change adaptation methods had been developed from their own knowledge and experiences with little support from NGOs. Most of the adaptations being used are primarily low-cost and low-tech. This may be questionable in terms of sustainability. For instance, the communities were found to change their fishing zones when fish became less abundant in the fishing zones near their villages. These fishers go further into the lake in order to find deeper pools to fish. This method is seen as not sustainable if the intensity of floods and droughts keeps increasing. Local adaptation and IK are essential for local communities to respond to such natural hazards. However, the local adaptation and IK have their limits if climate change continues. As noted by Nuorteva et al. 8.3 Final comments (2010), the communities’ capacity to respond to climate change is still limited due to the current severity of climate change. 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Climate Change and Natural Disasters: Transforming Economies and Policies for a Sustainable Future. New York: Routledge. Tracy, S. J. (2013). Qualitative Research Methods: Collecting Evidence, Crafting Analysis, Communicating Impact. West Sussex: John Wiley & Sons. Trenberth, K. E. (2005). The impact of climate change and variability on heavy precipitation, floods, and droughts. Encyclopedia of Hydrological Sciences, 1- 11. Udmale, P. D., Ichikawa, Y., Kiem, A. S., & Panda, S. N. (2014). Drought impacts and adaptation strategies for agriculture and rural livelihood in the Maharashtra State of India. The Open Agriculture Journal, 8(1), 41-47. 116 Uk, S., Yoshimura, C., Siev, S., Try, S., Yang, H., Oeurng, C., . . . Hul, S. (2018). Tonle Sap Lake: Current status and important research directions for environmental management. Lakes & Reservoirs: Research & Management, 23(3), 177-189. Ultanir, E. (2012). An Epistemological Glance at the Constructivist Approach: Constructivist Learning in Dewey, Piaget, and Montessori. International Journal of Instruction, 5(2), 195-212. Un, S. (2018). References Review of Implementation Challenges Posed by the Climate Change Action Plan 2014-2018 in Cambodia: Case Studies of Three Ministries. Phnom Penh: Parliamentary Institute of Cambodia (PIC). NFCCC. (2019). What do adaptation to climate change and climate resilience mean? Retrieved from https://unfccc.int/adaptation/items/4159.php UNDP. (2009). Country Report on Global Assessment of Risk: ISDR Global Assessment Report on Poverty and Disaster Risk 2009. Kathmandu: United Nations Development Programme (UNDP). UNDP. (2016). Sustainable Development Goals. Retrieved from http://www.undp.org/content/undp/en/home/sustainable-development-goals.html UNDRR. (2019, 8 April 2019). Cambodia steps up fight against climate change. Retrieved from https://www.unisdr.org/archive/64687 UNESCO. (2009). Learning and Knowing in Indigenous Societies Today (M. C. P. Bates, S. Kube & D. Nakashima Eds.). Paris: UNESCO. UNESCO. (2017). Local and Indigenous Knowledge System. Retrieved from http://www unesco org/new/en/natural sciences/priority areas/links/related UNDP. (2016). Sustainable Development Goals. Retrieved from http://www.undp.org/content/undp/en/home/sustainable-development-goals.html UNDRR. (2019, 8 April 2019). Cambodia steps up fight against climate change. Retrieved from https://www.unisdr.org/archive/64687 UNESCO. (2009). Learning and Knowing in Indigenous Societies Today (M. C. P. Bates, S. Kube & D. Nakashima Eds.). Paris: UNESCO. UNESCO. (2017). Local and Indigenous Knowledge System. Retrieved from h // / / / l i / i i /li k / l d UNESCO. (2017). Local and Indigenous Knowledge System. Retrieved from http://www.unesco.org/new/en/natural-sciences/priority-areas/links/related- information/what-is-local-and-indigenous-knowledge/ UNESCO. (2019). Local and Indigenous Knowledge Systems (LINKS). Retrieved from https://en.unesco.org/links UNISDR. (2002). Impacts of Disasters Since the 1992 Rio de Janeiro Earth Summit. Retrieved from https://www.unisdr.org/files/27162_infographic.pdf UNISDR. (2015). Making Development Sustainable: The Future of Disaster Risk Management. Global Assessment Report on Disaster Risk Reduction. Geneva: United Nations Office for Disaster Risk Reduction (UNISDR). Retrieved from https://www.preventionweb.net/english/hyogo/gar/2015/en/gar- pdf/GAR2015_EN.pdf University of Notre Dame. (2019, 30 November 2019). Notre Dame Global Adaptation Initiative: country index, Cambodia. Retrieved from https://gain.nd.edu/our- work/country-index/ 117 USAID. (2019a, 23 August 2019). Cambodia: agriculture and food security. Retrieved from https://www.usaid.gov/cambodia/agriculture-and-food-security USAID. (2019b). Climate Risk Profile: Cambodia. Retrieved from https://www.climatelinks.org/sites/default/files/asset/document/2019_USAID_C ambodia%20CRP.pdf Van Aalst, M. K. (2006). The impacts of climate change on the risk of natural disasters. Disasters, 30(1), 5-18. Van den Ban, A. W., & Hawkins, H. S. (2000). Agricultural Extension (2nd Ed.). Oxford: Blackwell Science. Varis, O., Kummu, M., Keskinen, M., Sarkkula, J., Koponen, J., Heinonen, U., & Makkonen, K. (2006). Tonle Sap Lake, Cambodia: Nature’s Affluence Meets Human Poverty. Retreived from http://hdr.undp.org/sites/default/files/olli_varis_tonlesap_study.pdf Walliman, N. (2011). Research Methods: The Basics. London: Routledge. Wongchuig Correa, S., Paiva, R. C. D. d., Espinoza, J. C., & Collischonn, W. (2017). References Multi-decadal Hydrological Retrospective: Case study of Amazon floods and droughts. Journal of Hydrology, 549, 667-684. World Bank. (2011). Vulnerability, Risk Reduction, and Adaptation to Climate Change. Washington, USA. Retrieved from https://climateknowledgeportal.worldbank.org/sites/default/files/2018- 10/wb_gfdrr_climate_change_country_profile_for_KHM.pdf World Bank. (2017). Cambodia - Sustaining Strong Growth for the Benefit of All. Washington, D.C.: World Bank Group. World Bank. (2019a, 25 September 2019). Cambodia. Retrieved from https://data.worldbank.org/country/cambodia World Bank. (2019b, 25 September 2019). The World Bank in Cambodia. Retrieved from https://www.worldbank.org/en/country/cambodia/overview World Bank. (2019c, 30 September 2019). Cambodia: climate data. Retrieved from https://climateknowledgeportal.worldbank.org/country/cambodia/climate-data- historical Wuebbles, D. J. (2018). Climate Change in the 21st Century: Looking Beyond the Paris Agreement. In Climate Change and Its Impacts (pp. 15-38). Cham: Springer. Yohe, G. W., Lasco, R. D., Ahmad, Q. K., Arnell, N. W., Cohen, S. J., Hope, C., . . . Perez, R. T. (2007). Perspectives on climate change and sustainability. In M. L. Parry, O. F. Canziani, J. P. Palutikof, P. J. van der Linden, & C. E. Hanson 118 (Eds.), Climate Change 2007: Impacts, Adaptation and Vulnerability: Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. (pp. 811-841). Cambridge: Cambridge University Press. 119 Appendices Appendix 1. Ethical approval How can you help? If you agree to take part, I will interview you at your place or anywhere convenient for you. I will ask you questions about your perception of floods and droughts, how you prepare for floods and droughts, and what livelihood activities you use during floods and droughts. The interview will take between 30 to 60 minutes. I will audio record the interview with your permission and write it up later. You can choose to not answer any question or stop the interview at any time, without giving a reason. You can withdraw from the study by contacting me at any time within 7 days of the interview. If you withdraw, the information you provided will be destroyed or returned to you. What is the aim of the project? This project is going to explore the existing indigenous knowledge system in the Tonle Sap region and study how the people in this region use their knowledge to respond to floods and droughts. In this regard, the project will particularly look at how local people prepare for regular floods and droughts and what livelihood strategies they use in order to survive. Who am I? My name is Sopheak Seng and I am a master’s student in Development Studies at Victoria University of Wellington. This research project is work towards my thesis. INFORMATION SHEET FOR PARTICIPANTS You are invited to take part in this research. Please read this information before deciding whether or not to take part. If you decide to participate, thank you. If you decide not to participate, thank you for considering this request. 120 Appendix 2. Participant information sheet in English What will the project produce? What will the project produce? The information from my research will be used in my Master thesis. I may also use the findings from the project for conference presentations and academic publications. Your information will not be identified in any of these outputs. If you accept this invitation, what are your rights as a research participant? You do not have to accept this invitation if you don’t want to. If you do decide to participate, you have the right to: • choose not to answer any question; • ask for the recorder to be turned off at any time during the interview; • withdraw from the study up to one week after your interview; • ask any questions about the study at any time; • reflect whether it will be recorded; • read over and comment on a written summary of your interview; • be able to read reports of this research by emailing the researcher to request a copy. If you have any questions or problems, who can you contact? If you have any questions, either now or in the future, please feel free to contact either: Student: Name: Sopheak Seng School: School of Geography, Environment, and Earth Sciences Email: sengsoph@myvuw.ac.nz Phone: +64 211688331 (New Zealand) +855 16399066 (Cambodia) Supervisor: Name: Prof. John Overton Role: Programme Director and lecturer of Development Studies School: School of Geography, Environment, and Earth Sciences Phone: +64 04 4635281 Email: john.overton@vuw.ac.nz Human Ethics Committee information If you have any concerns about the ethical conduct of the research, you may contact the Victoria University HEC Convenor: Dr Judith Loveridge. Email hec@vuw.ac.nz or telephone +64-4-463 6028. The information from my research will be used in my Master thesis. I may also use the findings from the project for conference presentations and academic publications. Your information will not be identified in any of these outputs. What will happen to the information you give? This research is confidential. I will not use your name or the name of your village or organisation in any outputs, and I will not use the information that would identify you. Only my supervisor and I will read the notes or transcript of the interview. The 121 interview transcripts, summaries and any recordings will be kept securely and destroyed on 1 January 2021. If you accept this invitation, what are your rights as a research participant? You do not have to accept this invitation if you don’t want to. If you do decide to participate, you have the right to: If you have any questions or problems, who can you contact? If you have any questions, either now or in the future, please feel free to contact either: Student: Name: Sopheak Seng School: School of Geography, Environment, and Earth Sciences Email: sengsoph@myvuw.ac.nz Phone: +64 211688331 (New Zealand) +855 16399066 (Cambodia) ខgu5k1នj1ឯ? u ខgu5\1ទemn17 eសង សuភUpកe k1នiសiតqrs1ក:អនuបណtiត ក5ពuងសiកM1%ផOកh1រអភiវឌw ex1Q1កលវiទz1លUយវiកត.eរ$Nន ទA2កuង%វលAងតuង 2បeទសញ{ហq\|%ឡន។ គe2W1ង2Q1វ2k1វeន7eធ3eឡ3ងស2W1ប:នieកបបទរបស:ខgu5។ eត3អAk1e>1លប5ណងNនគe2W1ងសiកM1eន7? គe2W1ងeន7នyងeធ3h1រសiកM1eE1eល32បពUនច5eណ7ដyងម.ល/01នរបស:សហគមន-កOuងត5បន:ទeនCQ1ប eហ3យនyងសiកM1 ពAវiធA%ដលសហគមន-កOuងត5បន:eន7e2ប32\1ស:1eដ3ម@AeឆC3យតបeE1នyងe2>17ទyកជ5នន: នiងe2>17J15ងសKLត។ គe2W1ងeន7 នyងe1តk1ពieសសeE1eល3វiធA%ដល2បk1ជនកOuងត5បន:eន7eធ3h1រeរ$បច5ដ.ចeមeចស2W1ប:e2>17ទyកជ5នន: នiងe2>17 J15ងសKLត%ដលeក3តk12បi15 នiងយuទQ12សeចi]^yមជAវiត%ដលព<ក>1ត:e2ប3។ លiខiតបST1ក:ពUតVW1នស2W1ប:អOកច.លរYម អOក2តZវ\1នអe]^3ញឲaច.លរYមកOuងគe2W1ងសiកM1eន7។ ស.មc1នពUតVW1នeន7មuនeពលសe2មចចiតeច.លរYម ឬមiនច.លរY ម។ 2បសiនeប3អOកសe2មចចiតeច.លរYម ខgu5ស.មអរគuណ។ eប3សiនអOកសe2មចចiតeមiនច.លរYម ខgu5ស.មអរគuណស2W1ប:h1រ ពii1រj1eល3h1រeសO3រសu5របស:ខgu5។ Human Ethics Committee information If you have any concerns about the ethical conduct of the research, you may contact the Victoria University HEC Convenor: Dr Judith Loveridge. Email hec@vuw.ac.nz or telephone +64-4-463 6028. 122 Appendix 3. Participant information sheet in Khmer eត3អOកc1ចជ<យអA\1នសe2W1ប:h1រសiកM1eន7? 2បសiនeប3អOកយល:2ពមច.លរYម ខgu5នyងសW1សអOកex1ទAក%នCងរបស:អOក ឬក%នCងj1%ដល1យ2ស<លស2W1ប:អOក។ ខgu5នyងស<ស5ន<រ1ក:ទងeE1នyងh1រយល:eឃ3ញរបស:អOកeE1នyងe2>17ទyកជ5នន: នiងe2>17J15ងសKLត នiងវiធA%ដលអOក eរ$បច5ស2W1ប:e2>17ទyកជ5នន: នiងe2>17J15ងសKLត eហ3យនiងសកមI1ពចi]^yមជAវiត%ដលអOកe2ប3ex1eពលW1នe2>17 ទyកជ5នន: នiងe2>17J15ងសKLត។ h1រសW1សនyងeធ3eឡ3ងចe17ពA ៣០ eE1 ៦០1ទA។ ខgu5នyងសW1សe/1យថតសeមCង e/1យW1នh1រយល:2ពមពAអOក ស2W1ប:h1រកត:211eពលe2h1យ។ អOកc1ចe2ជ3សeរ3សមiនeឆC3យស5ន<រj1ក\1ន ឬប]ប:h1រសW1សeពលj1ក\1នe/1យមiនi15\1ច:W1នeហតuផលអAeឡ3យ។ អOកc1ចដកខCLនeចញពAh1រសiកM1 123 eន7e/1យ1ក:ទងមកខgu5កOuងរយ7eពល៧NថKNនh1រeធ3សW1ស។ eប3សiនk1អOកដកខCLន ពUតVW1ន%ដលអOក\1នផeល:នyង 2តZវប51ញei1ល ឬប]{ន2តលប:eE1អOកវiញ។ eត3នyងW1នអAeក3តeឡ3ងស2W1ប:ពUតVW1ន%ដលអOកផeល:ឲa h1រ2Q1វ2k1វeន7គ\|k1h1រសW1ត:។ ខgu5នyងមiនe2ប3emn17អOក ឬemn17ភ.មi ឬQ1បUនអOកex1កOuងរ\1យh1រណ-j1ម<យ eឡ3យ eហ3យខgu5កនyងe2ប32\1ស:ពUតVW1នj1%ដលc1ចក5ណត:អតeស1ណអOក%ដរ។ W1ន%តQ12Q1i1រដyក15ខgu5 នiងខgu5%តបuej17នyងc1នក5ណត:21 នiងអតបទសW1សន-eន7។ អតបទសW1សន- eសចកeAសeងប នiង ឯកQ1រស5 eលង15ងអស: នyងទuក/1ក:e/1យសuវតI1ព eហ3យនyង2តZវប51ញei1លex1NថKទA១ %ខមកJ1 s15២០២១។ eត3នAតiវiធANនh1រសiកM1នyងW1នអAខC7? ពUតVW1នពAh1រ2Q1វ2k1វរបស:ខgu5នyង2តZវ\1នe2ប3ex1កOuងនieកបបទរបស:ខgu5។ ខgu5កc1ចe2ប3h1ររកeឃ3ញពAគe2W1ង ស2W1ប:h1រeធ3បទប 1ញសនOiសiទ នiងh1រe\17ពuម¡ផM1យh1រសiកM1។ ពUតVW1នរបស:អOកនyងមiន2តZវ\1នក5ណត:អតe ស1ណex1កOuងលទផល15ងeន7eទ។ 2បសiនeប3អOកទទ<លយកh1រអe]3ញeន7eត3អOកW1នសiទiអAខC7កOuង1មk1អOកច.លរYមកOuងh1រ2Q1វ2k1វ អOកមiនi15\1ច:ទទ<លយកh1រអe]3ញeន7eទ 2បសiនeប3អOកមiនចង:។ 2បសiនeប3អOកសe2មចចiតeច.លរYមអOកW1នសiទiដ.ច k1៖ ពUតVW1នពAh1រ2Q1វ2k1វរបស:ខgu5នyង2តZវ\1នe2ប3ex1កOuងនieកបបទរបស:ខgu5។ ខgu5កc1ចe2ប3h1ររកeឃ3ញពAគe2W1ង ស2W1ប:h1រeធ3បទប 1ញសនOiសiទ នiងh1រe\17ពuម¡ផM1យh1រសiកM1។ ពUតVW1នរបស:អOកនyងមiន2តZវ\1នក5ណត:អតe ស1ណex1កOuងលទផល15ងeន7eទ។ 2បសនeបអOកទទ<លយកh1រអe]ញeន7eតអOកW1នសទអខC7កOuង1មk1អOកច.លរYមកOuងh1រ2Q1វ2k1វ អOកមiនi15\1ច:ទទ<លយកh1រអe]3ញeន7eទ 2បសiនeប3អOកមiនចង:។ 2បសiនeប3អOកសe2មចចiតeច.លរYមអOកW1នសiទiដ k1៖ • e2ជ3សeរ3សមiនeឆC3យស5ណ£រ • សu5ឱaបiទW¥1សuAនថតសeមCងex1eពលj1ម<យកOuងក5ឡuងeពលសW1សន- • ដកខCLនeចញពAh1រសiកM1រហ.តដល:ម<យស\1ហ-ប¦1ប:ពAh1រសW1សរបស:អOក • ស<រស5ន<រeផqងៗអ5ពAh1រសiកM1ex1eពលj1ម<យ • ដyងr1eត3នyងW1នh1រថតស5eលង • c1ន នiងផeល:e¨1បល:អ5ពAh1រសeងប NនបទសW1សរបស:អOក 124 Appendix 4. Participant consent form in English Appendix 4. Participant consent form in English Appendix 4. Participant consent form in English Appendix 5. Participant consent form in Khmer Appendix 5. Participant consent form in Khmer អOក2Q1វ2k1វ៖ eសង សuភUpកe Q11ភ.មiQ12សe បរiQ1ន នiង%ផនដAវiទz1 NនQ1កលវiទz1លUយវiកត.eរ$ NនទA2កuង %វលលAងតuន 2បeទសញ{ហq\|%ឡន។ • ខgu5\1នc1នលiខiតបST1ក:ពUតVW1ន eហ3យខgu5ទទ<ល\1នh1រពនaល:ពAគe2W1ងសiកM1eន7 ។ ស5ណ£ររបស:ខgu52តZវ \1នeឆC3យតបeE1នyងh1រeពញចiតeរបស:ខgu5។ ខgu5ដyងr1 ខgu5c1ចស<រស5ន<រប%នមex1eពលj1ក\1ន។ u u u • ខgu5យល:2ពមច.លរYមh1រសW1ស%ដលW1នថតk1ស5eលង។ u u u • ខgu5យល:2ពមច.លរYមh1រសW1ស%ដលW1នថតk1ស5eលង។ CONSENT TO INTERVIEW This consent form will be held for 5 years. Researcher: Sopheak Seng, School of Geography, Environment and Earth Sciences, Victoria University of Wellington. Researcher: Sopheak Seng, School of Geography, Environment and Earth Sciences, Victoria University of Wellington. • I have read the Information Sheet and the project has been explained to me. My questions have been answered to my satisfaction. I understand that I can ask further questions at any time. • I have read the Information Sheet and the project has been explained to me. My questions have been answered to my satisfaction. I understand that I can ask further questions at any time. • I agree to take part in audio recorded interview. • I agree to take part in audio recorded interview. I agree to take part in audio recorded interview. • I may withdraw from this study up to one week after the interview, and any information that I have provided will be returned to me or destroyed. The identifiable information I have provided will be destroyed 18 months after the research ends. My name will not be used in reports, nor will any information that would identify me. • I would like a summary of my interview: Yes □ No □ • I would like to receive a summary of the final report and have added my email address below. Yes □ No □ I would like a summary of my interview: Yes □ N I would like to receive a summary of the final report and have added my email address below. Yes □ No □ 125 Appendix 5. Participant consent form in Khmer Preparation for floods and/or droughts 10. Given the impact of floods and/or droughts on your daily livelihood, how do you prepare for them (before they occur)? What strategies do you use? 11. How long have you been practicing these strategies/activities? 12. What strategies are being practicing now which were not done in the past (or vice versa)? 13. How did you know/learn about these strategies/activities? 14. How effective are these strategies? 13. How did you know/learn about these strategies/activities? Question guide for local communities Date: ……………………. Name of respondent: …………………. Gender: ………. Occupation: …………………………… Climate Change and its impacts 1. What changes in climate patterns have you noticed in your area? 2. What about floods and/or droughts? Compared to the past 10 years, are they more serious? 3. What have been the major changes in floods and/or droughts? 4. How long have you noticed the changes? 5. How do they impact on your family and community? 6. What are your primary livelihood activities? 7. What are your secondary livelihood activities? 8. How do floods and/or droughts impact on your primary livelihood? 9. How do floods and/or droughts impact on your secondary livelihood? ខgu5ដyងr1៖ ខgu5ដyងr1៖ u • ខgu5c1ចដកខCLនeចញពAh1រសiកM1eន7រហ.តដល:ម<យស\1ហ-ប¦1ប:ពAh1រសW1ស eហ3យពUតVW1ន%ដលខgu5\1នផe ល:នyង2តZវប]{នមកខgu5វiញឬប51ញei1ល។ លនង2តZវប]{នមកខguវញឬប1ញei1ល។ • ពUតVW1ន%ដលខgu5\1នផeល:នyង2តZវ\1នប51ញei1ល 18 កOuងរយ7eពល %ខប¦1ប:ពAh1រ2Q1វ2k1វ\1ន ប]^ប:។ • ពUតVW1ន%ដលខgu5ផ³ល:នyង2តZវរកM1ទuកk1h1រសW1ត:eE1ដល:អOក2Q1វ2k1វ នiងQ12Q1i1រដyក15។ • ខgu5ដyងr1លទផលនyង2តZវ\1នe2ប3ស2W1ប:នieកបបទរបស: e1ក សuភUpកe eហ3យeសចក³AសeងបNនលទផល c1ច2តZវ\1នe2ប3ex1កOuងរ\1យh1រណ-សiកM1 ឬប 1ញex1សនOiសAទ។ { u • ពUតVW1ន%ដលខgu5\1នផeល:នyង2តZវ\1នប51ញei1ល 18 កOuងរយ7eពល %ខប¦1ប:ពAh1រ2Q1វ2k1វ\1ន ប]^ប • ពUតVW1ន%ដលខgu5ផ³ល:នyង2តZវរកM1ទuកk1h1រសW1ត:eE1ដល:អOក2Q1វ2k1វ នiងQ12Q1i1រដyក15។ • ពUតVW1ន%ដលខgu5ផ³ល:នyង2តZវរកM1ទuកk1h1រសW1ត:eE1ដល:អOក2Q1វ2k1វ នiងQ12Q1i1រដyក15។ • ខgu5ដyងr1លទផលនyង2តZវ\1នe2ប3ស2W1ប:នieកបបទរបស: e1ក សuភUpកe eហ3យeសចក³AសeងបNនលទផល c1ច2តZវ\1នe2ប3ex1កOuងរ\1យh1រណ-សiកM1 ឬប 1ញex1សនOiសAទ។ • emn17របស:ខgu5នyងមiន2តZវ\1នe2ប3ex1កOuងរ\1យh1រណ-eទ eហ3យកមiនW1នពUតVW1នj1ម<យ%ដលc1ច ក5ណត:អតeស1ណ\1នខgu5%ដរ។ 126 • ខgu5យល:2ពមច5e´17ពUតVW1ន ឬមតi%ដលខgu5\1នបe]^ញ 2តZវ\1នផeល:e/1យខgu5 ex1កOuងនieកបបទNនគe2W1ង2Q1វ2k1វeន7។ \1ទ/i1 □ eទ □ • ខgu5ចង:\1នeសចកeAសeងបNនh1រសW1ស \1ទ/i1 □ eទ □ • ខgu5ចង:\1នeសចកeAសeងបNនh1រសW1ស 126 ហតeលµ1អOកច.លរYម: ________________________________ emn17អOកច.លរYម: ________________________________ h1លបរieច¶ទ: ________________________________ ពUតVW1នទ51ក:ទ5នងលម·iត: ________________________________ h1លបរieច¶ទ: ពUតVW1នទ51ក:ទ5នងលម·iត: 127 Appendix 6. Question guide for local communities Appendix 6. Question guide for local communities Question guide for local communities Date: ……………………. Name of respondent: …………………. Gender: ………. Occupation: …………………………… Climate Change and its impacts 1. What changes in climate patterns have you noticed in your area? 2. What about floods and/or droughts? Compared to the past 10 years, are they more serious? 3. What have been the major changes in floods and/or droughts? 4. How long have you noticed the changes? 5. How do they impact on your family and community? 6. What are your primary livelihood activities? 7. What are your secondary livelihood activities? 8. How do floods and/or droughts impact on your primary livelihood? 9. How do floods and/or droughts impact on your secondary livelihood? Preparation for floods and/or droughts 10. Given the impact of floods and/or droughts on your daily livelihood, how do you prepare for them (before they occur)? What strategies do you use? 11. How long have you been practicing these strategies/activities? 12. What strategies are being practicing now which were not done in the past (or vice versa)? 13. How did you know/learn about these strategies/activities? 14. How effective are these strategies? Strategies to respond to floods and/or droughts 15. In order to survive, what strategies do you use during floods and/or droughts? 16. How long have you been practicing these strategies? 17. What strategies are being practicing now which were not done in the past (or vice versa)? Strategies to respond to floods and/or droughts 15. In order to survive, what strategies do you use during floods and/or droughts? 16. How long have you been practicing these strategies? 15. In order to survive, what strategies do you use during floods and/or droughts? 16. How long have you been practicing these strategies? 17. What strategies are being practicing now which were not done in the past (or vice versa)? 128 18. How did you know/learn about these strategies? 19. How effective are these strategies? 20. Do the strategies improve your livelihood situation? If yes, how? 21. Have this strategies/knowledge been shared in the family or community? If yes, how? 22. Have these strategies been changing due to the pattern of floods and/or droughts? If yes, how? 22. Have these strategies been changing due to the pattern of floods and/or droughts? If yes, how? 22. Have these strategies been changing due to the pattern of floods and/or droughts? If yes, how? Preparation for floods and/or droughts 9. Given the impact of floods and/or droughts on community’s daily livelihood, how do they prepare for them (before they occur)? What strategies do they use? 10. How long have the community been practicing these strategies/activities? 11. What strategies are being practicing now which were not done in the past (or vice versa)? 9. Given the impact of floods and/or droughts on community’s daily livelihood, how do they prepare for them (before they occur)? What strategies do they use? 10. How long have the community been practicing these strategies/activities? 11. What strategies are being practicing now which were not done in the past (or vice versa)? 11. What strategies are being practicing now which were not done in the past (or vice versa)? 12. How did the community know/learn about these strategies/activities? 13. How effective are these strategies? Appendix 7. Question guide for local authorities Appendix 7. Question guide for local authorities Future hazards and solutions/support 23. What do you think about possible changes in floods and/or droughts that may occur in the future? 24. How should you and the community prepare for the changes? 25. Do you think the livelihood strategy solutions should be done collectively in the community? If yes, how? 25. Do you think the livelihood strategy solutions should be done collectively in the community? If yes, how? 26. Any support that you think you would need from the government, local authorities or other development agencies? If yes, what are they? 129 Appendix 7. Question guide for local authorities Question guide for local authorities Date: ……………………. Name of respondent: …………………. Gender: ………. Occupation: …………………………… Climate Change and its impacts 1. What changes in climate patterns have you noticed in your area? 2. What about floods and/or droughts? Compare to the past 10 years, are they more serious? 3. What have been the major changes of floods and/or droughts? 4. How long have you noticed the changes? 5. How do they impact on this community? 6. What are the primary livelihoods of this community? 7. What are the secondary livelihoods of this community? 8. How do floods and/or droughts impact on communities’ livelihood? Strategies to respond to floods and/or droughts 14. In order to survive, what strategies do the community use during floods and/or droughts? 14. In order to survive, what strategies do the community use during floods and/or droughts? 15. How long have the community been practicing these strategies? 16. What strategies are being practicing now which were not done in the past (or vice versa)? 130 17. How did the community know/learn about these strategies? 18. How effective are these strategies? 19. Do the strategies improve communities’ livelihood situation? If yes, how? 20. Have this knowledge been shared in the family or community? If yes, how? 21. Have these strategies been changing due to the pattern of floods and/or droughts? If yes, how? 17. How did the community know/learn about these strategies? 18. How effective are these strategies? 21. Have these strategies been changing due to the pattern of floods and/or droughts? If yes, how? Future hazards and solutions/support 22. What do you think about possible changes in floods and/or droughts that may occur in the future? 23. How should the community prepare for the changes? 24. Do you think that the livelihood strategy solutions should be done collectively in the community? If yes, how? 24. Do you think that the livelihood strategy solutions should be done collectively in the community? If yes, how? 25. Does the commune/village have any plan to support communities’ livelihood in this village? If yes, how? 25. Does the commune/village have any plan to support communities’ livelihood in this village? If yes, how? 26. Does the government have any plan to support communities’ livelihood in this village? If yes, how? 26. Does the government have any plan to support communities’ livelihood in this village? If yes, how? 27. Are there any NGO working to improve local livelihood in this village? What are their activities? 28. Have there been activities of NGOs to improve communities’ understanding on livelihood activities to adapt to natural hazards? If yes, what are the activities? 29. Should indigenous knowledge be integrated into adaptation policy of the government? If yes, how? 131 Appendix 8. Question guide for NGO staff Question guide for NGO staff Date: ……………………. Name of respondent: …………………. Gender: ………. Occupation: …………………………… 1. How long have you been working in this area? 2. What are the project activities that your NGO is involved in in this area? 3. What changes in climate pattern have you noticed in your area? 4. Are floods and/or droughts serious problems in this community? Why? 5. Do you think the local farmers and fishermen are seriously affected by floods and/or droughts? If yes, in what ways are they affected? 6. What are the methods used by local communities to combat floods and/or droughts? 7. Which of the above methods involves indigenous knowledge and which methods provided by outsiders, such as development agencies and government officials? 8. What are the livelihood strategies developed by local people using their indigenous knowledge? 9. Do you think these livelihood strategies are effective enough for them to survive? If yes, why? 10. Does your NGO provide new knowledge for local people to use in order to improve their livelihood? What are they? 11. Is the new knowledge integrated with indigenous knowledge of local community? If yes, how? 12. Does your organisation have adaptation policy for local community who are Appendix 8. Question guide for NGO staff Appendix 8. Question guide for NGO staff Question guide for NGO staff Question guide for NGO staff Name of respondent: …………………. Gender: ………. Occupation: …………………………… 1. How long have you been working in this area? 2. What are the project activities that your NGO is involved in in this area? 3. What changes in climate pattern have you noticed in your area? 4. Are floods and/or droughts serious problems in this community? Why? 5. Do you think the local farmers and fishermen are seriously affected by floods and/or droughts? If yes, in what ways are they affected? 6. What are the methods used by local communities to combat floods and/or droughts? 7. Which of the above methods involves indigenous knowledge and which methods provided by outsiders, such as development agencies and government officials? 8. What are the livelihood strategies developed by local people using their indigenous knowledge? 9. Do you think these livelihood strategies are effective enough for them to survive? If yes, why? 10. Does your NGO provide new knowledge for local people to use in order to improve their livelihood? What are they? 11. Is the new knowledge integrated with indigenous knowledge of local community? If yes, how? 12. Does your organisation have adaptation policy for local community who are affected by floods or droughts? If yes, what are they? 13. How should indigenous knowledge should be integrated in adaptation policy? 14. How long has your NGO supported the local communities? 15. How long has this support been planned to last? 16. Do you think the NGO support will be sustainable? Why? 7. Which of the above methods involves indigenous knowledge and which methods provided by outsiders, such as development agencies and government officials? 132

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